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WO2021175192A1 - Treatment methods and biomarkers for mdm2 inhibitors - Google Patents

Treatment methods and biomarkers for mdm2 inhibitors Download PDF

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Publication number
WO2021175192A1
WO2021175192A1 PCT/CN2021/078476 CN2021078476W WO2021175192A1 WO 2021175192 A1 WO2021175192 A1 WO 2021175192A1 CN 2021078476 W CN2021078476 W CN 2021078476W WO 2021175192 A1 WO2021175192 A1 WO 2021175192A1
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WIPO (PCT)
Prior art keywords
cancer
stk11
lkb1
subject
ferroptosis
Prior art date
Application number
PCT/CN2021/078476
Other languages
French (fr)
Inventor
Yifan Zhai
Dajun Yang
Yilong WU
Hao Sun
Douglas Dong Fang
Qiuqiong TANG
Original Assignee
Ascentage Pharma (Suzhou) Co., Ltd.
Ascentage Pharma Group Corp Limited
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Application filed by Ascentage Pharma (Suzhou) Co., Ltd., Ascentage Pharma Group Corp Limited filed Critical Ascentage Pharma (Suzhou) Co., Ltd.
Publication of WO2021175192A1 publication Critical patent/WO2021175192A1/en

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Definitions

  • the present invention relates to treatment methods and biomarkers with MDM2 inhibitors to treat conditions and diseases wherein inhibition of MDM2 and MDM2-related proteins provides a benefit.
  • MDM2 Human murine double minute 2
  • p53 mediates growth arrest, senescence and apoptosis in response to a broad array of cellular damage and thereby prevents cancer.
  • MDM2 directly interacts with p53 and inactivates p53 through multiple mechanisms. Blocking the MDM2-p53 interaction to reactivate the p53 function is therefore a promising anticancer therapeutic strategy (Chesse P, Nature Reviews Cancer, 2003, 3: 102) .
  • a, ” “an, ” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • a method means one method or more than one method.
  • an MDM2 inhibitor or a pharmaceutically acceptable salt thereof is particularly effective in cancer patients with certain biomarker characteristics.
  • treatment with MDM2 inhibitor in a subject with a cancer characterized by certain biomarker can lead to an increase in response rate, more complete regression responders, delay in tumor growth, as well as conversion of resistance tumors into responding ones.
  • the present disclosure provides a method of identifying a subject with cancer as likely to respond to treatment with an MDM2 inhibitor, the method comprising:
  • the method further comprising:
  • the present disclosure provides a method of treating a subject with cancer, the method comprising:
  • the present disclosure provides a method of treating a subject with cancer, the method comprising administering to the subject with a therapeutically effective amount of an MDM2 inhibitor, wherein the subject has been determined to have deficiency in functional LKB1/STK11 in a biological sample from the subject.
  • the subject is relapsed from or refractory to an immunotherapy or chemotherapy.
  • the immunotherapy is PD-1/PD-L1 blockade therapy.
  • the subject has been determined to further have KRAS mutation.
  • said determining comprises detecting the presence of one or more inactivating mutations in LKB1/STK11 in the biological sample, wherein the presence of the one or more inactivating mutations in LKB1/STK11 is indicative of the deficiency in functional LKB1/STK11.
  • the one or more inactivating mutations in LKB1/STK11 comprises deletion, insertion, substitution or any combination thereof that reduces serine/threonine kinase activity of LKB1/STK11.
  • the one or more inactivating mutations in LKB1/STK11 comprises a mutation selected from the group of mutations as listed in Table 1.
  • said determining comprises determining if level of LKB1/STK11 is reduced in the biological sample relative to a reference level, and the reduced level of LKB1/STK11 is indicative of the deficiency in functional LKB1/STK11
  • said determining comprises determining if promoter of LKB1/STK11 is hypermethylated in the biological sample, wherein the hypermethylated LKB1/STK11 promoter is indicative of the deficiency in functional LKB1/STK11.
  • said determining further comprises determining in the biological sample presence or absence of functional p53, wherein the presence of functional p53 is indicative of likelihood to respond to the treatment with an MDM2 inhibitor.
  • the subject identified as likely to respond to the treatment with an MDM2 inhibit or the subject to be administered with the MDM2 inhibitor has, or is further determined to have, functional p53 in the biological sample.
  • the functional p53 comprises wild-type p53.
  • said determining further comprises determining in the biological sample presence of one or more mutations in KRAS, wherein the presence of one or more mutations in KRAS is indicative of likelihood to respond to the treatment an MDM2 inhibitor.
  • the subject to be administered with an MDM2 inhibitor has, or is further determined to have, one or more mutations in KRAS in the biological sample.
  • i) the deficiency in functional LKB1/STK11, ii) the presence or absence of functional p53, and/or iii) the one or more mutations in KRAS is measured by an amplification assay, a hybridization assay, a sequencing assay, or an immunoassay.
  • the biological sample comprises a cancer cell or a non-cancer cell.
  • the cancer is solid tumor or hematologic malignancy.
  • the cancer is gastric cancer (e.g. stomach cancer) , cholangiocarcinoma, lung cancer, melanoma, breast cancer (e.g. invasive breast carcinoma) , colon cancer, ovarian cancer, prostate cancer, liver cancer (e.g. hepatocellular carcinoma) , bladder cancer, pancreatic cancer, renal cancer, esophageal cancer, head and neck cancer, thyroid cancer, cutaneous squamous cell carcinoma, glioblastoma.
  • gastric cancer e.g. stomach cancer
  • cholangiocarcinoma cholangiocarcinoma
  • lung cancer e.g. invasive breast carcinoma
  • colon cancer ovarian cancer
  • prostate cancer e.g. hepatocellular carcinoma
  • bladder cancer e.g. hepatocellular carcinoma
  • pancreatic cancer renal cancer
  • renal cancer esophageal cancer
  • head and neck cancer thyroid cancer
  • thyroid cancer cutaneous squamous cell carcinoma
  • glioblastoma e.g
  • lymphoma e.g., mantel cell lymphoma, diffuse large B cell lymphoma
  • leukemia e.g., T-cell prolymphocytic leukemia, chronic lymphocytic leukemia, or acute myeloid leukemia
  • multiple myeloma uterine cancer, colorectal cancer, lung adenocarcinoma, uterine carcinosarcoma, lung squamous cell carcinoma, cervical cancer, esophagus cancer, sarcoma, chromophobe, renal cell carcinoma (RCC) , clear cell RCC, papillary RCC, uveal melanoma, testicular germ cell tumor, low grade glioma (LGG) , mesothelioma, pheochromocytoma and paraganglioma (PCPG
  • the cancer is selected from small cell lung carcinoma and non-small cell lung carcinoma (e.g. lung adenocarcinoma, lung squamous cell carcinoma, or lung large cell carcinoma) .
  • non-small cell lung carcinoma e.g. lung adenocarcinoma, lung squamous cell carcinoma, or lung large cell carcinoma
  • an immune therapy can comprise a modulator of immune checkpoint molecule.
  • immune checkpoint molecule include, without limitation, PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG3, CD160, 2B4, TGF ⁇ , VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CDS, ICAM-1, NKG2C, SLAMF7, NKp80, B7-H3, LFA-1, 1COS, 4-1BB, GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT or CD83 ligand, and preferably, the immune checkpoint molecule is PD-1, PD-L1 or CTLA-4.
  • the modulator of an immune checkpoint molecule can be an antibody, an antibody Fab fragment, a bivalent antibody, an antibody-drug conjugate, an scFv, a fusion protein, or a tetravalent antibody, and preferably, the modulator of an immune checkpoint molecule is a monoclonal antibody or an antigen-binding fragment thereof.
  • the modulator of an immune checkpoint molecule is pembrolizumab, ipilimumab, nivolumab, atezolizumab, avelumab, durvalumab, AGEN-1884, BMS-986016, CS1001 (WO2017020858A1, all of which is incorporated herein to its entirety) , CS-1002, LAG525, MBG453, MEDI-570, OREG-103/BY40, lirilumab, tremelimumab, JS001, SHR-1210, BGB-A317, IBI-308, REGN2810, JS003, SHR-1316, KN035 or BMS-936559, and preferably, the modulator of an immune checkpoint molecule is pembrolizumab.
  • the immunotherapy is PD-1/PD-L1 blockade therapy.
  • PD-1/PD-L1 blockade therapy can include, for example, anti-PD-1 antibody, or anti-PD-L1 antibody.
  • the subject has been determined to further have KRAS mutation.
  • the MDM2 inhibitors provided herein e.g. compounds of Formula (I) , and in particular Compound C
  • KRAS is also frequently co-mutated with LKB1/STK11 and the co-mutation has been implicated as a major driver of primary resistance to PD-1 blockade therapy (Skoulidis F, et al, Cancer Discov, (2016) 8: 822) .
  • the subject has been determined to have a wild-type or functional p53.
  • the present disclosure provides a method of inducing ferroptosis in a subject in need thereof comprising administering to the subject an effective amount of a MDM2 inhibitor.
  • the present disclosure provides a method for treating or ameliorating the effects of a condition that would benefit from ferroptosis in a subject in need thereof comprising administering to the subject a MDM2 inhibitor in an effective amount to induce ferroptosis.
  • the present disclosure provides a method of treating a subject identified as having ferroptosis-sensitive condition or cancer , comprising administering to the subject a MDM2 inhibitor in an effective amount to induce ferroptosis.
  • the induction of ferroptosis is determined by increase in lipid reactive oxygen species (ROS) .
  • the subject is identified as having a wild-type p53 or a p53 variant capable of regulating SLC7A11 expression.
  • the ferroptosis-sensitive condition or cancer is characterized in active or overactive lipid ROS production.
  • the ferroptosis-sensitive condition or cancer is characterized in having one or more functional or overactive genes or gene products selected from the group consisting of: heme carrier protein 1, integrin, ferroprotin, stimulator of Fe transport (SFT) , iron responsive element binding protein (IRP) , transferrin, DMT1, ACSL4, CARS, ALOXs, ATP5G3, CHAC1, CS, DPP4, GLS2, LPCAT3, NCOA4, RPL8, SAT1, TFRC, and TTC35/EMC2.
  • SFT stimulator of Fe transport
  • IRP iron responsive element binding protein
  • transferrin transferrin
  • the overactive genes or gene products are over-expressed or having an activating mutation.
  • the ferroptosis-sensitive condition or cancer is characterized in having reduced activity in one or more genes or gene products selected from the group consisting of: ZEB2, AKR1C1-3, SLC7A11, SLC3A2, GPX4, CD44v, CBS, CISD1, FANCD2, GCLC/GCLM, GSS, HMGCR, HSPB1/5, NFE2L2, and SQS.
  • the ferroptosis-sensitive cancer is characterized in having reduced activity in SLC7A11 and/or GPX4.
  • the genes or gene products having reduced activity are under-expressed or having an inactivating mutation.
  • the ferroptosis-sensitive condition or cancer is characterized in a high-mesenchymal cell state.
  • the high-mesenchymal cell state is characterized in upregulation of one or more genes selected from the group consisting of: CD133, CD44, VIM, FN1, ZEB1, TWIST1, SNAI2, and CDH2.
  • the ferroptosis-sensitive condition or cancer is characterized in one or more of the following: over-expression or activating mutation of RAS (e.g. KRAS and/or HRAS) , TFRC or MRP1, .
  • RAS e.g. KRAS and/or HRAS
  • TFRC TFRC or MRP1
  • the subject is further identified as having functional p53 (e.g. wild-type 53) and/or one or more mutations in KRAS in the biological sample.
  • functional p53 e.g. wild-type 53
  • one or more mutations in KRAS in the biological sample.
  • the subject is identified as having deficiency in functional LKB1/STK11.
  • the ferroptosis-sensitive condition or cancer comprises lung cancer, neuroblastoma, pancreatic cancer, acute myeloid leukemia, hepatocellular carcinoma, rhadomyoscarcoma, diffuse large B-cell lymphoma, renal cell carcinoma, prostate cancer, melanoma, fibrosarcoma, ovarian cancer, brain cancer or breast cancer.
  • the MDM2 inhibitor has an IC50 of no more than 1 ⁇ M (e.g. no more than 500 nM, 400 nM, 300 nM, 200 nM, 150 nM, 100 nM, 50 nM, 20 nM, 10 nM or 5 nM) in inhibiting the binding of MDM2 to p53 as determined by a fluorescence-polarization MDM2 binding assay.
  • 1 ⁇ M e.g. no more than 500 nM, 400 nM, 300 nM, 200 nM, 150 nM, 100 nM, 50 nM, 20 nM, 10 nM or 5 nM
  • the MDM2 inhibitor comprises a compound of formula (I) :
  • B is a C 4-7 carbocyclic ring
  • R 1 is H, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, OR a , or NR a R b ;
  • n 0, 1, or 2;
  • R 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 , and R 10 are selected from the group consisting of H, F, Cl, CH 3 , and CF 3 ;
  • R a is hydrogen or substituted or unsubstituted C 1-4 alkyl
  • R b is hydrogen or substituted or unsubstituted C 1-4 alkyl
  • R c and R d are substituents on one carbon atom of ring B, wherein
  • R c is H, C 1-3 alkyl, C 1-3 alkylene-OR a , OR a , or halo;
  • R d is H, C 1-3 alkyl, C 1-3 alkylene-OR a , OR a , or halo; or
  • R c and R d are taken together with the carbon to which they are attached to form a 4 to 6-membered Spiro substituent, optionally containing an oxygen atom;
  • R c and R d are F and F, H and H, OH and CH 3 , OH and H, CH 3 and CH 3 , CH 3 and OH, H and OH, CH 2 CH 3 and CH 2 CH 3 , or CH 2 OH and CH 2 OH.
  • R 2 is H; R 3 is halo; R 4 and R 5 are H.
  • the MDM2 inhibitor is a compound selected from:
  • the MDM2 inhibitor is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-oxidethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the MDM2 inhibitor is selected from the group consisting of idasanutlin (RG7388) , RG7112, HDM201, KRT-232, AMG 232, BI907828, SAR-405838 (MI-77301) , MK-8242 (SCH 900242) , DS3032-b, ALRN-6924 and CGM097; or a pharmaceutically acceptable salt of any of the foregoing.
  • the methods provided herein further comprise further administering an effective amount of one or more additional therapies.
  • the one or more additional therapies comprise a radiotherapy, chemotherapy, a targeted cancer therapy, or a therapy with a modulator of an immune checkpoint molecule.
  • the one or more additional therapies comprise administering an anti-PD-1 antibody, a Bcl-2 inhibitor, a FAK inhibitor, a MEK inhibitor, or a MET inhibitor.
  • the Bcl-2 inhibitor is compound D,
  • the FAK inhibitor is Compound E,
  • the MEK inhibitor is trametinib.
  • the additional therapy comprises an anti-cancer agent that does not induce ferroptosis.
  • the additional therapy comprises a ferroptosis inducing agent.
  • the ferroptosis inducing agent comprises RSL3, altretamine, artesunate, buthioninesulfoximine, BAY 87-2243, cyct (e) inase, DP17 erastin, FIN56, lanperisone, piperazine-coupled erastin, imidazole-ketone erastin, statins, sulfasalazine, sorafenib or withaferin A.
  • the ferroptosis inducing agent is lenvatinib
  • the statins is Atorvastatin.
  • the present disclosure provides a kit for predicting responsiveness of a subject with cancer to treatment with an MDM2 inhibitor, comprising
  • the kit further comprises:
  • one or more reagents for detecting presence or absence of functional p53 e.g. wild-type p53
  • the kit further comprises:
  • Figure 1A shows LKB1/STK11 mutations occur at a high frequency in lung adenocarcinoma and Figure 1B shows TP53 may co-mutate with LKB1/STK11 in lung adenocarcinoma.
  • FIG. 1 shows LKB1/STK11 mutants A549, NCI-H2122 and NCI-H460 are sensitive to Compound C.
  • Figure 3A-3H show treatment with Compound C increased lipid ROS level ( Figures 3A-3E) and suppressed the expression of SCL7A11 in cancer cells ( Figures 3F-3H) .
  • Figure 4 shows the results of flow cytometry.
  • Figure 5 shows the results of cell apoptosis after compound treatment.
  • Figure 6A shows the WB results of A549 cell line
  • figure 6B shows the cell survival rate
  • Figure 6C shows the flow cytometry results
  • Figure 6D shows the results of lipid ROS detected by attune NXT flow cytometry.
  • Figure 7 shows the results of cell apoptosis and relative lipid ROS.
  • Figure 8 shows the cell viability test results of compound C, trametinib alone or in combination for 72 hours.
  • Figure 9 shows the cell viability test results of compound C, trametinib alone or in combination for 72 hours.
  • Figure 10 shows the cell viability test results of compound C, trametinib alone or in combination for 72 hours.
  • Figure 11 are cell viability test results of compound C and compound D treated alone or in combination for 72 hours.
  • Figure 12 shows the results of cell viability test of compound C and compound D treated alone or in combination for 72 hours.
  • Figure 13 shows the cell viability test results of compound C and compound D treated alone or in combination for 72 hours.
  • Figure 14 shows the tumor volume changes of compound C in the treatment of lu5209.
  • Figure 15 shows the change of tumor volume after treatment with C, atorvastatin and lenvatinib alone or in combination.
  • Figure 16 shows the weight changes of experimental animals treated with compound C, atorvastatin and lenvatinib alone or in combination.
  • Figure 17 shows the change of tumor volume after compound C and compound E were treated alone or in combination.
  • Figure 18 shows the weight changes of experimental animals after compound C and compound E were treated alone or in combination.
  • Figure 19 shows the tumor volume changes of compounds C, RSL3 and atorvastain after treatment alone or in combination.
  • Figure 20 shows the weight changes of experimental animals treated with compounds C, rsl3 and atorvastain alone or in combination.
  • Figure 21 shows DNA coding sequences and protein (amino acids) sequences of biomarkers with SEQ ID numbers mentioned in the present disclosure
  • biomarker refers to a biological molecule that is a measurable indicator of some biological state or condition.
  • biomarker used herein is intended to encompass a polynucleotide of interest, or a polypeptide (for example encoded by the polynucleotide of interest) .
  • examples of biomarker provided herein can be a gene (e.g. genomic DNA, cDNA) or a product of the gene such as an mRNA transcribed from the gene, and a protein encoded by the gene.
  • specific examples of the biomarkers provided herein include LKB1/STK11, p53 and KRAS.
  • MDM2 as used herein is short for Murine Double Minute 2.
  • the term MDM2 is intended to encompass the MDM2 gene, as well as the MDM2 gene product (e.g. mRNA, protein) .
  • Exemplary sequence of human MDM2 is available under the NCBI accession number of ABT17086, ABT17084.1, ABT17085.1, or ABT17083.1.
  • level refers to the amount or quantity of the biomarker of interest present in a sample. Such amount or quantity may be expressed in the absolute terms, i.e., the total quantity of the biomarker in the sample, or in the relative terms, i.e., the concentration or percentage of the biomarker in the sample. Level of a biomarker can be measured at DNA level (for example, as represented by the amount or quantity or copy number of the gene in a chromosomal region) , at RNA level (for example as mRNA amount or quantity) , or at protein level (for example as protein or protein complex amount or quantity) .
  • DNA level for example, as represented by the amount or quantity or copy number of the gene in a chromosomal region
  • RNA level for example as mRNA amount or quantity
  • protein level for example as protein or protein complex amount or quantity
  • activity refers to the biological activity (e.g. catalytic or regulatory ability) of the proteins as described here.
  • “likelihood” and “likely” with respect to response of a subject to a treatment is a measurement of how probable the therapeutic response is to occur in the subject. It may be used interchangeably with “probability” . Likelihood refers to a probability that is more than speculation, but less than certainty. Thus, a therapeutic response is likely if a reasonable person using common sense, training or experience concludes that, given the circumstances, a therapeutic response is probable. In one embodiment, the term “likelihood” and “likely” denotes a chance in percent of how probable a therapeutic response is to occur.
  • a subject with cancer identified as “likely to respond” refers to a subject with cancer who has more than 30%chance, more than 40%chance, more than 50%chance, more than 60%chance, more than 70%chance, more than 80%chance, more than 90%chance of responding to a treatment.
  • beneficial response can be expressed in terms of a number of clinical parameters, including loss of detectable tumor (complete response) , decrease in tumor size and/or cancer cell number (partial response) , tumor growth arrest (stable disease) , enhancement of anti-tumor immune response, possibly resulting in regression or rejection of the tumor; relief, to some extent, of one or more symptoms associated with the tumor; increase in the length of survival following treatment; and/or decreased mortality at a given point of time following treatment. Continued increase in tumor size and/or cancer cell number and/or tumor metastasis is indicative of lack of beneficial response to treatment, and therefore decreased responsiveness.
  • cancer or “tumor” is a generic name for a wide range of cellular malignancies characterized by unregulated growth, lack of differentiation, and the potential or ability to invade local tissues and metastasize. These neoplastic malignancies affect, with various degrees of prevalence, every tissue and organ in the body. Cancer involves presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone or may circulate in the blood stream as independent cells, such as leukemic cells.
  • relapse refers to the regression of the subject’s illness back to its former diseased state, especially the return of symptoms following an apparent recovery or partial recovery. Unless otherwise indicated, relapsed state refers to the process of returning to or the return to illness before the previous treatment including, but not limited to, chemotherapies or immunotherapies.
  • refractory refers to the resistance or non-responsiveness of a disease or condition to a treatment (e.g., the number of neoplastic cells increases even though treatment if given) . Unless otherwise indicated, the term “refractory” refers a resistance or non-responsiveness to any previous treatment including, but not limited to, chemotherapies or immunotherapies.
  • immunotherapy refers to a therapy (e.g. anti-cancer therapy) comprising a modulator of an immune checkpoint molecule.
  • a “pharmaceutically acceptable” component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.
  • determining can be used interchangeably and refer to both quantitative and semi-quantitative determinations or qualitative determinations.
  • hybridizing refers to the binding, duplexing or pairing of at least partially complementary strands of nucleic acid molecules.
  • a nucleic acid strand can specifically hybridize to a target nucleic acid strand when there is sufficient degree of complementarity to avoid non-specific binding to non-target nucleic acid sequences.
  • nucleic acid and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA) , transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, single-stranded short or long RNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers.
  • the nucleic acid molecule may be linear or circular.
  • complementarity refers to the ability of baseparing between a nucleic acid sequence and another nucleic acid sequence via either traditional Watson-Crick or other non-traditional types. Complementarity can be partial or total. Partial complementarity occurs when one or more nucleic acid bases is not matched according to the base pairing rules. A percent complementarity indicates the percentage of nucleic acid base in a nucleic acid molecule which can form basepairs (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 base pairing out of 10 bases being 50%, 60%>, 70%>, 80%>, 90%, and 100%complementary) .
  • basepairs e.g., Watson-Crick base pairing
  • second nucleic acid sequence e.g., 5, 6, 7, 8, 9, 10 base pairing out of 10 bases being 50%, 60%>, 70%>, 80%>, 90%, and 100%complementary
  • prognosis refers to the prediction or forecast of the future course or outcome of a disease or condition.
  • a “protein” is a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds) . Proteins may include moieties other than amino acids (e.g., may be glycoproteins) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence) , or can be a functional portion thereof. Those of ordinary skill will further appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.
  • treating means reversing, alleviating, inhibiting the progress of, or preventing, either partially or completely, the growth of tumors, tumor metastases, or other cancer-causing or neoplastic cells in a subject.
  • “combination therapy” is understood as administration of two or more active agents using separate formulations or a single pharmaceutical formulation, or consecutive administration in any order such that, there is a time period while both (or all) active agents simultaneously exert their biological activities. It is contemplated herein that one active agent (e.g., an MDM2 inhibitor) can improve the activity of a second agent, for example, can sensitize target cells, e.g., cancer cells, to the activities of the second agent. Combination therapy does not require that the agents are administered at the same time, at the same frequency, or by the same route of administration.
  • one active agent e.g., an MDM2 inhibitor
  • target cells e.g., cancer cells
  • administer include any method of delivery of a pharmaceutical composition or agent into a subject's system or to a particular region in or on a subject.
  • the administration may be local or systemic.
  • the administration is by oral route or parental route (e.g. intravenous, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intraperitoneal, intranasal, or intraocular injection; direct injection to a tumor; or intravenous infusion) .
  • routes of administration include pulmonary administration, suppositories, and transdermal or transcutaneous applications. Administering an agent can be performed by a number of people working in concert.
  • Administering an agent includes, for example, prescribing an agent to be administered to a subject and/or providing instructions, directly or through another, to take a specific agent, either by self-delivery, e.g., as by oral delivery, subcutaneous delivery, intravenous delivery through a central line, etc.; or for delivery by a trained professional, e.g., intravenous delivery, intramuscular delivery, intratumoral delivery, continuous infusion, etc.
  • the term “subject” refers to a human or any non-human animal or mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) .
  • a subject is a human being.
  • a subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.
  • the term “subject” is used herein interchangeably with “individual” or “patient. ”
  • a subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • terapéuticaally effective amount or “effective amount” means the amount of a compound that that produces some desired local or systemic therapeutic effect at a reasonable benefit/risk ratio applicable to any treatment. When administered for preventing a disease, the amount is sufficient to avoid or delay onset of the disease. A therapeutically effective amount or an effective amount need not be curative or prevent a disease or condition from ever occurring. In certain embodiments, a therapeutically-effective amount of a compound will depend on its therapeutic index, solubility, and the like.
  • alkyl refers to straight chained and branched saturated C 1-10 hydrocarbon groups, including but not limited to methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, t-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 2, 2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 2-dimethylbutyl, 2, 3-dimethylbutyl, 3, 3-dimethylbutyl, and 2-ethylbutyl.
  • C m-n means the designated group has a range of carbon atoms from “m” to “n” , including the endpoints “m” and “n” .
  • alkylene refers to an alkyl group having a substituent.
  • An alkyl, e.g., methyl, or alkylene, e.g., -CH 2 -, group can be substituted with one or more, and typically one to three, of independently selected halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro, cyano, alkylamino, or amino groups, for example.
  • substituted as used herein with respect to a chemical group means that one or more hydrogen atoms on the chemical group is independently replaced with one or more substituents.
  • substituted as used herein, has the ordinary meaning known in the art and refers to a chemical moiety that is covalently attached to, or if appropriate, fused to, a parent group.
  • spiro refers to a ring system having two rings connected through only one carbon atom in common. Such cyclic moiety may essentially be a carbocyclic or heterocyclic ring. Spiro systems exclude other bicyclic compounds such as naphthalene which have two or more carbon atoms in common.
  • halo or halogen is defined as fluoro, chloro, bromo, or iodo.
  • cycloalkyl means a monocyclic or bicyclic, saturated or partially unsaturated, ring system containing three to eight carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, optionally substituted with one or more, and typically one to three, of independently selected halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro, cyano, alkylamino, or amino groups, for example.
  • heterocycloalkyl means a monocyclic or a bicyclic, saturated or partially unsaturated, ring system containing 4 to 12 total atoms, of which one to five of the atoms are independently selected from nitrogen, oxygen, and sulfur and the remaining atoms are carbon.
  • heterocycloalkyl groups are azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, dihydropyrrolyl, morpholinyl, thiomorpholinyl, dihydropyridinyl, oxacycloheptyl, dioxacycloheptyl, thiacycloheptyl, diazacycloheptyl, each optionally substituted with one or more, and typically one to three, of independently selected halo, C 1-6 alkyl, C 1-6 alkoxy, cyano, amino, carbamoyl, nitro, carboxy, C 2-7 alkenyl, C 2-7 alkynyl, or the like on an atom of the ring.
  • any of the recited numerical values may be the upper limit or lower limit of a numerical range. It is to be further understood that the invention encompasses all such numerical ranges, i.e., a range having a combination of an upper numerical limit and a lower numerical limit, wherein the numerical value for each of the upper limit and the lower limit can be any numerical value recited herein. Ranges provided herein are understood to include all values within the range. For example, 1-10 is understood to include all of the values 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and fractional values as appropriate. Similarly, ranges delimited by “at least” are understood to include the lower value provided and all higher numbers.
  • an element means one element or more than one element.
  • Described herein are methods of identifying a subject with cancer as likely to respond to treatment with an MDM2 inhibitor, methods of treating a subject with cancer with an MDM2 inhibitor, and kits for predicting responsiveness of a subject with cancer to treatment with an MDM2 inhibitor.
  • MDM2 inhibitors have been described previously as an anti-cancer therapeutic agent (See, e.g, U.S. Patent No. 9,745,314, the entire contents of which are incorporated herein by reference) , and are being evaluated in humans as mono-therapy or in combination with standard of care anticancer agents for treatment of diseases and conditions wherein inhibition of MDM2 and MDM2-related proteins activity provides a benefit.
  • an anti-cancer therapeutic agent See, e.g, U.S. Patent No. 9,745,314, the entire contents of which are incorporated herein by reference
  • MDM2 inhibitors have been described previously as an anti-cancer therapeutic agent (See, e.g, U.S. Patent No. 9,745,314, the entire contents of which are incorporated herein by reference) , and are being evaluated in humans as mono-therapy or in combination with standard of care anticancer agents for treatment of diseases and conditions wherein inhibition of MDM2 and MDM2-related proteins activity provides a benefit.
  • a method of identifying a subject with cancer as likely to respond to treatment with an MDM2 inhibitor comprising: providing a biological sample from the subject; determining in the biological sample if there is deficiency in functional LKB1/STK11; and identifying the subject as likely to respond to the treatment with an MDM2 inhibitor based on the deficiency in functional LKB1/STK11 found in the biological sample.
  • the method further comprises administering the MDM2 inhibitor in a therapeutically effective amount to the subject identified as likely to respond to the treatment with an MDM2 inhibitor.
  • a method of selecting a subject with cancer for treatment with an MDM2 inhibitor comprising: providing a biological sample from the subject; determining in the biological sample: if there is deficiency in functional LKB1/STK11; and selecting the subject for the treatment with an MDM2 inhibitor based on the deficiency in functional LKB1/STK11 found in the biological sample.
  • the method further comprises administering the MDM2 inhibitor in a therapeutically effective amount to the selected subject.
  • a method of predicting likelihood of responsiveness of a subject with cancer to treatment with an MDM2 inhibitor comprises: providing a biological sample from the subject; determining in the biological sample if there is deficiency in functional LKB1/STK11; and predicting the subject as likely to be responsive to the treatment with an MDM2 inhibitor based on the deficiency in functional LKB1/STK11 found in the biological sample.
  • a method of treating a subject with cancer with an MDM2 inhibitor comprises: determining in a biological sample from the subject if there is deficiency in functional LKB1/STK11; and administering the subject with an MDM2 inhibitor in a therapeutically effective amount based on the deficiency in functional LKB1/STK11 found in the biological sample.
  • a method of treating a subject with cancer with an MDM2 inhibitor comprising administering to the subject with a therapeutically effective amount of an MDM2 inhibitor, wherein the subject has been determined to have deficiency in functional LKB1/STK11 in a biological sample from the subject.
  • LKB1/STK11 refers to serine/threonine-protein kinase STK11, which is also known as, e.g., polarization-related protein LKB1, liver kinase B1, renal carcinoma antigen NY-REN-19, PJS.
  • LKB1/STK11 is intended to encompass the LKB1/STK11gene (e.g. genomic DNA, cDNA) , as well as the LKB1/STK11 gene product (e.g. mRNA transcribed from the gene, a protein encoded by the gene) .
  • LKB1/STK11 e.g. genomic DNA, cDNA
  • LKB1/STK11 gene product e.g. mRNA transcribed from the gene, a protein encoded by the gene
  • Exemplary sequence of human LKB1/STK11 is available in UniProtKB database under the accession number of Q15831 (STK11-HUMAN) , in the GenBank database under the NCBI accession number of AAB97833.1.
  • LKB1/STK11 as used herein is intended to encompass both wild-type LKB1/STK11 and variants of LKB1/STK11.
  • the gene of wild-type LKB1/STK11 comprises a DNA sequence of SEQ ID NO: 1.
  • the protein of wild-type LKB1/STK11 comprises an amino acid sequence of SEQ ID NO: 2.
  • variant refers to a gene or a gene product having substantially homologous sequences (e.g., coding sequences) to the corresponding wild-type gene or the product thereof, and having substantially similar function as that of the wild-type counterpart.
  • variants of a particular biomarker disclosed herein will have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more sequence identity to the sequence of the biomarker as determined by sequence alignment programs.
  • Variants can include, for example, allelic variants, conservative substitution variants and homologs that can be isolated/generated and characterized by methods known in the field.
  • LKB1/STK11 plays roles in various process such as cell metabolism, cell polarity, apoptosis and DNA damage response by activating targets including 5’-adenosine monophosphate-activated protein kinase (AMPK) and the AMPK-related kinases by direct phosphorylation (Williams T., et al., Trends in cell Biology, (2008) 18: 193) .
  • AMPK 5’-adenosine monophosphate-activated protein kinase
  • AMPK-related kinases by direct phosphorylation
  • the term “functional LKB1/STK11” means wild-type LKB1/STK11 and any LKB1/STK11 variant having at least 30%, 40%, 50%, 60%, preferably at least 70%, 80%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more activity of wild-type LKB1/STK11.
  • “deficiency” or “deficient” with respect to a biomarker refers to insufficiency in activity or level of the biomarker, and can include, for example, being less than normal activity or level, or being absent or null in activity or level of the biomarker.
  • the term “deficiency in functional LKB1/STK11” as used herein refers to insufficiency in level or activity of functional LKB1/STK11 in the biological sample.
  • the methods provided herein comprises providing a biological sample from the subject.
  • biological sample refers to a biological specimen taken by sampling from a subject, optionally with additional processing.
  • the sample can be a biological sample comprising cancer cells, or non-cancer cells.
  • non-cancer cells can be from the same tissue or organ as the cancer cells are also found.
  • the biological sample is a fresh or archived sample obtained from a tumor tissue, e.g., by a tumor biopsy or fine needle aspirate.
  • the sample can be any biological fluid containing cancer cells or non-cancer cells (e.g. peripheral blood mononuclear cells (PBMC) ) .
  • PBMC peripheral blood mononuclear cells
  • a sample from a subject is performed in accordance with the standard protocol generally followed by hospital or clinics, such as during a biopsy.
  • a biological sample include without limitation, bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, uterine or vaginal flushing fluids, plural fluid, ascetic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchioalveolar lavage fluid, etc., and tissues, such as biopsy tissue (e.g.
  • a biological sample comprises cells, tissue, blood, plasma, serum, urine, mouthwash, stool, saliva, and any combination thereof.
  • a biological sample is blood, plasma, serum, or urine.
  • a biological sample is blood.
  • a biological sample is tumor tissue.
  • the sample can be further processed by a desirable method for determining the genetic status, activity or level of the at least one biomarker (such as LKB1/STK11) .
  • a desirable method for determining the genetic status, activity or level of the at least one biomarker such as LKB1/STK11
  • the method further comprises isolating or extracting cancer cell (such as circulating tumor cell) from the biological fluid sample (such as peripheral blood sample) or the tissue sample obtained from the subject.
  • cancer cells can be separated by immunomagnetic separation technology such as that available from Immunicon (Huntingdon Valley, Pa. ) .
  • a tissue sample can be processed to perform in situ hybridization.
  • the tissue sample can be paraffin-embedded before fixing on a glass microscope slide, and then deparaffinized with a solvent, typically xylene.
  • the method further comprises isolating the nucleic acid from the sample, if RNA or DNA level of the biomarker is to be measured.
  • RNA or DNA level of the biomarker is to be measured.
  • Various methods of extraction are suitable for isolating the DNA or RNA from cells or tissues, such as phenol and chloroform extraction, and various other methods as described in, for example, Ausubel et al., Current Protocols of Molecular Biology (1997) John Wiley &Sons, and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3 rd ed. (2001) .
  • kits can also be used to isolate RNA, including for example, the NucliSens extraction kit (Biomerieux, Marcy l'Etoile, France) , QIAamp TM mini blood kit, Agencourt Genfind TM , mini columns (Qiagen) , RNA mini kit (Thermo Fisher Scientific) , and Eppendorf Phase Lock Gels TM .
  • NucliSens extraction kit Biomerieux, Marcy l'Etoile, France
  • QIAamp TM mini blood kit Agencourt Genfind TM , mini columns (Qiagen)
  • RNA mini kit Thermo Fisher Scientific
  • Eppendorf Phase Lock Gels TM Eppendorf Phase Lock Gels TM .
  • a skilled person can readily extract or isolate RNA or DNA following the manufacturer’s protocol.
  • the methods provided herein comprise determining in the biological sample if there is deficiency in functional LKB1/STK11.
  • Deficiency in functional LKB1/STK11 can be resulted from, for example, presence of one or more inactivating mutations in LKB1/STK11, presence of negative alteration (e.g. hypermethylation) in the promoter or other cis-regulatory elements of LKB1/STK11, reduced copy number of LKB1/STK11, reduced mRNA level or protein level of LKB1/STK11, and/or reduced protein activity of LKB1/STK11.
  • the deficiency in functional LKB1/STK11 can be indicated by the presence of one or more inactivating mutations in LKB1/STK11.
  • the methods provided herein can comprise the step of detecting the presence of one or more inactivating mutations in LKB1/STK11 in the biological sample, wherein the presence of the inactivating mutations in STK 11 is indicative of the deficiency in functional STK 11.
  • activating mutation refers to a mutation that results in at least partial (or complete) loss of function or activity of the gene or of the gene product of the biomarker, or results in a nonfunctional gene or gene product of the biomarker, or results in decreased gene expression (e.g. decrease LKB1/STK11 mRNA or protein abundance) .
  • the activity and/or level of the affected gene or gene product of the biomarker would be significantly lower than wild-type counterpart or even be eliminated.
  • the inactivating mutation reduces activity of LKB1/STK11, for example, serine/threonine kinase activity of LKB1/STK11. In certain embodiments, the inactivating mutation reduces LKB1/STK11 mRNA expression or protein expression.
  • an inactivating mutation in LKB1/STK11 can be a deletion, insertion, substitution or any combination thereof.
  • the inactivating mutation can affect, e.g., coding sequence, RNA splicing sites, promoter or other cis-regulatory elements.
  • the mutation is a large deletion of chromosome 19p13, where LKB1/STK11 resides. These large deletions can span the entire chromosome or an arm of the chromosome.
  • the mutation can be smaller, e.g., a deletion of less than 1,000 base pairs.
  • the smaller deletion can target one or only a few exons of LKB1/STK11 or can be a deletion in the promoter of LKB1/STK11 that does not target the coding sequence of LKB1/STK11.
  • the inactivating mutations can be point mutations and small insertion/deletion mutations.
  • substitution is a mutation that exchanges one nucleobase for another in a polynucleotide sequence, or that substitutes one amino acid residue for another in a polypeptide sequence.
  • Substitution in a polynucleotide sequence can: 1) change a codon to one that encodes a different amino acid residue, and therefore will cause change in amino acid sequence in the protein produced ( “missense mutation” ) , or 2) change to a codon that encodes the same amino acid residue thereby causing no change in the protein produced ( “synonymous mutation” ) ; or 3) change an amino-acid- coding codon to a single “stop” codon and cause an incomplete protein (an incomplete protein is usually nonfunctional) ( “nonsense mutation” ) .
  • an “insertion” is a mutation in which one or more extra nucleobase pairs are inserted into a place in a polynucleotide sequence, or in which one or more amino acid residue is inserted into a polypeptide sequence.
  • a “deletion” is a mutation in which one or more nucleobase pairs are lost or deleted from a polynucleotide sequence, or in which one or more amino acid residue are deleted from a polypeptide sequence.
  • insertion or deletion in a polynucleotide sequence may cause frame shift, which changes the reading frame of the codons and results in a completely different translated gene product from the original. This often generates truncated proteins that result in loss of function.
  • inactivating mutations in LKB1/STK11 include, without limitation, one or more mutations in Table 1.
  • the inactivating mutation in LKB1/STK11 comprises a mutation selected from the group of mutations as listed in Table 1.
  • substitution in a polypeptide sequence can be denoted as AnB, where “n” is a number indicating the n th amino acid residue in the polypeptide sequence, “A” is the amino acid residue at the n th residue in the wild-type polypeptide sequence, and “B” is the mutated amino acid residue at the n th residue.
  • “*” when the mutated residue is shown as “*” , it means a mutation leading to a nonsense codon in a nucleotide sequence that results in a truncated, incomplete polypeptide.
  • S19P denotes that the 19 th amino acid residue Serine (S) is changed to a Proline (P)
  • E33* denotes that the nucleotides encoding amino acid residue 33 (Glutamic acid, E) is changed to a stop codon and the resultant polypeptide is truncated.
  • Frame shift in a polypeptide is denoted by “AnBfs*m” , indicating a shift in the reading frame starting at the n th amino acid residue and terminating at the m th residues downstream that causes a premature termination of the protein, where “A” and “B” have the same meaning as described above.
  • “I35Lfs*127” denotes a frame shifting starting at the amino acid residue 35 (Isoleucine, I) as the first affected amino acid residue and terminating 127 residues downstream.
  • Deletion in a polypeptide is denoted by “del” after the amino acid residue number (s) flanking the deletion site.
  • “E98_G155del” denotes that amino acid residues 98-155 are deleted.
  • Insertion in a polypeptide sequence can be denoted by as “ins” after the amino acid residue number (s) flanking the insertion site, followed by the amino acid residue (s) inserted.
  • the inactivating mutation can be a single-copy or two-copy mutation.
  • the mutation can be inactivating even if it only causes haploinsufficiency.
  • the inactivating mutation can have a homozygous deletion mutation of LKB1/STK11, a deletion mutation of one allele, a deletion mutation of one allele and a point mutation of another allele of LKB1/STK11, or heterozygous mutations of LKB1/STK11.
  • the deficiency in functional LKB1/STK11 can be indicated by the presence of negative alteration (e.g. hypermethylation) in the promoter or other cis-regulatory elements of LKB1/STK11 in the biological sample.
  • the methods provided herein can comprise the step of determining if promoter or other cis-regulatory elements of LKB1/STK11 is negatively alternated in such a way that would lead to reduced expression of LKB1/STK11.
  • the methods of the present disclosure include determining if promoter of LKB1/STK11 is hypermethylated in the biological sample, wherein hypermethylation in the promoter is indicative of the deficiency in functional LKB1/STK11.
  • the deficiency in functional LKB1/STK11 can be indicated by the level of functional LKB1/STK11 in the biological sample. Accordingly, the methods provided herein can comprise the step of determining if level of functional LKB1/STK11 is reduced in the biological sample relative to a reference level, wherein the reduced level of LKB1/STK11 is indicative of the deficiency in functional LKB1/STK11.
  • the level of functional LKB1/STK11 includes gene copy number, mRNA expression level, or protein expression level of functional LKB1/STK11.
  • functional LKB1/STK11 comprises wild-type LKB1/STK11.
  • Copy number refers to the number of copies of a particular gene or a particular genomic sequence in the genome of an individual.
  • CNV Copy number variation
  • CNA copy number alteration
  • the methods of the present disclosure include measuring expression level or gene copy number variation of LKB1/STK11.
  • LKB1/STK11 mutations can lead to a reduction or loss of LKB1/STK11 mRNA or protein expression, and in some other cases, hypermethylation of the LKB1/STK11 promoter may also result in decreased mRNA or protein levels.
  • the methods of the present disclosure include measuring biological activity of LKB1/STK11. Methods of assaying, monitoring and modulating activity are described in e.g. US patent application US20050026233A1 or PCT application WO2004113562A1.
  • LKB1/STK11 activity in a biological sample can be assayed by contacting the sample with a substrate kinase under conditions that permit phosphorylation, and monitoring incorporation of phosphate into the substrate kinase, wherein the incorporation of phosphate into the substrate kinase indicates LKB1/STK11 activity.
  • the substrate kinase is or is derived from AMPK.
  • the step of determining further comprises determining in the biological sample if p53 is a functional p53, wherein presence of functional p53 is indicative of likelihood to respond to an MDM2 inhibitor.
  • functional p53 comprises wild-type p53.
  • p53 and “TP53” are used interchangeably herein, and are short for tumor protein p53.
  • Alternative names include, e.g., antigen NY-CO-13, phosphorprotein p53, tumor suppressor p53 and cellular tumor antigen p53.
  • Both TP53 and p53 can refer to the protein or the DNA or RNA sequence of the biomarker p53.
  • Exemplary sequence of human p53 is available in UniProtKB database under the accession number of P04637 (P53-HUMAN) , and in Genbank under the NCBI accession number of AYF55702.1, or AXU92429.1.
  • p53 is a transcription factor capable of regulating a number of genes that regulate e.g. cell cycle and apoptosis.
  • p53 protein is controlled by MDM2.
  • MDM2 By binding to p53, MDM2 inhibits p53 transactivation.
  • MDM2 as E3 ubiquitin ligase, also targets p53 to proteosomal cytosol degradation. Blocking p53-MDM2 interaction therefore can reduce the negative regulation on p53 function, and enable p53 to mediate its downstream functions. Presence of functional p53 is therefore suggested to be beneficial to treatment response to MDM2 inhibitor.
  • p53 used as the biomarker herein can be p53 protein as well as a polynucleotide (e.g.
  • DNA or RNA encoding the p53 protein.
  • the gene encoding p53 in certain embodiments can be referred to as TP53 in the present disclosure.
  • the gene of p53 comprises a gene sequence of SEQ ID NO: 3.
  • the protein of p53 comprises an amino acid sequence of SEQ ID NO: 4.
  • the term “functional p53” refers to wild-type p53 and mutant or allelic variants of p53 that retain at least about 5%of the activity of wild-type p53, e.g., at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of wild-type p53 activity.
  • the methods provided herein further comprise determining in the biological sample presence or absence of a functional p53 (e.g. wild-type p53 protein or TP53 gene) .
  • the methods of the present disclosure include determining in the biological sample if p53 is wild-type (e.g. wild-type p53 protein or TP53 gene) .
  • the subject to be administered with the MDM2 inhibitor has, or is further determined to have, functional p53 (e.g. wild-type p53 protein or TP53 gene) in the biological sample.
  • functional p53 e.g. wild-type p53 protein or TP53 gene
  • the step of determining further comprises determining in the biological sample if there is one or more inactivation mutations in KRAS.
  • KRAS GTPase KRas.
  • the term KRAS is intended to encompass the KRAS gene, as well as the KRAS gene product (e.g. mRNA, protein) .
  • Exemplary sequence of human KRAS is available in UniProtKB database under the accession number of P01116 (RASK-HUMAN) , in the GenBank database under the NCBI accession number of AAM12631.1.
  • KRAS protein converts GTP to GDP in order to modify transductive signals from the cytoplasm to the nucleus and is part of the RAS/MAPK signaling pathway that regulates cells proliferation and differentiation.
  • KRAS mutation has been considered as an indicator of poor response to cancer therapies, such as treatment with EGFR tyrosine kinase inhibitor in patient with non-small-cell lung cancer (Massarelli E, et al., Clinical Cancer Research (2007) 13: 2890) .
  • KRAS measured in the methods provided herein can be KRAS protein as well as a polynucleotide (e.g. DNA or RNA) encoding the KRAS protein.
  • the gene of KRAS comprises a gene sequence of SEQ ID NO: 5.
  • the protein of KRAS comprises an amino acid sequence of SEQ ID NO: 6.
  • the determining step further comprises determining in the biological sample presence of one or more mutations in KRAS, wherein the presence of one or more mutations in KRAS is indicative of likelihood to respond to the treatment an MDM2 inhibitor.
  • the subject to be administered with an MDM2 inhibitor has, or is further determined to have, one or more mutations in KRAS in the biological sample.
  • the biomarker LKB1/STK11 as provided herein, and in certain embodiments other biomarkers such as p53 and/or KRAS, are intended to encompass different forms including mRNA, protein and also DNA (e.g. genomic DNA) . Therefore, the level and/or activity of these biomarkers can be measured with RNA (e.g. mRNA) , protein or DNA (e.g. genomic DNA) of the respective biomarker. Similarly, mutation status and/or wild-type status of the biomarkers can also be measured with DNA (e.g. genomic DNA) , RNA (e.g. mRNA) , or protein (for example by measuring for an altered protein product encoded by the mutated gene) .
  • DNA e.g. genomic DNA
  • RNA e.g. mRNA
  • protein e.g. genomic DNA
  • Mutation status of a biomarker (including LKB1/STK11, p53 and/or KRAS as provided herein) at DNA or RNA level can be measured by any methods known in the art, for example, without limitation, an amplification assay, a hybridization assay, or a sequencing assay. Mutation status at protein level can be measured by any methods known in the art, for example, without limitation, immunoassays.
  • Expression level of a biomarker (including LKB1/STK11, p53 and/or KRAS as provided herein) at DNA or RNA level can be measured by any methods known in the art, for example, without limitation, an amplification assay, a hybridization assay, or a sequencing assay.
  • Expression level of a biomarker (including LKB1/STK11, p53 and/or KRAS as provided herein) at protein level can be measured by any methods known in the art, for example, without limitation, immunoassays.
  • Activity level of a biomarker can be measured by a suitable functional assay known in the art, for example, without limitation, by a phosphorylation assay.
  • a nucleic acid amplification assay involves copying a target nucleic acid (e.g. DNA or RNA) , thereby increasing the number of copies of the amplified nucleic acid sequence. Amplification may be exponential or linear. Exemplary nucleic acid amplification methods include, but are not limited to, amplification using the polymerase chain reaction ( “PCR” , see U.S.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • the target RNA of the biomarker is reverse transcribed to cDNA before the amplification.
  • Various reverse transcriptases may be used, including, but not limited to, MMLV RT, RNase H mutants of MMLV RT such as Superscript and Superscript II (Life Technologies, GIBCO BRL, Gaithersburg, Md. ) , AMV RT, and thermostable reverse transcriptase from Thermus thermophilus.
  • one method which may be used to convert RNA to cDNA is the protocol adapted from the Superscript II Preamplification system (Life Technologies, GIBCO BRL, Gaithersburg, Md.; catalog no. 18089-011) , as described by Rashtchian, A., PCR Methods Applic., 4: S83-S91, (1994) .
  • the expression level of RNA (e.g. mRNA) or the copy number variation of DNA of the biomarkers is quantified after the nucleic acid amplification assay.
  • the amplified products can be separated on an agarose gel and stained with ethidium bromide followed by detection and quantification using standard gel electrophoresis methods.
  • the amplified products can be integrally labeled with a suitable detectable label (e.g. a radio-or fluorescence nucleotide) and then visualized using x-ray film or under the appropriate stimulating spectra.
  • the expression level of RNA (e.g. mRNA) or the copy number variation of DNA of the biomarkers is quantified during the nucleic acid amplification assay, which is also known as real-time amplification or quantitative amplification.
  • Methods of quantitative amplification are disclosed in, e.g., U.S. Patent Nos. 6,180,349; 6,033,854; and 5,972,602, as well as in, e.g., Gibson et al., Genome Research (1996) 6: 995-1001; DeGraves, et al., Biotechniques (2003) 34 (1) : 106-10, 112-5; Deiman B, et al., Mol Biotechnol.
  • Quantification is usually based on the monitoring of the detectable signal representing copies of the template in cycles of an amplification (e.g., PCR) reaction.
  • Detectable signals can be generated by intercalating agents (e.g. SYBR GREEN TM and SYBR GOLD TM ) or labeled primer or labeled probes used during the amplification.
  • the labeled primer or labeled probe comprise a detectable label comprising a fluorophore.
  • the labeled primer or labeled probe may further comprise a quencher substance. Presence of both a fluorophore and a quencher substance ( “dual labeled” ) in one primer or probe could be helpful to provide for a self-quenching probe such as a TaqMan (U.S. Pat. Nos. 5,210,015 and 5,538,848) or Molecular Beacon probe (U.S. Pat. Nos.
  • the quencher substance and the fluorophore are in close proximity, such that when the fluorophore is excited by irradiation, it transfers energy to the quencher substance in the same probe via fluorescence resonance energy transfer (FRET) , thereby does not emit a signal.
  • FRET fluorescence resonance energy transfer
  • RNA e.g. mRNA
  • the copy number variation of DNA of the biomarkers can be quantified using methods known in the art.
  • the fluorescence signal can be monitored and calculated during each PCR cycle.
  • the threshold cycle, or Ct value can be further calculated.
  • Ct value is the cycle at which fluorescence intersects a predetermined value.
  • the Ct can be correlated to the initial amount of nucleic acids or number of starting cells using a standard curve.
  • a standard curve is constructed to correlate the differences between the Ct values and the logarithmic level of the measured biomarker.
  • the expression level or copy number variation of an internal control biomarker may be measured.
  • an internal control biomarker can be inherently present in the sample and its expression level or copy number variation can be used to normalize the measured expression level or copy number variation of the biomarkers of interest, to offset any difference in the absolute amount of the sample.
  • Nucleic acid hybridization assays use probes to hybridize to the target nucleic acid, thereby allowing detection of the target nucleic acid.
  • Non-limiting examples of hybridization assay include Northern blotting, Southern blotting, in situ hybridization, microarray analysis, and multiplexed hybridization-based assays.
  • the probes for hybridization assay are detectably labeled.
  • the nucleic acid-based probes for hybridization assay are unlabeled. Such unlabeled probes can be immobilized on a solid support such as a microarray, and can hybridize to the target nucleic acid molecules which are detectably labeled.
  • hybridization assays can be performed by isolating the nucleic acids (e.g. RNA or DNA) , separating the nucleic acids (e.g. by gel electrophoresis) followed by transfer of the separated nucleic acid on suitable membrane filters (e.g. nitrocellulose filters) , where the probes hybridize to the target nucleic acids and allows detection.
  • suitable membrane filters e.g. nitrocellulose filters
  • the hybridization of the probe and the target nucleic acid can be detected or measured by methods known in the art.
  • autoradiographic detection of hybridization can be performed by exposing hybridized filters to photographic film. Densitometric scanning of the photographic films exposed by the hybridized filters provides an accurate measurement of the target nucleic acid levels.
  • Computer imaging systems can also be used to quantify the level of the biomarker.
  • hybridization assays can be performed on microarrays.
  • Microarrays provide a method for the simultaneous measurement of the levels of large numbers of target nucleic acid molecules.
  • the target nucleic acids can be RNA, DNA, cDNA reverse transcribed from mRNA, or chromosomal DNA.
  • the target nucleic acids can be allowed to hybridize to a microarray comprising a substrate having multiple immobilized nucleic acid probes arrayed at a density of up to several million probes per square centimeter of the substrate surface.
  • the RNA or DNA in the sample is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative levels of the RNA or DNA. See, U.S. Patent Nos. 6,040,138, 5,800,992 and 6,020,135, 6,033,860, and 6,344,316.
  • arrays may be peptides or nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Patent Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992.
  • Arrays may be packaged in such a manner as to allow for diagnostics or other manipulation of an all-inclusive device.
  • Useful microarrays are also commercially available, for example, microarrays from Affymetrix, from Nano String Technologies, QuantiGene 2.0 Multiplex Assay from Panomics.
  • hybridization assays can be in situ hybridization assay.
  • In situ hybridization assay is useful to detect the presence of copy number variation (e.g. increase or amplification) at the locus of the biomarker of interest (e.g. LKB1/STK11) .
  • Probes useful for in situ hybridization assay can be locus specific probes, which hybridize to a specific locus on a chromosome to detect the presence or absence of a specific locus of interest (e.g. LKB1/STK11) .
  • Other types of probes may also be useful, for example, chromosome enumeration probes (e.g.
  • chromosome arm probes e.g. hybridizable to a chromosomal region and indicate the presence or absence of an arm of a specific chromosome.
  • Sequencing methods allow determination of the nucleic acid sequence of the target nucleic acid, and can also permit enumeration of the sequenced target nucleic acid, thereby measures the level of the target nucleic acid.
  • sequence methods include, without limitation, RNA sequencing, pyrosequencing, and high throughput sequencing.
  • High throughput sequencing involves sequencing-by-synthesis, sequencing-by-ligation, and ultra-deep sequencing (such as described in Marguiles et al., Nature 437 (7057) : 376-80 (2005) ) .
  • Sequence-by-synthesis involves synthesizing a complementary strand of the target nucleic acid by incorporating labeled nucleotide or nucleotide analog in a polymerase amplification. Immediately after or upon successful incorporation of a label nucleotide, a signal of the label is measured and the identity of the nucleotide is recorded. The detectable label on the incorporated nucleotide is removed before the incorporation, detection and identification steps are repeated.
  • Sequencing-by-synthesis may be performed on a solid surface (or a microarray or a chip) using fold-back PCR and anchored primers.
  • Target nucleic acid fragments can be attached to the solid surface by hybridizing to the anchored primers, and bridge amplified. This technology is used, for example, in the sequencing platform.
  • Pyrosequencing involves hybridizing the target nucleic acid regions to a primer and extending the new strand by sequentially incorporating deoxynucleotide triphosphates corresponding to the bases A, C, G, and T (U) in the presence of a polymerase. Each base incorporation is accompanied by release of pyrophosphate, converted to ATP by sulfurylase, which drives synthesis of oxyluciferin and the release of visible light. Since pyrophosphate release is equimolar with the number of incorporated bases, the light given off is proportional to the number of nucleotides adding in any one step. The process is repeated until the entire sequence is determined.
  • the detection of mutation and/or wild-type status and the measurement of level of biomarkers of interest described herein is by whole transcriptome sequencing, or RNA sequencing (e.g. RNA-Seq) .
  • RNA sequencing e.g. RNA-Seq
  • the method of RNA sequencing has been described (see Wang Z, Gerstein M and Snyder M, Nature Review Genetics (2009) 10: 57-63; Maher CA et al., Nature (2009) 458: 97-101; Kukurba K &Montgomery SB, Cold Spring Harbor Protocols (2015) 2015 (11) : 951-969) .
  • mRNA extracted from a sample is reverse transcribed into cDNA and sheared into fragments. Fragments within proper length ranges are selected and ligated with sequencing adaptors, followed by amplification, sequencing, and mapping reads to a reference genome.
  • the CNV of a biomarker is determined using whole exome sequencing (WES) .
  • WES involves sequencing DNA exons (i.e. protein encoding regions) using high-throughput sequencing technology. More details of WES can be found, for example, in Ng SB et al, Nature. 461 (7261) : 272–276 (2009) , and Bao R et al, Cancer Inform. 2014; 13 (Suppl 2) : 67–82, which are incorporated herein to their entirety.
  • Immunoassays typically involves using antibodies that specifically bind to the biomarker polypeptide or protein (e.g. the LKB1/STK11, KRAS, and/or p53 protein as provided herein) to detect or measure the presence or level of the target polypeptide or protein.
  • biomarker polypeptide or protein e.g. the LKB1/STK11, KRAS, and/or p53 protein as provided herein
  • Such antibodies can be obtained using methods known in the art (see, e.g., Huse et al., Science (1989) 246: 1275-1281; Ward et al, Nature (1989) 341 : 544-546) , or can be obtained from commercial sources.
  • immunoassays include, without limitation, Western blotting, enzyme-linked immunosorbent assay (ELISA) , enzyme immunoassay (EIA) , radioimmunoassay (RIA) , sandwich assays, competitive assays, immunofluorescent staining and imaging, immunohistochemistry (IHC) , and fluorescent activating cell sorting (FACS) .
  • ELISA enzyme-linked immunosorbent assay
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • sandwich assays sandwich assays
  • competitive assays sandwich assays
  • immunofluorescent staining and imaging immunohistochemistry
  • IHC immunohistochemistry
  • FACS fluorescent activating cell sorting
  • the immunoassays can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980) ; and Harlow &Lane, supra.
  • Enzyme Immunoassay Maggio, ed., 1980
  • Harlow &Lane supra.
  • Methods in Cell Biology Antibodies in Cell Biology, volume 37 (Asai, ed. 1993) ; Basic and Clinical Immunology (Stites &Terr, eds., 7 th ed. 1991) .
  • the antibodies are detectably labeled, or alternatively are not labeled but can react with a second molecule which is detectably labeled (e.g. a detectably labeled secondary antibody) .
  • a detectably labeled secondary antibody e.g. a detectably labeled secondary antibody
  • Other detection systems such as time-resolved fluorescence, internal-reflection fluorescence, amplification (e.g., polymerase chain reaction) and Raman spectroscopy are also useful.
  • the antibodies may be immobilized on a solid substrate.
  • the immobilization can be via covalent linking or non-covalent attachment (e.g. coating) .
  • solid substrate include porous and non-porous materials, latex particles, magnetic particles, microparticles, strips, beads, membranes, microtiter wells and plastic tubes. The choice of solid phase material and method of detectably labeling the antigen or antibody reagent are determined based upon desired assay format performance characteristics.
  • the biological activity of a protein can be measured using a bioassay.
  • LKB1/STK11 activities in a biological sample can be determined by contacting the sample with a substrate kinase (e.g. AMPK) under conditions that permit phosphorylation, and monitoring incorporation of phosphate into the substrate kinase, wherein the incorporation of phosphate into the substrate kinase indicates LKB1/STK11 activity;
  • the activity of p53 can be measured by detecting the phosphorylation of the amino acid residue at position 15 of p53, or by detecting the change in expression level of the downstream target genes of p53. Due to a protein’s ability to exert multiple biological activities, several acceptable bioassays may exist for a particular protein.
  • Exemplary functional assays for measuring the activity of LKB1/STK11 or p53 can be found in Thompson T, et al, Journal Biological Chemistry, 279: 53015-53022 (2004) , US patent application US20050026233A1 or PCT application WO2004113562A1.
  • the method further comprises identifying the subject as likely to respond to the treatment with an MDM2 inhibitor based on the deficiency in functional LKB1/STK11 found in the biological sample.
  • detection of one or more inactivating mutation in LKB1/STK11 in the biological sample indicates deficiency of LKB1/STK11.
  • the subject is identified as likely to respond to the treatment of MDM2 inhibitor based on having one or more inactivating mutations in LKB1/STK11.
  • the one or more inactivating mutations are selected from mutations shown in Table 1.
  • the subject is identified as likely to respond to the treatment of MDM2 inhibitor based on a decrease (e.g. at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%decrease) in expression level or activity level of LKB1/STK11 relative to a reference level of LKB1/STK11 respectively.
  • the subject is identified as likely to respond to the treatment of MDM2 inhibitor based on decrease in copy number of LKB1/STK11 (e.g. 0, 1) relative to a reference copy number of LKB1/STK11.
  • the expression level or activity level or the copy number of LKB1/STK11 can be normalized to an internal control value or to a standard curve.
  • the expression level or activity level or the copy number of LKB1/STK11 can be normalized to a standard level for a standard marker.
  • the standard level of the standard marker can be predetermined, determined concurrently, or determined after a sample is obtained from the subject.
  • the standard marker can be run in the same assay or can be a known standard marker from a previous assay.
  • sequencing assay such as RNA sequencing
  • the term “reference level” of LKB1/STK11 can be the normal or baseline level (e.g. expression level, activity level or copy number) of LKB1/STK11, for example, a level of LKB1/STK11 in the healthy cell or tissue sample, or an average level of LKB1/STK11 in a general cancer patient population or in a cancer patient population of a particular cancer of interest.
  • the reference level can be a typical level, a measured level, or a range of the level of LKB1/STK11 that would normally be observed in one or more healthy cell or tissue samples, or in one or more control cell or tissue samples.
  • the reference level can be an average level of LKB1/STK11 in a healthy subject population, or in a general cancer patient population or in a cancer patient population of a particular cancer of interest. For example, it can be an empirical level of LKB1/STK11 that is considered to be representative of a control sample or a general cancer sample.
  • the reference level of LKB1/STK11 is obtained using the same or comparable measurement method or assay as used in the measurement of the level of LKB1/STK11 in the biological sample.
  • a “general cancer patient population” as used herein, refers to a population of cancer subjects or patients having different kinds of cancers.
  • a general cancer patient population may be a group of at least three (four, five, six, seven, eight, nine, ten, or more) types of cancer patients, with some patients having the first type of cancer, some having the second type of cancer, some having the third type of cancer, and so on.
  • a general cancer patient population can be a population having all kinds of cancers or a variety of cancer types.
  • the reference level can also be an empirical level considered representative of a general cancer patient population.
  • the reference level can be predetermined.
  • the reference level can be calculated or generalized based on measurements of LKB1/STK11 level in a collection of control biological samples (e.g. samples from healthy subjects, or samples from control cancer patients) .
  • the reference level can be based on statistics of the level of LKB1/STK11 generally observed in healthy subjects, or in general cancer patient population.
  • the method further comprises determining in the biological sample presence or absence of a functional p53. In certain embodiments, the method further comprises determining in the biological sample if p53 is wild-type. In certain embodiments, the method further comprises determining expression level of p53 or activity level of p53.
  • the method further comprises determining in the biological sample presence of one or more mutations KRAS.
  • the methods provided herein further comprises administering the MDM2 inhibitor to the subject identified as likely to respond to the treatment with an MDM2 inhibitor.
  • the MDM2 inhibitor is administered at a therapeutically effective amount to the subject.
  • the present disclosure provides methods of treating a subject with cancer with an MDM2 inhibitor, wherein the subject has been identified as likely to respond to the treatment with MDM2 inhibitors by any of the methods provided herein.
  • the step of treating comprising administering a therapeutically effective amount of the MDM2 inhibitor to the subject having been identified as likely to respond to the treatment with the MDM2 inhibitors.
  • the subject to be administered with the MDM2 inhibitor is determined to have, deficiency in functional LKB1/STK11 in the biological sample.
  • the subject to be administered with the MDM2 inhibitor has, or is determined to have, functional p53 in the biological sample.
  • the subject to be administered with the MDM2 inhibitor has, or is determined to have, one or more mutations in KRAS in the biological sample.
  • the subject is relapsed from or refractory to an immunotherapy.
  • the immunotherapy is PD-1/PD-L1 blockade therapy.
  • the subject has been determined to further have a KRAS mutation.
  • the present disclosure also provides methods of inducing ferroptosis in a subject in need thereof comprising administering to the subject an effective amount of a MDM2 inhibitor provided herein, and optionally, the MDM2 inhibitor has a compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • the present disclosure also provides methods for treating or ameliorating the effects of a condition that would benefit from ferroptosis in a subject in need thereof comprising administering to the subject a MDM2 inhibitor provided herein in an effective amount to induce ferroptosis, and optionally, the MDM2 inhibitor has a compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • the present disclosure also provides methods for treating a subject identified as having ferroptosis-sensitive condition or cancer comprising administering to the subject a MDM2 inhibitor provided herein in an effective amount to induce ferroptosis, and optionally, the MDM2 inhibitor has a compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • ferroptosis refers to a type of programmed cell death dependent on iron and is genetically and biochemically distinct from other forms of regulated cell death such as apoptosis, necrosis, and autophagy (see e.g. Dixon SJ et al, Cell (2012) 149: 1060-72) .
  • Ferroptosis is characterized by the overwhelming, iron- dependent accumulation of lethal lipid reactive oxygen species (lipid ROS) , a process in which free radicals “steal” electrons from the lipids in cell membranes and promotes their oxidation by oxygen, resulting in cell damage.
  • lipid ROS lethal lipid reactive oxygen species
  • the subject is identified as having a wild-type p53 or a p53 variant capable of regulating SLC7A11 expression.
  • MDM2 increases expression of p53, which in turn can regulate expression of SLC7A11.
  • some p53 variants or mutants can also regulate SLC7A11, for example, the acetylation-defective mutant p53 3KR (see, e.g. L. Jiang et al., Nature. 520 (7545) : 57-62 (2015) ) , and such p53 variants or mutants are also encompassed in the present disclosure.
  • the MDM2 inhibitors provided herein are administered at an effective amount to induce ferroptosis.
  • Ferroptosis can be detected by the measurement of lipid ROS. Induction of ferroptosis is determined by increase in lipid ROS. Any suitable methods for detecting lipid ROS can be used to detect ferroptosis.
  • An exemplary method uses C11-BODIPY TM 581/591, which is a fluorescent fatty acid analogue that allows the quantification of lipid peroxidation by indirect measure of ROS production in cells and membranes. Upon free radical-induced oxidation, its fluorescent properties shift from red to green.
  • the measurement of lipid ROS by C11-BODIPY TM 581/591 can be carried out following the manufacturer’s instructions.
  • GSH depletion, glutamate release, and NADPH and cystine uptake assay can also be used as the measurement for ferroptosis (Xie Y, et al, Cell Death Differ., (2016) 23: 369) .
  • ferroptosis-sensitive means, that the cell or condition that is susceptible to or treatable by ferroptosis-induced cell death.
  • the ferroptosis-sensitive condition or cancer is characterized in active or overactive lipid ROS production.
  • active lipid ROS production it is intended to mean that the biological pathway for lipid ROS production is not significantly inhibited or suppressed, relative to a control (e.g., in a normal cell) .
  • overactive lipid ROS production it is intended to mean that the lipid ROS production pathway has an elevated activity than a control (e.g. in a normal cell) , for example, due to suppression or inhibition or absence of a negative regulator in the pathway, or due to an increased expression level or increased activity of a gene or a protein that is not a negative regulator in the pathway.
  • ferroptosis sensitivity is modulated by several pathways and processes, for example glutathione metabolism (e.g. cysteine transportation and biosynthesis of glutathione) , lipid metabolism (e.g. biosynthesis and peroxidation of polyunsaturated fatty acids (PUFAs) ) , iron metabolism (e.g.
  • glutathione metabolism e.g. cysteine transportation and biosynthesis of glutathione
  • lipid metabolism e.g. biosynthesis and peroxidation of polyunsaturated fatty acids (PUFAs)
  • iron metabolism e.g.
  • ferroptosis is an iron-dependent cell death, without having to be bound by theory, sensitivity to ferroptosis can be at least partly attributable to the amount of iron that is available in cells. Iron can generate reactive oxygen species (ROS) that can damage organelles. Proteins or genes that regulate iron availability by uptake, export, or a shift from storage to the labile iron pool (LIP) can affect the sensitivity of cells to ferroptosis. Such proteins or genes include, without limitation, heme carrier protein 1, integrin, ferroprotin, stimulator of Fe transport (SFT) , iron responsive element binding protein (IRP) , TFRC, transferrin, and DMT1. For example, over-expression or activating mutation of TFRC, DMT1, and/or transferrin can be indicative of sensitivity to ferroptosis.
  • ROS reactive oxygen species
  • proteins or genes include, without limitation, heme carrier protein 1, integrin, ferroprotin, stimulator of Fe transport (SFT) , iron responsive element binding protein
  • proteins or genes that regulate generation of lipid ROS or lipid synthesis or metabolism can also affect the sensitivity of cells to ferroptosis.
  • proteins or genes include, without limitation, SLC7A11, GPX4, FSP1/AIFM2, and SAT1.
  • underexpression or inactivating mutation of SLC7A11 or GPX4 can be indicative of sensitivity to ferroptosis (see, e.g. Kim JKM, et al, PNAS (2019) 116: 9433) ) .
  • the ferroptosis-sensitive condition or cancer is characterized in having one or more functional or overactive genes or gene products selected from the group consistingf: heme carrier protein 1, integrin, ferroprotin, stimulator of Fe transport (SFT) , iron responsive element binding protein (IRP) , transferrin, DMT1, ACSL4, CARS, ALOXs, ATP5G3, CHAC1, CS, DPP4, GLS2, LPCAT3, NCOA4, RPL8, SAT1, TFRC, and TTC35/EMC2.
  • SFT stimulator of Fe transport
  • IRP iron responsive element binding protein
  • transferrin transferrin
  • DMT1, ACSL4, CARS, ALOXs ATP5G3, CHAC1, CS, DPP4, GLS2, LPCAT3, NCOA4, RPL8, SAT1, TFRC, and TTC35/EMC2.
  • the overactive genes or gene products are over-expressed or having an activating mutation.
  • the ferroptosis-sensitive cancer is characterized in having reduced activity in one or more genes or gene products selected from the group consisting of: ZEB2, AKR1C1-3, SLC7A11, SLC3A2, GPX4, CD44v, CBS, CISD1, FANCD2, GCLC/GCLM, GSS, HMGCR, HSPB1/5, NFE2L2, and SQS.
  • the ferroptosis-sensitive cancer is characterized in having reduced level or activity in SLC7A11 and/or GPX4.
  • the genes or gene products having reduced activity are under-expressed or having an inactivating mutation.
  • underexpression refers to the presence of lower amount of the gene products, usually in a cancer cell, in comparison to a control (e.g. non-cancer cell) .
  • Underexpression of a gene or a gene product may be due to alteration (e.g. decrease) at the level of transcription, post transcriptional processing, translation, post-translational processing, cellular localization protein stability, as compared to a control.
  • Underexpression can be detected using conventional techniques for detecting gene products such as mRNA or proteins.
  • the underexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or less, or 1-fold, 2-fold, 3-fold, 4-fold or more fold lower as compared to a control.
  • overexpression refers to the presence of increased amount of the gene product, usually in a cancer cell, in comparison to a control (e.g. non-cancer cell) .
  • Overexpression of a gene or gene product (e.g. protein) may be due to alteration (e.g. increase) at the level of transcription, post transcriptional processing, translation, post-translational processing, cellular localization protein stability, as compared to a control.
  • Overexpression can be detected using conventional techniques for detecting gene products such as mRNA or proteins.
  • the overexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or more, or 1-fold, 2-fold, 3-fold, 4-fold or more fold higher as compared to a control.
  • activating mutation refers to a mutation that results in constitutive activation of a protein, and constitutive activation of a signaling pathway.
  • cancer biomarkers are also identified as potentially indicative of sensitivity to ferroptosis.
  • Such cancer biomarkers include, without limitation, RAS (e.g. HRAS and/or KRAS) , NRF2, MRP1 (multidrug resistance protein 1) .
  • RAS e.g. HRAS and/or KRAS
  • NRF2 nuclear factor receptor 1
  • MRP1 multidrug resistance protein 1
  • over-expression or activating mutation of RAS (e.g. HRAS and/or KRAS) , NRF2, and/or MRP1 can be indicative of sensitivity to ferroptosis (see, e.g., C. M .Bebber et al, Cancers (2020) : 12, 164) .
  • Cancer cells harboring oncogenic Ras are reported to be more sensitive to ferroptosis induction. (see e.g., Bebber CM et al, Cancers (2020) 12, 164, Yang WS, et al, Cell (2014) 156: 317-
  • cancers having a high-mesenchymal cell state are also ferroptosis-sensitive.
  • High-mesenchymal cell state as used herein is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids, as disclosed by V.S. Viswanathan et al. in Nature 547 (7664) , 453-457 (2017) .
  • Cancers having a high-mesenchymal cell state are dependent on GPX4 for survival.
  • cancers having a high-mesenchymal cell state are characterized in upregulation of one or more genes (e.g.
  • stemness markers or mesenchymal markers selected from the group consisting of: CD133, CD44, VIM, FN1, ZEB1, TWIST1, SNAI2, and CDH2. These markers have been disclosed in detail in V. S. Viswanathan et al., Nature 547 (7664) , 453-457 (2017) , and M.J. Hangauer et al., Nature 551, 247-250 (2017) , which are incorporated herein to its entirety. Without being limited to any theories, it is believed that the subjects are particularly sensitive or vulnerable to ferroptosis, thereby are likely to respond to the MDM2 inhibitors provided herein.
  • the subject is identified as having wild-type 53 or a p53 variant capable of regulating SLC7A11 expression, and optionally one or more mutations in KRAS in the biological sample.
  • the subject is identified as having wild-type 53 or a p53 variant capable of regulating SLC7A11 expression, and optionally as having high-mesenchymal cell state.
  • the high-mesenchymal cell state is characterized in upregulation of one or more genes selected from the group consisting of: CD133, CD44, VIM, FN1, ZEB1, TWIST1, SNAI2, and CDH2.
  • a ferroptosis-sensitive condition can be any condition that can be treated by inducing ferroptosis in the subject.
  • suitable conditions may involve elimination or growth arrest of unwanted cells (such as cancer) .
  • ferroptosis-sensitive conditions or cancers are characteristic of having over-expression or activating mutation of RAS (e.g. KRAS and/or HRAS) .
  • ferroptosis-sensitive conditions or cancers are characteristic of having functional p53 (e.g. wild-type 53) and/or one or more mutations in KRAS.
  • ferroptosis-sensitive conditions or cancers are further characterized in having deficiency in functional LKB1/STK11.
  • the MDM2 inhibitors disclosed in the present invention inhibit the interaction between p53 or p53-related proteins and MDM2 or MDM2-related proteins.
  • the MDM2 inhibitors of the present invention sensitize cells to inducers of apoptosis and/or cell cycle arrest, and/or ferroptosis.
  • the MDM2 inhibitors of the present invention induce apoptosis and/or cell cycle arrest, and/or ferroptosis.
  • MDM2 inhibitors can be determined by fluorescence-polarization MDM2 binding assay, a competitive binding assay between MDM2 inhibitors and a p53-based peptidomimetic compound competing for binding to a MDM2 protein as described in US patent 9,745,314B2.
  • Fluorescence polarization measurement of competitive binding works by titrating a mixture of a protein of interest and a fluorescently labeled probe with an unlabeled competitor and demonstrating that the fluorescence polarization decreases to the value observed with the free fluorescently labeled probe (Moerke N, Current Protocols in Chemical Biology, (2009) 1: 1) .
  • a fluorescence-polarization MDM2 binding assay a recombinant human His-tagged MDM2 protein (residue 1-118) and a fluorescently tagged p53-based peptide called PMDM6-F (Garcia-Echeverria et al., J. Med. Chem.
  • the MDM2 inhibitors has an IC50 of no more than 1 ⁇ M, e.g. no more than 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 150 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, in inhibiting the binding of MDM2 to P53 as determined by the fluorescence-polarization MDM2 binding assay.
  • IC50 of no more than 1 ⁇ M, e.g. no more than 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM,
  • MDM2 inhibitor is selected from idasanutlin (RG7388) , RG7112 (PubChem Compound CID: 57406853) , HDM201 (PubChem Compound CID: 71678098) , KRT-232 (also known as AMG232, PubChem Compound CID: 58573469) , AMG 232 (PubChem Compound CID: 58573469) , BI907828 (accessible in NCI Thesaurus (version: 19.10d) under code C156709) , SAR-405838 (also known as MI-77301, PubChem Compound CID: 53476877) , MK-8242 (also known as SCH 900242, accessible in NCI Thesaurus (version: 19.10d) under code C116867) , DS3032-b (PubChem Compound CID: 9051550) , ALRN-6924 (PubChem Compound CID: 38
  • the MDM2 inhibitor comprises a compound represented by formula (I) :
  • B is a C 4-7 carbocyclic ring
  • R 1 is H, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, OR a , or NR a R b ;
  • n 0, 1, or 2;
  • R 2 , R 3 , R 4 , R 5 , R 7 , R 8 , R 9 , and R 10 are selected from the group consisting of H, F, Cl, CH 3 , and CF 3 ;
  • R a is hydrogen or substituted or unsubstituted C 1-4 alkyl
  • R b is hydrogen or substituted or unsubstituted C 1-4 alkyl
  • R c and R d are substituents on one carbon atom of ring B, wherein
  • R c is H, C 1-3 alkyl, C 1-3 alkylene-OR a , OR a , or halo;
  • R d is H, C 1-3 alkyl, C 1-3 alkylene-OR a , OR a , or halo; or
  • R c and R d are taken together with the carbon to which they are attached to form a 4 to 6-membered Spiro substituent, optionally containing an oxygen atom;
  • B is
  • n is 0 or 1 and R 1 is H or CH 3 .
  • R 2 is H.
  • R 3 is halo, and preferably chloro.
  • R 4 is H, R 5 is H, or both R 4 and R 5 are H.
  • R 7 is halo, and more preferably is fluoro.
  • each of R 8 , R 9 , and R 10 are H.
  • R a and R b individually, are H, CH 3 , or CH 2 CH 3 .
  • R c and R d individually, are H, halo, OH, CH 3 , CH 2 CH 3 , or CH 2 OH.
  • R c and R d are F and F, H and H, OH and CH 3 , OH and H, CH 3 and CH 3 , CH 3 and OH, H and OH, CH 2 CH 3 and CH 2 CH 3 , and CH 2 OH and CH 2 OH.
  • the MDM2 inhibitor is a compound selected from
  • the MDM2 inhibitor is a compound having the following structure
  • the MDM2 inhibitor is a compound having the following structure (also known as Compound C)
  • MDM2 inhibitors More MDM2 inhibitors and the synthesis of the MDM2 inhibitors that can be used in the present application are further disclosed in U.S. Patent No. 9,745,314, which is incorporated herein by reference.
  • the MDM2 inhibitors provided herein can exist as salts.
  • Pharmaceutically acceptable salts of the MDM2 inhibitors provided herein often are preferred in the methods of the invention.
  • pharmaceutically acceptable salts refers to salts or zwitterionic forms of the compounds of structural formula (I) .
  • Salts of compounds of formula (I) can be prepared during the final isolation and purification of the compounds or separately by reacting the compound with an acid having a suitable cation, such as, but not limited to, alkali and alkaline earth metal ions, e.g., Na + , K + , Ca 2+ , and Mg 2+ well as organic cations such as, but not limited to, ammonium and substituted ammonium ions, e.g., NH 4 + , NHMe 3 + , NH 2 Me 2 + , NHMe 3 + and NMe 4 + . Examples of monovalent and divalent pharmaceutically acceptable cations are discussed, e.g., in Berge et al. J. Pharm. Sci., 66: 1-19 (1997) .
  • the pharmaceutically acceptable salts of compounds of structural formula (I) can be acid addition salts formed with pharmaceutically acceptable acids.
  • acids which can be employed to form pharmaceutically acceptable salts include inorganic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric.
  • Nonlimiting examples of salts of compounds of the invention include, but are not limited to, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2-hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerolphsphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pi
  • available amino groups present in the compounds of the invention can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides.
  • any reference to compounds of the present invention appearing herein is intended to include compounds of structural formula (I) as well as pharmaceutically acceptable salts thereof.
  • Stereoisomers are compounds that differ only in their spatial arrangement. Stereoisomers include all diastereomeric, enantiomeric, and epimeric forms as well as racemates and mixtures thereof.
  • geometric isomer refers to cyclic compounds having at least two substituents, wherein the two substituents are both on the same side of the ring (cis) or wherein the substituents are each on opposite sides of the ring (trans) .
  • a disclosed compound is named or depicted by structure without indicating stereochemistry, it is understood that the name or the structure encompasses one or more of the possible stereoisomers, or geometric isomers, or a mixture of the encompassed stereoisomers or geometric isomers.
  • geometric isomer When a geometric isomer is depicted by name or structure, it is to be understood that the named or depicted isomer exists to a greater degree than another isomer, that is that the geometric isomeric purity of the named or depicted geometric isomer is greater than 50%, such as at least 60%, 70%, 80%, 90%, 99%, or 99.9%pure by weight. Geometric isomeric purity is determined by dividing the weight of the named or depicted geometric isomer in the mixture by the total weight of all of the geomeric isomers in the mixture.
  • Racemic mixture means 50%of one enantiomer and 50%of is corresponding enantiomer.
  • a compound with one chiral center is named or depicted without indicating the stereochemistry of the chiral center, it is understood that the name or structure encompasses both possible enantiomeric forms (e.g., both enantiomerically-pure, enantiomerically-enriched or racemic) of the compound.
  • Enantiomeric and diastereomeric mixtures can be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent.
  • Enantiomers and diastereomers also can be obtained from diastereomerically-or enantiomerically-pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.
  • a compound When a compound is designated by a name or structure that indicates a single enantiomer, unless indicated otherwise, the compound is at least 60%, 70%, 80%, 90%, 99%or 99.9%optically pure (also referred to as “enantiomerically pure” ) .
  • Optical purity is the weight in the mixture of the named or depicted enantiomer divided by the total weight in the mixture of both enantiomers.
  • stereochemistry of a disclosed compound is named or depicted by structure, and the named or depicted structure encompasses more than one stereoisomer (e.g., as in a diastereomeric pair)
  • the stereoisomeric purity of the named or depicted stereoisomers at least 60%, 70%, 80%, 90%, 99%or 99.9%by weight.
  • the stereoisomeric purity in this case is determined by dividing the total weight in the mixture of the stereoisomers encompassed by the name or structure by the total weight in the mixture of all of the stereoisomers.
  • the MDM2 inhibitor is administered as a pharmaceutical composition.
  • the pharmaceutical composition can comprise an MDM2 inhibitor or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.
  • “Pharmaceutically acceptable carrier” and “pharmaceutically acceptable diluent” refer to a substance that aids the formulation and/or administration of an active agent to and/or absorption by a subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the subject.
  • Non-limiting examples of pharmaceutically acceptable carriers and/or diluents include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer’s solution) , alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, hydroxymethycellulose, fatty acid esters, polyvinyl pyrrolidine, and colors, and the like.
  • the carriers, diluents and/or excipients are “acceptable” in the sense of being compatible with the other ingredients of the pharmaceutical composition and not deleterious to the recipient thereof.
  • an effective, non-toxic amount of an MDM2 inhibitor e.g., Compound C
  • a therapeutically active amount of MDM2 inhibitor may vary according to factors such as the disease stage (e.g., stage I versus stage IV) , age, sex, medical complications (e.g., immunosuppressed conditions or diseases) and weight of the subject, and the ability of the MDM2 inhibitor (e.g., Compound C) to elicit a desired response in the subject.
  • a therapeutically active amount is a safe amount of MDM2 inhibitor which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this disclosure.
  • the dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or administered by continuous infusion or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • an MDM2 inhibitor e.g., Compound C
  • MDM2 inhibitor e.g., Compound C
  • acts as a therapeutic anti-cancer agent and not predominantly as an agent to ameliorate side effects of other chemotherapy or other cancer treatments.
  • an MDM2 inhibitor e.g., Compound C
  • an MDM2 inhibitor is administered in an amount that would be effective to improve or augment the immune response to the tumor.
  • the dosages provided below may be used for any mode of administration of MDM2 inhibitor (e.g., Compound C) , including topical administration, administration by inhalation, and intravenous administration (e.g. continuous infusion) .
  • the pharmaceutical composition comprises Compound C or a pharmaceutically acceptable salt thereof.
  • the pharmaceutical composition is in a solid dose form.
  • the solid dose form is capsules.
  • the solid dose form is dry-filled capsules.
  • the solid dose form is dry-filled size 1 gelatin capsules.
  • the capsule comprises from about 10-500 mg of an MDM2 inhibitor, such as Compound C.
  • the pharmaceutical composition or capsule comprises silicified microcrystalline cellulose.
  • the MDM2 inhibitor (e.g. Compound C) or a pharmaceutically acceptable salt thereof is administered in the range of about 0.5mg/kg to about 10000 mg/kg. In certain embodiments, the MDM2 inhibitor (e.g. Compound C) or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 mg/kg/day (24 hours) to about 150 mg/kg/day (24 hours) . In certain embodiments, the MDM2 inhibitor (e.g. Compound C) or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 mg/kg/week to about 700 mg/kg/week. In certain embodiments, the MDM2 inhibitor (e.g. Compound C) or a pharmaceutically acceptable salt thereof is administered orally every other day (QOD)
  • compositions or dosage regimens of MDM2 inhibitors contemplated for the treatment have been described previously (See, e.g, U.S. Patent No. 9,745,314, the entire contents of which are incorporated herein by reference) ..
  • the MDM2 inhibitor may also be administered in combination with one or more additional therapy to the subject identified as likely to respond to the treatment with an MDM2 inhibitor by any of the methods provided herein.
  • the method of treating a subject with cancer comprises to administering to the subject with a therapeutically effective amount of an MDM2 inhibitor in combination with one or more additional therapy, wherein the subject has been determined to have deficiency in functional LKB1/STK11 in a biological sample from the subject.
  • the one or more additional therapy comprises a radiotherapy, a chemotherapy, a targeted cancer therapy, or a therapy with a modulator of an immune checkpoint molecule.
  • the one or more additional therapy comprise administering an anti-PD-1 antibody, a Bcl-2 inhibitor, a FAK inhibitor, a MEK inhibitor, or a MET inhibitor. It is noted that the additional therapy may comprise administering traditional small organic chemical molecules or macromolecules such as a proteins, antibodies, peptibodies, DNA, RNA or fragments of such macromolecules.
  • the Bcl-2 inhibitor is compound D,
  • the FAK inhibitor is Compound E,
  • the MEK inhibitor is trametinib.
  • radiotherapy refers to the treatment of cancers with ionizing radiation.
  • chemotherapy refers to the treatment of cancers using specific chemical agents.
  • targeted cancer therapy refers to the treatment of cancers with agents (chemical compounds or macromolecules) that selectively interact with a chosen biomolecule.
  • an “immune checkpoint” or “immune checkpoint molecule” is a molecule in the immune system that modulates a signal.
  • An immune checkpoint molecule can be a co-stimulatory checkpoint molecule, i.e., turn up a signal, or an inhibitory checkpoint molecule, i.e., turn down a signal.
  • a “modulator of an immune checkpoint molecule” is an agent capable of altering the activity of an immune checkpoint in a subject.
  • methods as provided herein for inducing ferroptosis in a subject in need thereof further comprises administering to the subject an additional therapy.
  • the method as provided herein for treating or ameliorating the effects of a condition that would benefit from ferroptosis in a subject in need thereof further comprises administering to the subject an additional therapy.
  • the method as provided herein for treating a subject identified as having ferroptosis-sensitive condition or cancer further comprises administering to the subject an additional therapy.
  • the additional therapy comprises an anti-cancer agent that does not induce ferroptosis.
  • the MDM2 inhibitors can enhance the anti-cancer effects of such anti-cancer agents by providing a different mechanism to kill the cancer cells.
  • anti-cancer agents that do not induce ferroptosis include, for example, DNA damaging agents such as etoposide and doxorubicin. DNA damaging agents are believed to be unable to induce ferroptosis (see, e.g. L. Jiang et al, Nature. 2015 April 2; 520 (7545) : 57-62. )
  • the additional therapy comprises ferroptosis inducing agent.
  • ferroptosis inducing agent refers to agents that can either directly induce ferroptosis in cells or increase the sensitivity of cells to ferroptosis. Without being bound to any theory, it is believed that combination of the MDM2 inhibitors provided herein and additional ferroptosis inducing agent can provide additive or synergistic ferroptosis activity.
  • Ferroptosis inducing agents can act on multiple factors that are involved in the regulation of ferroptosis, for example, (i) RSL3 and/or other compounds which inhibit GPX4, (ii) erastin (e.g., and/or another compound which inhibits amino acid transporters system xc-) , and (iii) buthionine sulfoximine (BSO) which inhibits gamma-glutamylcysteine synthetase and production of glutathione.
  • RSL3 and/or other compounds which inhibit GPX4 erastin (e.g., and/or another compound which inhibits amino acid transporters system xc-)
  • BSO buthionine sulfoximine
  • ferroptosis inducing agents examples include RSL3, altretamine, artesunate, buthioninesulfoximine, BAY 87-2243, cyct (e) inase, DP17 erastin, FIN56, lanperisone, piperazine-coupled erastin, imidazole-ketone erastin, statins, sulfasalazine, sorafenib and withaferin A (Bebber CM et al, Cancers (2020) 12, 164, Xie Y, et al, Cell Death Differ., (2016) 23: 369) .
  • the ferroptosis inducing agent is lenvatinib
  • the statins is Atorvastatin.
  • the cancer is solid tumor or hematologic malignancy.
  • the cancer is selected from the group consisting of leukemia, a lymphoma, a melanoma, a carcinoma, and a sarcoma.
  • the cancer is selected from the group consisting of adrenal cortical cancer, advanced cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain/CNS tumors in adults, brain/CNS tumors in children, breast cancer, breast cancer in men, cancer in children, cancer of unknown primary, Castleman disease, cervical cancer, colon/rectum cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST) , gestational trophoblastic disease, head and neck cancer, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia-acute lymphocytic (ALL) in adults, leukemia-acute myeloid (AML) , leuhepatomakemia-chronic lymphocytic (CLL) , leukemia-
  • ALL
  • the cancer is selected from the group consisting of melanoma, Hodgkin lymphoma, lung cancer, kidney cancer, bladder cancer, head and neck cancer, Merkel cell carcinoma, urothelial carcinoma, solid tumors that are microsatellite instability-high or mismatch repair-deficient, sarcoma, colon cancer, prostate cancer, choriocarcinoma, breast cancer, retinoblastoma, stomach carcinoma, acute myeloid leukemia, lymphoma, multiple myeloma, and leukemia.
  • the cancer is selected from the group consisting of gastric cancer (e.g. stomach cancer) , cholangiocarcinoma, lung cancer, melanoma, breast cancer (e.g. invasive breast carcinoma) , colon cancer, ovian cancer, prostate cancer, liver cancer (e.g. hepatocellular carcinoma) , bladder cancer, pancreatic cancer, renal cancer, esophageal cancer, head and neck cancer, thyroid cancer, cutaneous squamous cell carcinoma, glioblastoma.
  • gastric cancer e.g. stomach cancer
  • cholangiocarcinoma cholangiocarcinoma
  • lung cancer e.g. invasive breast carcinoma
  • colon cancer e.g. hepatocellular carcinoma
  • ovian cancer e.g. hepatocellular carcinoma
  • bladder cancer e.g. hepatocellular carcinoma
  • pancreatic cancer renal cancer
  • renal cancer esophageal cancer
  • head and neck cancer thyroid cancer
  • thyroid cancer cutaneous squam
  • lymphoma e.g., mantel cell lymphoma, diffuse large B cell lymphoma
  • leukemia e.g., T-cell prolymphocytic leukemia, chronic lymphocytic leukemia, or acute myeloid leukemia
  • multiple myeloma ulterine cancel, colorectal cancer, lung adenocarcinoma, uterine carcinosarcoma, lung squamous cell carcinoma, cervical cancer, esophagus cancer, sarcoma, chromophobe, renal cell carcinoma (RCC) , clear cell RCC, papillary RCC, uveal melanoma, testicular germ cell tumor, low grade glioma (LGG) , mesothelioma, pheochromocytoma and paraganglioma (PC
  • the cancer is selected from small cell lung carcinoma and non-small cell lung carcinoma (e.g. lung adenocarcinoma, lung squamous cell carcinoma, lung large cell carcinoma) .
  • non-small cell lung carcinoma e.g. lung adenocarcinoma, lung squamous cell carcinoma, lung large cell carcinoma
  • the cancer is locally advanced or metastatic solid tumor or lymphoma.
  • the subject is treatment-experienced and shows disease progression. “Treatment-experienced” as used herein means that the subject has been treated with an anti-cancer therapy. Disease progression can be characterized by a sign of reduced responsiveness to the previous treatment, for example, increase in tumor size, increase in tumor cell number, or tumor growth.
  • the cancer is a ferroptosis-sensitive cancer comprising lung cancer, neuroblastoma, pancreatic cancer, acute myeloid leukemia, hepatocellular carcinoma, rhadomyoscarcoma, diffuse large B-cell lymphoma, renal cell carcinoma, prostate cancer, melanoma, fibrosarcoma, ovarian cancer, brain cancer or breast cancer.
  • ferroptosis-sensitive cancers can have a high-mesenchymal cell state.
  • the high-mesenchymal cell state is characterized in upregulation of one or more genes selected from the group consisting of: CD133, CD44, VIM, FN1, ZEB1, TWIST1, SNAI2, and CDH2.
  • administering results in one or more of, reducing tumor size, weight or volume, increasing time to progression, inhibiting tumor growth and/or prolonging the survival time of a subject having cancer.
  • the present disclosure further provides a kit for predicting responsiveness to treatment with MDM2 inhibitor, comprising one or more reagents for detecting deficiency in functional LKB1/STK11.
  • the kit comprises reagents for detecting presence of one or more inactivating mutation in LKB1/STK11, measuring expression or activity level of LKB1/STK11, measuring copy number of LKB1/STK11, or determining methylation status of the promoter of LKB1/STK11.
  • the kit further comprises one or more reagents for detecting presence or absence of functional p53 (e.g. wild-type p53) .
  • the kit further comprises one or more reagents for detecting the presence of one or more mutations in KRAS.
  • the kit for predicting responsiveness to treatment with MDM2 inhibitor comprises one or more reagents for detecting the function for production of lipid ROS.
  • the kit further comprises one or more reagents for detecting the over expression or activation mutation in the genes or gene products selected from the group consisting of: heme carrier protein 1, integrin, ferroprotin, stimulator of Fe transport (SFT) , iron responsive element binding protein (IRP) , transferrin, DMT1, ACSL4, CARS, ALOXs, ATP5G3, CHAC1, CS, DPP4, GLS2, LPCAT3, NCOA4, RPL8, SAT1, TFRC, and TTC35/EMC2.
  • heme carrier protein 1 integrin
  • ferroprotin stimulator of Fe transport
  • IRP iron responsive element binding protein
  • transferrin DMT1, ACSL4, CARS, ALOXs, ATP5G3, CHAC1, CS, DPP4, GLS2, LPCAT3, NCOA4, RPL8, SAT1, TFRC, and TTC35/EMC2.
  • the kit further comprises one or more reagents for detecting the underexpression or inactivating mutation in the genes or gene products selected from the group consisting of: ZEB2, AKR1C1-3, SLC7A11, SLC3A2, GPX4, CD44v, CBS, CISD1, FANCD2, GCLC/GCLM, GSS, HMGCR, HSPB1/5, NFE2L2, and SQS.
  • the kit further comprises one or more reagents for detecting the upregulation of genes or gene products selected from the group consisting of: CD133, CD44, VIM, FN1, ZEB1, TWIST1, SNAI2, and CDH2.
  • the kit further comprises one or more reagents for detecting the overexpression or activating mutation of RAS (e.g. KRAS and/or HRAS) , TFRC or MRP1.
  • RAS e.g. KRAS and/or HRAS
  • the measurement or detection can be at RNA level, DNA level and/or protein level. Suitable reagents for detecting target RNA, target DNA or target proteins can be used.
  • the detection reagents comprise primers or probes that can hybridize to the polynucleotide of the gene of interest (e.g., LKB1/STK11, p53 or KRAS) .
  • the detection reagents comprise antibodies that can specifically bind to the protein of interest (e.g., LKB1/STK11, p53 or KRAS) .
  • the primers, the probes, and/or the antibodies may or may not be detectably labeled.
  • kits may further comprise other reagents to perform the methods described herein.
  • the kits may include any or all of the following: suitable buffers, reagents for isolating nucleic acid, reagents for amplifying the nucleic acid (e.g. polymerase, dNTP mix) , reagents for hybridizing the nucleic acid, reagents for sequencing the nucleic acid, reagents for quantifying the nucleic acid (e.g. intercalating agents, detection probes) , reagents for isolating the protein, and reagents for detecting the protein (e.g. secondary antibody) .
  • suitable buffers e.g. polymerase, dNTP mix
  • reagents for hybridizing the nucleic acid e.g. polymerase, dNTP mix
  • reagents for sequencing the nucleic acid e.g. sequencing the nucleic acid
  • reagents for quantifying the nucleic acid e.g. inter
  • the reagents useful in any of the methods provided herein are contained in a carrier or compartmentalized container.
  • the carrier can be a container or support, in the form of, e.g., bag, box, tube, rack, and is optionally compartmentalized.
  • primer refers to oligonucleotides that can specifically hybridize to a target polynucleotide sequence, due to the sequence complementarity of at least part of the primer within a sequence of the target polynucleotide sequence.
  • a primer can have a length of at least 8 nucleotides, typically 8 to 70 nucleotides, usually of 18 to 26 nucleotides.
  • a primer can have at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%sequence complementarity to the hybridized portion of the target polynucleotide sequence.
  • Oligonucleotides useful as primers may be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts. (1981) 22: 1859-1862, using an automated synthesizer, as described in Needham-Van Devanter et al, Nucleic Acids Res. (1984) 12: 6159-6168.
  • Primers are useful in nucleic acid amplification reactions in which the primer is extended to produce a new strand of the polynucleotide.
  • Primers can be readily designed by a skilled artisan using common knowledge known in the art, such that they can specifically anneal to the nucleotide sequence of the target nucleotide sequence of the at least one biomarker provided herein.
  • the 3' nucleotide of the primer is designed to be complementary to the target sequence at the corresponding nucleotide position, to provide optimal primer extension by a polymerase.
  • probe refers to oligonucleotides or analogs thereof that can specifically hybridize to a target polynucleotide sequence, due to the sequence complementarity of at least part of the probe within a sequence of the target polynucleotide sequence.
  • exemplary probes can be, for example DNA probes, RNA probes, or protein nucleic acid (PNA) probes.
  • a probe can have a length of at least 8 nucleotides, typically 8 to 70 nucleotides, usually of 18 to 26 nucleotides.
  • a probe can have at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%sequence complementarity to hybridized portion of the target polynucleotide sequence.
  • antibody refers to an immunoglobulin or an antigen-binding fragment thereof, which can specifically bind to a target protein antigen.
  • Antibodies can be identified and prepared by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing animals such as rabbits or mice (see, e.g., Huse et al., Science (1989) 246: 1275-1281; Ward et al, Nature (1989) 341 : 544-546) .
  • the primes or probes provided herein comprise a polynucleotide sequence hybridizable to a portion within the sequence of SEQ ID NO: 1, 3 or 5.
  • the primes or probes provided herein comprise a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100%complementarity to a portion within the sequence of SEQ ID N.: 1, 3 or 5.
  • the antibodies provided herein comprise an antigen-binding region capable of specifically binding to an epitope within the protein or polypeptide having the sequence of SEQ ID NO: 2, 4 or 6.
  • the primers, the probes and the antibodies provided herein are detectably labeled.
  • the detectable label suitable for labeling primers, probes and antibodies include, for example, chromophores, radioisotopes, fluorophores, chemiluminescent moieties, particles (visible or fluorescent) , nucleic acids, ligand, or catalysts such as enzymes.
  • radioisotopes examples include, without limitation, 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C, 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi, and 32 P.
  • fluorophores examples include, without limitation, Acridine, 7-amino-4-methylcoumarin-3-acetic acid (AMCA) , BODIPY, Cascade Blue, Cy2, Cy3, Cy5, Cy7, Edans, Eosin, Erythrosin, Fluorescein, 6-FAM, TET, JOC, HEX, Oregon Green, Rhodamine, Rhodol Green, Tamra. Rox, and Texas Red TM (Molecular Probes, Inc., Eugene, Oreg. ) .
  • AMCA 7-amino-4-methylcoumarin-3-acetic acid
  • BODIPY Cascade Blue
  • enzymes include, without limitation, alkaline phosphatase, acid phosphatase, horseradish peroxidase, beta-galactosidase, and ribonuclease.
  • ligands include, without limitation, biotin, avidin, an antibody or an antigen.
  • a detectable label it may can react with a detectable partner or react with one or more additional compounds to generate a detectable signal.
  • the detectable label can be a ligand capable of functioning as a specific binding pair member for a labeled ligand (e.g. a secondary labeled antibody) .
  • enzymes are useful a detectable labels due to their catalytic activity to catalyze chromo-, fluoro-, or lumo-genic substrate which results in generation of a detectable signal.
  • the detectably labeled primers, probes or antibodies as provided herein can further comprise a quencher substance.
  • a quencher substance refers to a substance which, when present in sufficiently close proximity to a fluorescent substance, can quench the fluorescence emitted by the fluorescent substance as a result of, for example, fluorescence resonance energy transfer (FRET) .
  • FRET fluorescence resonance energy transfer
  • quencher substance examples include, without limitation, Tamra, Dabcyl, or Black Hole Quencher (BHQ, Biosearch Technologies) , DDQ (Eurogentec) , Iowa Black FQ (Integrated DNA Technologies) , QSY-7 (Molecular Probes) , and Eclipse quenchers (Epoch Biosciences) .
  • Primer and probes can be labeled to high specific activity by either the nick translation method or by the random priming method.
  • Useful probe labeling techniques are described in the literature (Fan, Y-S, Molecular cytogenetics: protocols and applications, Humana Press, Totowa, N.J. xiv, 411 (2002) ) .
  • kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods provided herein. While the instructional materials typically comprise written or printed materials they are not limited to such.
  • kits can further comprise a computer program product stored on a computer readable medium.
  • computer program product When computer program product is executed by a computer, it performs the step of identifying the subject as likely to respond to the treatment with an MDM2 inhibitor based on the deficiency in functional LKB1/STK11 found in the biological sample.
  • Any medium capable of storing such computer executable instructions and communicating them to an end user is contemplated by this invention.
  • Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips) , optical media (e.g., CD ROM) , and the like.
  • Such media may include addresses to internet sites that provide such instructional materials.
  • the computer programs may also be encoded and transmitted using carrier signals adapted for transmission via wired, optical, and/or wireless networks conforming to a variety of protocols, including the Internet.
  • a computer readable medium may be created using a data signal encoded with such programs.
  • Computer readable media encoded with the program code may be packaged with a compatible device or provided separately from other devices (e.g., via Internet download) . Any such computer readable medium may reside on or within a single computer product (e.g. a hard drive, a CD, or an entire computer system) , and may be present on or within different computer products within a system or network.
  • the present disclosure provides oligonucleotide probes attached to a solid support, such as an array slide or chip, e.g., as described in Eds., Bowtell and Sambrook DNA Microarrays: A Molecular Cloning Manual (2003) Cold Spring Harbor Laboratory Press. Construction of such devices are well known in the art, for example as described in US Patents and Patent Publications U.S. Patent No. 5,837,832; PCT application WO95/11995; U.S. Patent No. 5,807,522; US Patent Nos.
  • Nucleic acid arrays are also reviewed in the following references: Biotechnol Annu Rev (2002) 8: 85-101; Sosnowski et al. Psychiatr Genet (2002) 12 (4) : 181-92; Heller, Annu Rev Biomed Eng (2002) 4: 129-53; Kolchinsky et al., Hum.
  • LKB1/STK11 Mutations in LKB1/STK11 have been identified in various neoplasms, such as malignant melanoma (Guldberg, P. et al., (1999) 18: 1777) , breast cancer (Stephens P. et al., Nature (2005) 37: 590) , lung cancer (Shah U., et al, Cancer Res (2008) 68: 3562) and pancreatic cancer (Goggins, M. et al., (1999) 10: Suppl 4) .
  • LKB1/STK11 has been suggested to be relevant to resistance to immune checkpoint inhibitor therapy in patients with lung adenocarcinoma, especially when mutation (s) in KRAS are also present (Skoulidis F, et al., J Clin Oncol. 2017; 35 (suppl_15_abstr 9016) ) .
  • Patients with metastatic NSCLC characterized LKB1/STK11 mutations have also be found to be less likely to benefit from first-line chemotherapy (Shire N, et al. IASLC 2019 World Conference on Lung Cancer, September 7-10, 2019; Barcelona, Spain. Abstract OA07.02) .
  • FIG. 1A The data shown in Figure 1A demonstrates that LKB1/SKT11 mutations (including copy number variation (CNA) ) are most frequent in lung adenocarcinoma, occurring at a rate as high as 14%. Additionally, LKB1/STK11 mutations in lung adenocarcinoma may be accompanied with TP53 mutation, at a frequency of 36% ( Figure 1B) . In other words, the patients with LKB1/STK11 mutant and TP53 wild-type make up about 8.7%of the population of lung adenocarcinoma.
  • CNA copy number variation
  • the experiment is to determining the binding affinity of Compound C to MDM2 protein by a fluorescence polarization-based (FP-based) binding assay using a recombinant human His-tagged MDM2 protein (residues 1-118) and a fluorescently tagged p53-based peptide.
  • FP-based fluorescence polarization-based
  • the design of the fluorescence probe was based upon a previously reported high-affinity p53-based peptidomimetic compound called PMDM6-F (Garc ⁇ a-Echeverr ⁇ a et al., J. Med. Chem. 43: 3205-3208 (2000) ) .
  • the Kd value of PMDM6-F with the recombinant MDM2 protein was determined from the saturation curve.
  • MDM2 protein was serially double diluted in a Dynex 96-well, black, round-bottom plate, and the PMDM6-F peptide was added at 1 nM concentration.
  • the assay was performed in the buffer: 100 mM potassium phosphate, pH 7.5; 100 ⁇ g/mL bovine gamma globulin; 0.02%sodium azide, 0.01%Triton X-100) and the polarization values were measured after 3 h of incubation using an ULTRA READER (Tecan U.S. Inc., Research Triangle Park, NC) .
  • the IC50 value was obtained by fitting the mP values in a sigmoidal dose-response curve (variable slope) with a non-linear regression, and was determined to be 1.40 nM ⁇ 0.25.
  • L0/2 is the concentration of the free ligand (PMDM6-F) . Since PMDM6-F was used at a final concentration of 1 nM, L0/2 was 0.5 nM.
  • the controls included the MDM2 protein and PMDM6-F (equivalent to 0%inhibition) , PMDM6-F peptide alone (equivalent to 100%inhibition) .
  • the polarization values were measured after 3 h of incubation.
  • the IC50 values i.e., Compound C concentration at which 50%of bound peptide is displaced, were determined from a plot using nonlinear least-squares analysis. Curve fitting was performed using GRAPHPAD PRISM software (GraphPad Software, Inc., San Diego, Calif. ) .
  • the IC50 of Compound C was determined to be 3.8 nM.
  • Cell luminescence cell viability assay kit (Promega, Cat. #G7571) was used to quantify the anti-proliferative effect of Compound C.
  • Compound C (10 ⁇ M) was serially diluted in a 1: 3 ratios to obtain 5-7 series concentration. 10 ⁇ L/well of diluted Compound C solution was added into a 96-well plate while 10 ⁇ L/well of medium was added to control wells. Plates were incubated at 37°C in a 5%CO 2 incubator for 72 hours. Cell growth was observed daily under an inverted microscope.
  • 96-well plate was removed from the incubator and equilibrated to room temperature for 30 minutes before adding 30 ⁇ L Cell Titer- reagent (protected from light) to each well. Solutions were mixed thoroughly in each well to generate cell lysis and the 96-well plate was kept at room temperature for another 10 minutes to stabilize the luminescent signal. Luminescence signal was then detected using a Biotek synergy H1 microplate reader. Cell viability (%) was calculated using the mean luminescence (LN) value from control replicates (blank) using the following equation:
  • Cell proliferative (i.e., viability) curves were plotted using Graphpad Prism 6.0 software (Golden software, Golden, Colorado, USA) .
  • MDM2 inhibitor Compound C showed anti-cancer activity in all the cells having putative inactivating mutation in LKB1/STK11 and putative functional TP53.
  • KRAS is co-mutated in the cells having putative inactivating mutation in LKB1/STK11.
  • A549, NCI-H460, NCI-H292, NCI-H1944, NCI-H1666, DV90 and MOLM-13 cells were cultured and prepared for the treatment with Compound C as described in Example 2.
  • Compound C of different concentration and a vehicle control (DMSO) as indicated were tested.
  • the cells were harvested at the indicated times for the analysis.
  • Lipid ROS was detected by using BODIPY TM 581/591 C11 kit (Invitrogen, cat#D3861) according to the manufacturer’s instruction.
  • BODIPY 581/591 C11 staining the signals from both non-oxidized C11 (PE channel) and oxidized C11 (FITC channel) were monitored. The ratio of MFI of FITC to MFI of PE was calculated for each sample.
  • the PVDF membrane was then blocked with 1%BSA buffer for 1 hour at room temperature, and then it was incubated with the primary antibody anti-SLC7A11 (abcam, Cat. ab37185) , anti-p53 (CST, Cat. #2524S) or anti-p21 (CST, Cat. #2947S) diluted in 1%BSA TBST on a 4 °C shaker overnight. The membrane was washed for 10 minutes in 1 x TBST for 3 times. Then the PVDF membrane was incubated for 1 hour at room temperature with HRP (horse radish peroxidase) labeled secondary antibody (Yeasen, Cat. #33101ES60; Yeasen, Cat. #33201ES60) , which was prepared following the supplier's instruction. The membrane was washed for 10 minutes in 1 x TBST for 3 times again. HRP substrate was applied to the PVDF membrane. Signals were detected using ECL hypersensitive reagent.
  • Example 5 Responsiveness to the treatment with Compound C in LKB1/STK11 mutant PDX models.
  • the object of the study is to evaluate the anti-tumor activity of MDM2 inhibitor Compound C in LKB1/STK11 lung cancer PDX models.
  • mice female, 6-8W, 18-20g are inoculated subcutaneously at the right flank region with LKB1/STK11 mutant and TP53 wild-type A549 lung cancer cells (5 ⁇ 10 6 ) in Matrigel for tumor development.
  • An independent set of mice are inoculated H460 lung cancer cells. The treatments are initiated when the tumors reach suitable mean size. Both mice bearing A549 lung cancer cells and mice bearing H460 lung cancer cells are administered with vehicle or Compound C according to the experimental design shown in Table 3. After the treatment, fresh tumor tissues are collected for pharmacodynamic studies and serum samples are collected for pharmacokinetic studies.
  • Efficacy of the treatment with Compound C is further evaluated in additional LKB1/STK11 mutant and TP53 wild-type lung cancer PDX models available from Crowbio and Wuxi Biologics.
  • Compound C exerts effective anti-proliferative activity in LKB1/STK11 mutant and TP53 wild-type lung cancer PDX models.
  • Example 6 Compound C significantly induces apoptosis in STK11 mutant A549 and NCI-H460 cell lines
  • Apoptosis was detected using an Annexin V-PI (propidium iodide) staining kit. Briefly, cells were harvested 48 hours after the treatment and washed with PBS. Cells were then stained with Annexin-V and PI, analyzed by an Attune NxT flow cytometer following manufacturer’s instruction. Apoptosis data were obtained by analyzing 20,000 cells from each experimental condition. The data were analyzed by the FlowJo software.
  • Annexin V-PI propidium iodide
  • STK11 mutant A549 and NCI-H460 cell lines are sourced from ATCC.
  • Figure 6C detailed methods: for apoptosis analysis, STK11 null or overexpressed lung cancer cell lines were treated with COMPOUND C alone for 48 hours, then subjected to flow cytometry analysis using an Annexin V/fluorescein isothiocyanate (FITC) /propidium iodide (PI; Annexin V-FITC-PI) Apoptosis Detection Kit (BD Biosciences Cat#556547) . In brief, cells were washed once with cold PBS and incubated for 30 minutes on ice with Annexin V-FITC-PI in binding buffer.
  • FITC Annexin V/fluorescein isothiocyanate
  • PI idium iodide
  • BD Biosciences Cat#556547 Annexin V-FITC-PI
  • Figure 6 D detailed methods: Cells were seeded into 24-well plates and incubated overnight. Cells were treated with COMPOUND C for the indicated time, harvested by trypsinization and resuspended in 200 ⁇ L RPMI-1640 containing 5 ⁇ M C11-BODIPY 581/591 (Invitrogen, Cat#D3861, for lipid peroxidation detection) or 10 ⁇ M CM-H2DCFDA (Beyotime, Cat#S0033, for ROS detection) . Cells were incubated for 30 min at 37 °C in an incubator. Lipid peroxidation or ROS were assessed using the flow cytometer Attune NxT Flow Cytometer. A minimum of 3,0000 single cells were analyzed per well.
  • Compound C plus Compound D enhanced cell viability inhibition in A549, NCI-H1944, NCI-H2122 STK11 and KRAS co-mutated NSCLC cells after 72h combination treatment.
  • COMPOUND C single agent showed antitumor activity in LU5209 STK11mut LUAD PDX , T/C (%) value was 54%on Day 26.
  • Example 11 Combination treatment with COMPOUND C and other therapeutic agents in TP53 wt , STK11 mut LU5209 lung PDX model
  • COMPOUND C single agent showed minor antitumor activity
  • atorvastatin (selleck) single agent showed no antitumor activity
  • lenvatinib (selleck) single agent showed significantly antitumor activity.
  • COMPOUND C plus atorvastatin combination treatment achieved a synergistic antitumor effect.
  • COMPOUND C plus Lenvatinib combination treatment achieved an enhanced antitumor effect.
  • T/C (%) value of COMPOUND C and atorvastatin combination group was 41.32%on Day 42 compared to 74.04%or 92.86%from single agents groups, the synergy score was 1.66, indicating synergistic effects.
  • T/C (%) value of COMPOUND C and lenvastain combination group was 26.21%on Day 42 compared to 74.04%or 35.43%from single agents groups, the synergy score was 1.00.
  • T/C (%) value of COMPOUND C and COMPOUND E combination group was 43.85%on Day 53 compared to 78.91%or 71.66%from single agents groups, the synergy score was 1.29, indicating synergistic effects.
  • Example 13 Combination treatment with COMPOUND C and Compound E/RSL3/Atorvastatin in subcutaneous TP53 wt , KRAS mut , STK11 mut A549 lung cancer
  • A549 sourced from ATCC, animal model are sourced form Crownbio.
  • RSL3 100mg/kg intratumorally injected to the model BIW (twice per week) .
  • Atorvastain 100mg/kg, PO TIW (three times per week) .
  • COMPOUND C single agent showed antitumor activity
  • RSL3 (selleck) and atorvastatin (selleck) single agents showed minor antitumor activity.
  • COMPOUND C plus RSL3 or atorvastatin combination treatment achieved synergistic antitumor effect.
  • T/C (%) value of COMPOUND C and RSL3 combination group was 26.85%on Day 63 compared to 38.20%or 77.37%from single agents groups, the synergy score was 1.10, indicating synergistic effects.
  • T/C (%) value of COMPOUND C and atorvastatin combination group was 25.05%on Day 63 compared to 38.20%or 75.76%from single agents groups, the synergy score was 1.16, indicating synergistic effects.

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Abstract

Provided are biomarkers for predicting the efficacy of MDM2 inhibitors in treating cancer patients. Also provided are compositions, e.g., kits, for evaluating the biomarkers and methods of using the biomarkers to predict a cancer patient's response to the MDM2 inhibitors. Such information can be used in determining prognosis and treatment options for cancer patients.

Description

TREATMENT METHODS AND BIOMARKERS FOR MDM2 INHIBITORS FIELD OF THE INVENTION
The present invention relates to treatment methods and biomarkers with MDM2 inhibitors to treat conditions and diseases wherein inhibition of MDM2 and MDM2-related proteins provides a benefit.
BACKGROUND OF THE INVENTION
Human murine double minute 2 (MDM2) is an important negative regulator of the p53 tumor suppressor. p53 mediates growth arrest, senescence and apoptosis in response to a broad array of cellular damage and thereby prevents cancer. MDM2 directly interacts with p53 and inactivates p53 through multiple mechanisms. Blocking the MDM2-p53 interaction to reactivate the p53 function is therefore a promising anticancer therapeutic strategy (Chène P, Nature Reviews Cancer, 2003, 3: 102) .
However, clinical responses to anticancer therapies are often restricted to a subset of patients. To maximize the efficiency of anticancer therapy, personalized chemotherapy based on molecular biomarkers has been proposed. However, the identification of predicative biomarkers capable of predicting response to anticancer therapies still remains a challenge. Therefore, there is a continuing need for development of biomarkers for predicting anti-cancer efficacy of compounds targeting MDM2-p53 interaction.
SUMMARY OF THE INVENTION
Throughout the present disclosure, the articles “a, ” “an, ” and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a method” means one method or more than one method.
Throughout the present disclosure, all the references cited herein are incorporated in their entirety.
It has now been found by the inventors of the present application that the administration of an MDM2 inhibitor or a pharmaceutically acceptable salt thereof is particularly effective in cancer patients with certain biomarker characteristics. In particular, it is surprising to find that treatment with MDM2 inhibitor in a subject with a cancer characterized by certain biomarker can lead to an increase in response rate, more complete regression responders, delay in tumor growth, as well as conversion of resistance tumors into responding ones.
In one aspect, the present disclosure provides a method of identifying a subject with cancer as likely to respond to treatment with an MDM2 inhibitor, the method comprising:
a) providing a biological sample from the subject;
b) determining in the biological sample if there is deficiency in functional LKB/STK11; and
c) identifying the subject as likely to respond to the treatment with an MDM2 inhibitor based on the deficiency in functional LKB1/STK11 found in the biological sample.
In some embodiments, the method further comprising:
d) administering the MDM2 inhibitor in a therapeutically effective amount to the subject identified as likely to respond to the treatment with an MDM2 inhibitor.
In another aspect, the present disclosure provides a method of treating a subject with cancer, the method comprising:
a) determining in a biological sample from the subject if there is deficiency in functional LKB1/STK11; and
b) administering the subject with a therapeutically effective amount of an MDM2 inhibitor based on the deficiency in functional LKB1/STK11 found in the biological sample.
In another aspect, the present disclosure provides a method of treating a subject with cancer, the method comprising administering to the subject with a therapeutically effective amount of an MDM2 inhibitor, wherein the subject has been determined to have deficiency in functional LKB1/STK11 in a biological sample from  the subject. In some embodiments, the subject is relapsed from or refractory to an immunotherapy or chemotherapy. In some embodiments, the immunotherapy is PD-1/PD-L1 blockade therapy. In some embodiments, the subject has been determined to further have KRAS mutation.
In some embodiments, said determining comprises detecting the presence of one or more inactivating mutations in LKB1/STK11 in the biological sample, wherein the presence of the one or more inactivating mutations in LKB1/STK11 is indicative of the deficiency in functional LKB1/STK11.
In some embodiments, the one or more inactivating mutations in LKB1/STK11 comprises deletion, insertion, substitution or any combination thereof that reduces serine/threonine kinase activity of LKB1/STK11.
In some embodiments, the one or more inactivating mutations in LKB1/STK11 comprises a mutation selected from the group of mutations as listed in Table 1.
In some embodiments, said determining comprises determining if level of LKB1/STK11 is reduced in the biological sample relative to a reference level, and the reduced level of LKB1/STK11 is indicative of the deficiency in functional LKB1/STK11
In some embodiments, said determining comprises determining if promoter of LKB1/STK11 is hypermethylated in the biological sample, wherein the hypermethylated LKB1/STK11 promoter is indicative of the deficiency in functional LKB1/STK11.
In some embodiments, said determining further comprises determining in the biological sample presence or absence of functional p53, wherein the presence of functional p53 is indicative of likelihood to respond to the treatment with an MDM2 inhibitor.
In some embodiments, the subject identified as likely to respond to the treatment with an MDM2 inhibit or the subject to be administered with the MDM2 inhibitor has, or is further determined to have, functional p53 in the biological sample.
In some embodiments, the functional p53 comprises wild-type p53.
In some embodiments, said determining further comprises determining in the biological sample presence of one or more mutations in KRAS, wherein the presence of one or more mutations in KRAS is indicative of likelihood to respond to the treatment an MDM2 inhibitor.
In some embodiments, the subject to be administered with an MDM2 inhibitor has, or is further determined to have, one or more mutations in KRAS in the biological sample.
In some embodiments, i) the deficiency in functional LKB1/STK11, ii) the presence or absence of functional p53, and/or iii) the one or more mutations in KRAS, is measured by an amplification assay, a hybridization assay, a sequencing assay, or an immunoassay.
In some embodiments, the biological sample comprises a cancer cell or a non-cancer cell.
In some embodiments, the cancer is solid tumor or hematologic malignancy.
In some embodiments, the cancer is gastric cancer (e.g. stomach cancer) , cholangiocarcinoma, lung cancer, melanoma, breast cancer (e.g. invasive breast carcinoma) , colon cancer, ovarian cancer, prostate cancer, liver cancer (e.g. hepatocellular carcinoma) , bladder cancer, pancreatic cancer, renal cancer, esophageal cancer, head and neck cancer, thyroid cancer, cutaneous squamous cell carcinoma, glioblastoma. neuroblastoma, urinary bladder cancer, hysterocarcinoma, melanoma, osteosarcoma, lymphoma (e.g., mantel cell lymphoma, diffuse large B cell lymphoma) , leukemia (e.g., T-cell prolymphocytic leukemia, chronic lymphocytic leukemia, or acute myeloid leukemia) , multiple myeloma, uterine cancer, colorectal cancer, lung adenocarcinoma, uterine carcinosarcoma, lung squamous cell carcinoma, cervical cancer, esophagus cancer, sarcoma, chromophobe, renal cell carcinoma (RCC) , clear cell RCC, papillary RCC, uveal melanoma, testicular germ cell tumor, low grade glioma (LGG) , mesothelioma, pheochromocytoma and paraganglioma (PCPG) , thymoma adenoid cystic carcinoma (ACC) .
In some embodiments, the cancer is selected from small cell lung carcinoma and non-small cell lung carcinoma (e.g. lung adenocarcinoma, lung squamous cell carcinoma, or lung large cell carcinoma) .
In some embodiments, the subject is relapsed from or refractory to an immunotherapy. An immune therapy can comprise a modulator of immune checkpoint molecule. Examples of immune checkpoint molecule include, without limitation, PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG3, CD160, 2B4, TGFβ, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, CDS, ICAM-1, NKG2C, SLAMF7, NKp80, B7-H3, LFA-1, 1COS, 4-1BB, GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT or CD83 ligand, and preferably, the immune checkpoint molecule is PD-1, PD-L1 or CTLA-4. The modulator of an immune checkpoint molecule can be an antibody, an antibody Fab fragment, a bivalent antibody, an antibody-drug conjugate, an scFv, a fusion protein, or a tetravalent antibody, and preferably, the modulator of an immune checkpoint molecule is a monoclonal antibody or an antigen-binding fragment thereof.
In certain embodiments, the modulator of an immune checkpoint molecule is pembrolizumab, ipilimumab, nivolumab, atezolizumab, avelumab, durvalumab, AGEN-1884, BMS-986016, CS1001 (WO2017020858A1, all of which is incorporated herein to its entirety) , CS-1002, LAG525, MBG453, MEDI-570, OREG-103/BY40, lirilumab, tremelimumab, JS001, SHR-1210, BGB-A317, IBI-308, REGN2810, JS003, SHR-1316, KN035 or BMS-936559, and preferably, the modulator of an immune checkpoint molecule is pembrolizumab.
In some embodiments, the immunotherapy is PD-1/PD-L1 blockade therapy. PD-1/PD-L1 blockade therapy can include, for example, anti-PD-1 antibody, or anti-PD-L1 antibody.
In some embodiments, the subject has been determined to further have KRAS mutation. Without wishing to be bound by any theory, it is believed that the MDM2 inhibitors provided herein (e.g. compounds of Formula (I) , and in particular Compound C) , is effective to treat cancers having co-mutation of LKB1/STK11 and KRAS. In lung adenocarcinoma, KRAS is also frequently co-mutated with LKB1/STK11 and the co-mutation has been implicated as a major driver of primary resistance to PD-1 blockade therapy (Skoulidis F, et al, Cancer Discov, (2018) 8: 822) . In certain embodiments, the subject has been determined to have a wild-type or functional p53.
In another aspect, the present disclosure provides a method of inducing ferroptosis in a subject in need thereof comprising administering to the subject an effective amount of a MDM2 inhibitor.
In another aspect, the present disclosure provides a method for treating or ameliorating the effects of a condition that would benefit from ferroptosis in a subject in need thereof comprising administering to the subject a MDM2 inhibitor in an effective amount to induce ferroptosis.
In another aspect, the present disclosure provides a method of treating a subject identified as having ferroptosis-sensitive condition or cancer , comprising administering to the subject a MDM2 inhibitor in an effective amount to induce ferroptosis.
In some embodiments, the induction of ferroptosis is determined by increase in lipid reactive oxygen species (ROS) . In some embodiments, the subject is identified as having a wild-type p53 or a p53 variant capable of regulating SLC7A11 expression.
In some embodiments, the ferroptosis-sensitive condition or cancer is characterized in active or overactive lipid ROS production.
In some embodiments, the ferroptosis-sensitive condition or cancer is characterized in having one or more functional or overactive genes or gene products selected from the group consisting of: heme carrier protein 1, integrin, ferroprotin, stimulator of Fe transport (SFT) , iron responsive element binding protein (IRP) , transferrin, DMT1, ACSL4, CARS, ALOXs, ATP5G3, CHAC1, CS, DPP4, GLS2, LPCAT3, NCOA4, RPL8, SAT1, TFRC, and TTC35/EMC2.
In some embodiments, the overactive genes or gene products are over-expressed or having an activating mutation.
In some embodiments, the ferroptosis-sensitive condition or cancer is characterized in having reduced activity in one or more genes or gene products selected from the group consisting of: ZEB2, AKR1C1-3, SLC7A11, SLC3A2, GPX4, CD44v, CBS, CISD1, FANCD2, GCLC/GCLM, GSS, HMGCR, HSPB1/5, NFE2L2, and SQS.
In some embodiments, the ferroptosis-sensitive cancer is characterized in having reduced activity in SLC7A11 and/or GPX4.
In some embodiments, the genes or gene products having reduced activity are under-expressed or having an inactivating mutation.
In some embodiments, the ferroptosis-sensitive condition or cancer is characterized in a high-mesenchymal cell state.
In some embodiments, the high-mesenchymal cell state is characterized in upregulation of one or more genes selected from the group consisting of: CD133, CD44, VIM, FN1, ZEB1, TWIST1, SNAI2, and CDH2.
In some embodiments, the ferroptosis-sensitive condition or cancer is characterized in one or more of the following: over-expression or activating mutation of RAS (e.g. KRAS and/or HRAS) , TFRC or MRP1, .
In some embodiments, the subject is further identified as having functional p53 (e.g. wild-type 53) and/or one or more mutations in KRAS in the biological sample.
In some embodiments, the subject is identified as having deficiency in functional LKB1/STK11.
In some embodiments, the ferroptosis-sensitive condition or cancer comprises lung cancer, neuroblastoma, pancreatic cancer, acute myeloid leukemia, hepatocellular carcinoma, rhadomyoscarcoma, diffuse large B-cell lymphoma, renal cell carcinoma, prostate cancer, melanoma, fibrosarcoma, ovarian cancer, brain cancer or breast cancer.
In some embodiments, the MDM2 inhibitor has an IC50 of no more than 1μM (e.g. no more than 500 nM, 400 nM, 300 nM, 200 nM, 150 nM, 100 nM, 50 nM, 20 nM, 10 nM or 5 nM) in inhibiting the binding of MDM2 to p53 as determined by a fluorescence-polarization MDM2 binding assay.
In some embodiments, the MDM2 inhibitor comprises a compound of formula (I) :
Figure PCTCN2021078476-appb-000001
or a pharmaceutically acceptable salt thereof, wherein
Figure PCTCN2021078476-appb-000002
is selected from the group consisting of
Figure PCTCN2021078476-appb-000003
B is a C 4-7 carbocyclic ring;
R 1 is H, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, OR a, or NR aR b;
n is 0, 1, or 2;
R 2, R 3, R 4, R 5, R 7, R 8, R 9, and R 10, independently, are selected from the group consisting of H, F, Cl, CH 3, and CF 3;
R 6 is
Figure PCTCN2021078476-appb-000004
R a is hydrogen or substituted or unsubstituted C 1-4 alkyl;
R b is hydrogen or substituted or unsubstituted C 1-4 alkyl;
R c and R d are substituents on one carbon atom of ring B, wherein
R c is H, C 1-3 alkyl, C 1-3 alkylene-OR a, OR a, or halo;
R d is H, C 1-3 alkyl, C 1-3 alkylene-OR a, OR a, or halo; or
R c and R d are taken together with the carbon to which they are attached to form a 4 to 6-membered Spiro substituent, optionally containing an oxygen atom; and
R e is -C (=O) OR a, -C (=O) NR aR b, or -C (=O) NHSO 2CH 3.
In some embodiments, 
Figure PCTCN2021078476-appb-000005
is
Figure PCTCN2021078476-appb-000006
B is
Figure PCTCN2021078476-appb-000007
and
R c and R d are F and F, H and H, OH and CH 3, OH and H, CH 3 and CH 3, CH 3 and OH, H and OH, CH 2CH 3 and CH 2CH 3, or CH 2OH and CH 2OH.
In some embodiments, 
Figure PCTCN2021078476-appb-000008
is H, CH 3, or CH 2CH 3.
In some embodiments, R 2 is H; R 3 is halo; R 4 and R 5 are H.
In some embodiments, R 7 is fluoro; each of R 8, R 9, and R 10 is H; and R e is -C (=O) OH, -C (=O) NH 2, or -C (=O) NHSO 2CH 3.
In some embodiments, the MDM2 inhibitor is a compound selected from:
Figure PCTCN2021078476-appb-000009
Figure PCTCN2021078476-appb-000010
Figure PCTCN2021078476-appb-000011
Figure PCTCN2021078476-appb-000012
or a pharmaceutically acceptable salt thereof.
In some embodiments, the MDM2 inhibitor is
Figure PCTCN2021078476-appb-000013
or a pharmaceutically acceptable salt thereof.
In some embodiments, the MDM2 inhibitor is selected from the group consisting of idasanutlin (RG7388) , RG7112, HDM201, KRT-232, AMG 232, BI907828, SAR-405838 (MI-77301) , MK-8242 (SCH 900242) , DS3032-b, ALRN-6924 and CGM097; or a pharmaceutically acceptable salt of any of the foregoing.
In some embodiments, the methods provided herein further comprise further administering an effective amount of one or more additional therapies.
In some embodiments, the one or more additional therapies comprise a radiotherapy, chemotherapy, a targeted cancer therapy, or a therapy with a modulator of an immune checkpoint molecule.
In some embodiments, the one or more additional therapies comprise administering an anti-PD-1 antibody, a Bcl-2 inhibitor, a FAK inhibitor, a MEK inhibitor, or a MET inhibitor.
In some embodiments, the Bcl-2 inhibitor is compound D,
Figure PCTCN2021078476-appb-000014
or a pharmaceutically acceptable salt thereof.
Compound D: (R) -1 - (3 - (4 - (4 - (4 - (2 - (4-chlorophenyl) -1-isopropyl-5-methyl-4 - (methylsulfonyl) -1h-pyrrole-3-yl) -5-fluorophenyl) piperazine-1-yl) phenyl) -aminosulfonyl) -2 - (trifluoromethylsulfonyl) phenylamino) -4 - (phenylthio) butyl) piperidine-4-carboxylic acid. The synthesis method of compound D can be prepared according to the description of WO2014/113413A1.
In some embodiments, the FAK inhibitor is Compound E,
Figure PCTCN2021078476-appb-000015
or a pharmaceutically acceptable salt thereof.
Compound E : (5-chloro-N 2- (2-isopropoxy-5-methyl-4- (1- (tetrahydro-2H-pyran-4-yl) -1, 2, 3, 6-tetrahydropyridin-4-yl) phenyl) -N 4- (2- (isopropylsulfonyl) phenyl) pyrimidine-2, 4-diamine) are synthesized according to the  production methods described in WO 2018/044767, which is incorporated herein by reference in its entirety and for all purposes, or a method analogous thereto.
In some embodiments, the MEK inhibitor is trametinib.
In some embodiments, the additional therapy comprises an anti-cancer agent that does not induce ferroptosis.
In some embodiments, the additional therapy comprises a ferroptosis inducing agent.
In some embodiments, the ferroptosis inducing agent comprises RSL3, altretamine, artesunate, buthioninesulfoximine, BAY 87-2243, cyct (e) inase, DP17 erastin, FIN56, lanperisone, piperazine-coupled erastin, imidazole-ketone erastin, statins, sulfasalazine, sorafenib or withaferin A.
In some embodiments, the ferroptosis inducing agent is lenvatinib, the statins is Atorvastatin.
In another aspects, the present disclosure provides a kit for predicting responsiveness of a subject with cancer to treatment with an MDM2 inhibitor, comprising
a) one or more reagents for detecting presence of deficiency in functional LKB1/STK11.
In some embodiments, the kit further comprises:
b) one or more reagents for detecting presence or absence of functional p53 (e.g. wild-type p53) .
In some embodiments, the kit further comprises:
c) one or more reagents for detecting one or more mutations in KRAS.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1A shows LKB1/STK11 mutations occur at a high frequency in lung adenocarcinoma and Figure 1B shows TP53 may co-mutate with LKB1/STK11 in lung adenocarcinoma.
Figure 2 shows LKB1/STK11 mutants A549, NCI-H2122 and NCI-H460 are sensitive to Compound C.
Figure 3A-3H show treatment with Compound C increased lipid ROS level (Figures 3A-3E) and suppressed the expression of SCL7A11 in cancer cells (Figures 3F-3H) . Figure 4 shows the results of flow cytometry.
Figure 5 shows the results of cell apoptosis after compound treatment.
Figure 6A shows the WB results of A549 cell line, figure 6B shows the cell survival rate, and Figure 6C shows the flow cytometry results, Figure 6D shows the results of lipid ROS detected by attune NXT flow cytometry.
Figure 7 shows the results of cell apoptosis and relative lipid ROS.
Figure 8 shows the cell viability test results of compound C, trametinib alone or in combination for 72 hours.
Figure 9 shows the cell viability test results of compound C, trametinib alone or in combination for 72 hours.
Figure 10 shows the cell viability test results of compound C, trametinib alone or in combination for 72 hours.
Figure 11 are cell viability test results of compound C and compound D treated alone or in combination for 72 hours.
Figure 12 shows the results of cell viability test of compound C and compound D treated alone or in combination for 72 hours.
Figure 13 shows the cell viability test results of compound C and compound D treated alone or in combination for 72 hours.
Figure 14 shows the tumor volume changes of compound C in the treatment of lu5209.
Figure 15 shows the change of tumor volume after treatment with C, atorvastatin and lenvatinib alone or in combination.
Figure 16 shows the weight changes of experimental animals treated with compound C, atorvastatin and lenvatinib alone or in combination.
Figure 17 shows the change of tumor volume after compound C and compound E were treated alone or in combination.
Figure 18 shows the weight changes of experimental animals after compound C and compound E were treated alone or in combination.
Figure 19 shows the tumor volume changes of compounds C, RSL3 and atorvastain after treatment alone or in combination.
Figure 20 shows the weight changes of experimental animals treated with compounds C, rsl3 and atorvastain alone or in combination.
Figure 21 shows DNA coding sequences and protein (amino acids) sequences of biomarkers with SEQ ID numbers mentioned in the present disclosure
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
In accordance with the present disclosure and as used herein, the following terms are defined with the following meanings, unless explicitly stated otherwise. Unless otherwise defined, all terms of art, notations and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the chemical and medical arts.
The term “biomarker” as used here refers to a biological molecule that is a measurable indicator of some biological state or condition. The term “biomarker” used herein is intended to encompass a polynucleotide of interest, or a polypeptide (for example encoded by the polynucleotide of interest) . Examples of biomarker provided herein can be a gene (e.g. genomic DNA, cDNA) or a product of the gene such as an mRNA transcribed from the gene, and a protein encoded by the gene. Specific examples of the biomarkers provided herein include LKB1/STK11, p53 and KRAS.
“MDM2” as used herein is short for Murine Double Minute 2. The term MDM2 is intended to encompass the MDM2 gene, as well as the MDM2 gene product (e.g. mRNA, protein) . Exemplary sequence of human MDM2 is available under the NCBI accession number of ABT17086, ABT17084.1, ABT17085.1, or ABT17083.1.
The term “level” with respect to a biomarker refers to the amount or quantity of the biomarker of interest present in a sample. Such amount or quantity may be expressed in the absolute terms, i.e., the total quantity of the biomarker in the sample, or in the relative terms, i.e., the concentration or percentage of the biomarker in the sample. Level of a biomarker can be measured at DNA level (for example, as represented by the  amount or quantity or copy number of the gene in a chromosomal region) , at RNA level (for example as mRNA amount or quantity) , or at protein level (for example as protein or protein complex amount or quantity) .
The term “activity” with respect to a biomarker refers to the biological activity (e.g. catalytic or regulatory ability) of the proteins as described here.
As used herein, “likelihood” and “likely” with respect to response of a subject to a treatment is a measurement of how probable the therapeutic response is to occur in the subject. It may be used interchangeably with “probability” . Likelihood refers to a probability that is more than speculation, but less than certainty. Thus, a therapeutic response is likely if a reasonable person using common sense, training or experience concludes that, given the circumstances, a therapeutic response is probable. In one embodiment, the term “likelihood” and “likely” denotes a chance in percent of how probable a therapeutic response is to occur. In some embodiments, a subject with cancer identified as “likely to respond” refers to a subject with cancer who has more than 30%chance, more than 40%chance, more than 50%chance, more than 60%chance, more than 70%chance, more than 80%chance, more than 90%chance of responding to a treatment.
The term “responsive” or “responsiveness” as used in the context of a subject’s therapeutic response to a cancer therapy, are used interchangeably and refer to a beneficial response of a subject to a treatment as opposed to unfavorable responses, i.e. adverse events. In a subject, beneficial response can be expressed in terms of a number of clinical parameters, including loss of detectable tumor (complete response) , decrease in tumor size and/or cancer cell number (partial response) , tumor growth arrest (stable disease) , enhancement of anti-tumor immune response, possibly resulting in regression or rejection of the tumor; relief, to some extent, of one or more symptoms associated with the tumor; increase in the length of survival following treatment; and/or decreased mortality at a given point of time following treatment. Continued increase in tumor size and/or cancer cell number and/or tumor metastasis is indicative of lack of beneficial response to treatment, and therefore decreased responsiveness.
As used herein, “cancer” or “tumor” is a generic name for a wide range of cellular malignancies characterized by unregulated growth, lack of differentiation, and  the potential or ability to invade local tissues and metastasize. These neoplastic malignancies affect, with various degrees of prevalence, every tissue and organ in the body. Cancer involves presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Often, cancer cells will be in the form of a tumor, but such cells may exist alone or may circulate in the blood stream as independent cells, such as leukemic cells.
As used herein, “relapse” refers to the regression of the subject’s illness back to its former diseased state, especially the return of symptoms following an apparent recovery or partial recovery. Unless otherwise indicated, relapsed state refers to the process of returning to or the return to illness before the previous treatment including, but not limited to, chemotherapies or immunotherapies.
As used herein, "refractory" refers to the resistance or non-responsiveness of a disease or condition to a treatment (e.g., the number of neoplastic cells increases even though treatment if given) . Unless otherwise indicated, the term "refractory" refers a resistance or non-responsiveness to any previous treatment including, but not limited to, chemotherapies or immunotherapies.
As used herein, “immunotherapy” refers to a therapy (e.g. anti-cancer therapy) comprising a modulator of an immune checkpoint molecule.
As used herein, a “pharmaceutically acceptable” component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.
The terms “determining” , “measuring” and “detecting” can be used interchangeably and refer to both quantitative and semi-quantitative determinations or qualitative determinations.
The term “hybridizing” refers to the binding, duplexing or pairing of at least partially complementary strands of nucleic acid molecules. A nucleic acid strand can specifically hybridize to a target nucleic acid strand when there is sufficient degree of complementarity to avoid non-specific binding to non-target nucleic acid sequences.
The term “nucleic acid” and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA) , transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, single-stranded short or long RNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.
The term “complementarity” refers to the ability of baseparing between a nucleic acid sequence and another nucleic acid sequence via either traditional Watson-Crick or other non-traditional types. Complementarity can be partial or total. Partial complementarity occurs when one or more nucleic acid bases is not matched according to the base pairing rules. A percent complementarity indicates the percentage of nucleic acid base in a nucleic acid molecule which can form basepairs (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 base pairing out of 10 bases being 50%, 60%>, 70%>, 80%>, 90%, and 100%complementary) .
The term “prognosis” as used herein refers to the prediction or forecast of the future course or outcome of a disease or condition.
In general, a “protein” is a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds) . Proteins may include moieties other than amino acids (e.g., may be glycoproteins) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence) , or can be a functional portion thereof. Those of ordinary skill will further appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means.
The term “treating” or “treatment” of cancer as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progress of, or preventing, either partially or completely, the growth of tumors, tumor metastases, or other cancer-causing or neoplastic cells in a subject.
As used herein, “combination therapy” is understood as administration of two or more active agents using separate formulations or a single pharmaceutical formulation, or consecutive administration in any order such that, there is a time period while both (or all) active agents simultaneously exert their biological activities. It is contemplated herein that one active agent (e.g., an MDM2 inhibitor) can improve the activity of a second agent, for example, can sensitize target cells, e.g., cancer cells, to the activities of the second agent. Combination therapy does not require that the agents are administered at the same time, at the same frequency, or by the same route of administration.
The terms “administer” , “administering” or “administration” include any method of delivery of a pharmaceutical composition or agent into a subject's system or to a particular region in or on a subject. The administration may be local or systemic. In certain embodiments, the administration is by oral route or parental route (e.g. intravenous, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intraperitoneal, intranasal, or intraocular injection; direct injection to a tumor; or intravenous infusion) . In certain embodiments, routes of administration include pulmonary administration, suppositories, and transdermal or transcutaneous applications. Administering an agent can be performed by a number of people working in concert. Administering an agent includes, for example, prescribing an agent to be administered to a subject and/or providing instructions, directly or through another, to take a specific agent, either by self-delivery, e.g., as by oral delivery, subcutaneous delivery, intravenous delivery through a central line, etc.; or for delivery by a trained professional, e.g., intravenous delivery, intramuscular delivery, intratumoral delivery, continuous infusion, etc.
As used herein, the term “subject” refers to a human or any non-human animal or mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) . In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient. ” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
The term “therapeutically effective amount” or “effective amount” means the amount of a compound that that produces some desired local or systemic therapeutic effect at a reasonable benefit/risk ratio applicable to any treatment. When administered for preventing a disease, the amount is sufficient to avoid or delay onset of the disease. A therapeutically effective amount or an effective amount need not be curative or prevent a disease or condition from ever occurring. In certain embodiments, a therapeutically-effective amount of a compound will depend on its therapeutic index, solubility, and the like.
As used herein, the term “alkyl” refers to straight chained and branched saturated C 1-10 hydrocarbon groups, including but not limited to methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, t-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 2, 2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 2-dimethylbutyl, 2, 3-dimethylbutyl, 3, 3-dimethylbutyl, and 2-ethylbutyl.
The term C m-n means the designated group has a range of carbon atoms from “m” to “n” , including the endpoints “m” and “n” .
The term “alkylene” refers to an alkyl group having a substituent. An alkyl, e.g., methyl, or alkylene, e.g., -CH 2-, group can be substituted with one or more, and typically one to three, of independently selected halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro, cyano, alkylamino, or amino groups, for example.
The term “substituted” as used herein with respect to a chemical group means that one or more hydrogen atoms on the chemical group is independently replaced with one or more substituents. The term “substituent” , as used herein, has the ordinary meaning known in the art and refers to a chemical moiety that is covalently attached to, or if appropriate, fused to, a parent group.
As used herein, the term “spiro” refers to a ring system having two rings connected through only one carbon atom in common. Such cyclic moiety may essentially be a carbocyclic or heterocyclic ring. Spiro systems exclude other bicyclic compounds such as naphthalene which have two or more carbon atoms in common.
As used herein, the term “halo” or “halogen” is defined as fluoro, chloro, bromo, or iodo.
As used herein, the term “cycloalkyl” means a monocyclic or bicyclic, saturated or partially unsaturated, ring system containing three to eight carbon atoms, including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, optionally substituted with one or more, and typically one to three, of independently selected halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, nitro, cyano, alkylamino, or amino groups, for example.
As used herein, the term “heterocycloalkyl” means a monocyclic or a bicyclic, saturated or partially unsaturated, ring system containing 4 to 12 total atoms, of which one to five of the atoms are independently selected from nitrogen, oxygen, and sulfur and the remaining atoms are carbon. Non-limiting examples of heterocycloalkyl groups are azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, dihydropyrrolyl, morpholinyl, thiomorpholinyl, dihydropyridinyl, oxacycloheptyl, dioxacycloheptyl, thiacycloheptyl, diazacycloheptyl, each optionally substituted with one or more, and typically one to three, of independently selected halo, C 1-6 alkyl, C 1-6 alkoxy, cyano, amino, carbamoyl, nitro, carboxy, C 2-7 alkenyl, C 2-7 alkynyl, or the like on an atom of the ring.
In all occurrences in this application where there are a series of recited numerical values, it is to be understood that any of the recited numerical values may be the upper limit or lower limit of a numerical range. It is to be further understood that the invention encompasses all such numerical ranges, i.e., a range having a combination of an upper numerical limit and a lower numerical limit, wherein the numerical value for each of the upper limit and the lower limit can be any numerical value recited herein. Ranges provided herein are understood to include all values within the range. For example, 1-10 is understood to include all of the  values  1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and fractional values as appropriate. Similarly, ranges delimited by “at least” are understood to include the lower value provided and all higher numbers.
As used herein, “about” is understood to include within three standard deviations of the mean or within standard ranges of tolerance in the specific art. In certain embodiments, about is understood a variation of no more than 0.5.
The articles “a” and “an” are used herein to refer to one or more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an  element” means one element or more than one element.
The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to” . Similarly, “such as” is used herein to mean, and is used interchangeably, with the phrase “such as but not limited to” .
The term “or” is used inclusively herein to mean, and is used interchangeably with, the term “and/or, ” unless context clearly indicates otherwise.
II. Methods for Patient Identification, Treatment and Prognosis
Described herein are methods of identifying a subject with cancer as likely to respond to treatment with an MDM2 inhibitor, methods of treating a subject with cancer with an MDM2 inhibitor, and kits for predicting responsiveness of a subject with cancer to treatment with an MDM2 inhibitor.
MDM2 inhibitors have been described previously as an anti-cancer therapeutic agent (See, e.g, U.S. Patent No. 9,745,314, the entire contents of which are incorporated herein by reference) , and are being evaluated in humans as mono-therapy or in combination with standard of care anticancer agents for treatment of diseases and conditions wherein inhibition of MDM2 and MDM2-related proteins activity provides a benefit. As surprisingly discovered by the inventors, subjects with cancer who has deficiency in functional LKB1/STK11 are likely to respond to treatment of an MDM2 inhibitor.
In one aspect, provided herein is a method of identifying a subject with cancer as likely to respond to treatment with an MDM2 inhibitor. In certain embodiments, the method comprising: providing a biological sample from the subject; determining in the biological sample if there is deficiency in functional LKB1/STK11; and identifying the subject as likely to respond to the treatment with an MDM2 inhibitor based on the deficiency in functional LKB1/STK11 found in the biological sample. In certain embodiments, the method further comprises administering the MDM2 inhibitor in a therapeutically effective amount to the subject identified as likely to respond to the treatment with an MDM2 inhibitor.
In another aspect, provided herein is a method of selecting a subject with cancer for treatment with an MDM2 inhibitor. In certain embodiments, the method comprising: providing a biological sample from the subject; determining in the  biological sample: if there is deficiency in functional LKB1/STK11; and selecting the subject for the treatment with an MDM2 inhibitor based on the deficiency in functional LKB1/STK11 found in the biological sample. In certain embodiments, the method further comprises administering the MDM2 inhibitor in a therapeutically effective amount to the selected subject.
In another aspect, provided herein is a method of predicting likelihood of responsiveness of a subject with cancer to treatment with an MDM2 inhibitor. In certain embodiments, the method comprises: providing a biological sample from the subject; determining in the biological sample if there is deficiency in functional LKB1/STK11; and predicting the subject as likely to be responsive to the treatment with an MDM2 inhibitor based on the deficiency in functional LKB1/STK11 found in the biological sample.
In another aspect, provided herein is a method of treating a subject with cancer with an MDM2 inhibitor. In certain embodiments, the method comprises: determining in a biological sample from the subject if there is deficiency in functional LKB1/STK11; and administering the subject with an MDM2 inhibitor in a therapeutically effective amount based on the deficiency in functional LKB1/STK11 found in the biological sample.
In another aspect, provided herein is a method of treating a subject with cancer with an MDM2 inhibitor, the method comprising administering to the subject with a therapeutically effective amount of an MDM2 inhibitor, wherein the subject has been determined to have deficiency in functional LKB1/STK11 in a biological sample from the subject.
“LKB1/STK11” as used herein refers to serine/threonine-protein kinase STK11, which is also known as, e.g., polarization-related protein LKB1, liver kinase B1, renal carcinoma antigen NY-REN-19, PJS. The term LKB1/STK11 is intended to encompass the LKB1/STK11gene (e.g. genomic DNA, cDNA) , as well as the LKB1/STK11 gene product (e.g. mRNA transcribed from the gene, a protein encoded by the gene) . The term “LKB1/STK11” , “LKB1” and “STK11” can be used interchangeably. Exemplary sequence of human LKB1/STK11 is available in  UniProtKB database under the accession number of Q15831 (STK11-HUMAN) , in the GenBank database under the NCBI accession number of AAB97833.1.
The term LKB1/STK11 as used herein is intended to encompass both wild-type LKB1/STK11 and variants of LKB1/STK11. In certain embodiments, the gene of wild-type LKB1/STK11 comprises a DNA sequence of SEQ ID NO: 1. In certain embodiments, the protein of wild-type LKB1/STK11 comprises an amino acid sequence of SEQ ID NO: 2.
The term “variant” as used herein refers to a gene or a gene product having substantially homologous sequences (e.g., coding sequences) to the corresponding wild-type gene or the product thereof, and having substantially similar function as that of the wild-type counterpart. Generally, variants of a particular biomarker disclosed herein will have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more sequence identity to the sequence of the biomarker as determined by sequence alignment programs. Variants can include, for example, allelic variants, conservative substitution variants and homologs that can be isolated/generated and characterized by methods known in the field.
LKB1/STK11 plays roles in various process such as cell metabolism, cell polarity, apoptosis and DNA damage response by activating targets including 5’-adenosine monophosphate-activated protein kinase (AMPK) and the AMPK-related kinases by direct phosphorylation (Williams T., et al., Trends in cell Biology, (2008) 18: 193) .
The term “functional LKB1/STK11” , means wild-type LKB1/STK11 and any LKB1/STK11 variant having at least 30%, 40%, 50%, 60%, preferably at least 70%, 80%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more activity of wild-type LKB1/STK11.
As used herein, “deficiency” or “deficient” with respect to a biomarker (such as LKB1/STK11) refers to insufficiency in activity or level of the biomarker, and can include, for example, being less than normal activity or level, or being absent or null in activity or level of the biomarker. The term “deficiency in functional LKB1/STK11” as used herein refers to insufficiency in level or activity of functional LKB1/STK11 in the biological sample.
i.  Sample Preparation
In certain embodiments, the methods provided herein comprises providing a biological sample from the subject.
Any biological sample suitable for conducting the methods provided herein can be obtained from the subject. As used herein, “biological sample” refers to a biological specimen taken by sampling from a subject, optionally with additional processing. In certain embodiments, the sample can be a biological sample comprising cancer cells, or non-cancer cells. For example, non-cancer cells can be from the same tissue or organ as the cancer cells are also found. In some embodiments, the biological sample is a fresh or archived sample obtained from a tumor tissue, e.g., by a tumor biopsy or fine needle aspirate. In some embodiments, the sample can be any biological fluid containing cancer cells or non-cancer cells (e.g. peripheral blood mononuclear cells (PBMC) ) . The collection of a sample from a subject is performed in accordance with the standard protocol generally followed by hospital or clinics, such as during a biopsy. Examples of a biological sample include without limitation, bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, uterine or vaginal flushing fluids, plural fluid, ascetic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchioalveolar lavage fluid, etc., and tissues, such as biopsy tissue (e.g. biopsied bone tissue, bone marrow, breast tissue, gastroinstetinal tract tissue, lung tissue, liver tissue, prostate tissue, brain tissue, nerve tissue, meningeal tissue, renal tissue, endometrial tissue, cervical dittuse, lymph node tissue, muscle tissue, or skin tissue) , a paraffin embedded tissue. In a further embodiment, a biological sample comprises cells, tissue, blood, plasma, serum, urine, mouthwash, stool, saliva, and any combination thereof. In a further embodiment, a biological sample is blood, plasma, serum, or urine. In a preferred embodiment, a biological sample is blood. In another preferred embodiment, a biological sample is tumor tissue.
In certain embodiments, the sample can be further processed by a desirable method for determining the genetic status, activity or level of the at least one biomarker (such as LKB1/STK11) .
In certain embodiments, the method further comprises isolating or extracting cancer cell (such as circulating tumor cell) from the biological fluid sample (such as  peripheral blood sample) or the tissue sample obtained from the subject. The cancer cells can be separated by immunomagnetic separation technology such as that available from Immunicon (Huntingdon Valley, Pa. ) .
In certain embodiments, a tissue sample can be processed to perform in situ hybridization. For example, the tissue sample can be paraffin-embedded before fixing on a glass microscope slide, and then deparaffinized with a solvent, typically xylene.
In certain embodiments, the method further comprises isolating the nucleic acid from the sample, if RNA or DNA level of the biomarker is to be measured. Various methods of extraction are suitable for isolating the DNA or RNA from cells or tissues, such as phenol and chloroform extraction, and various other methods as described in, for example, Ausubel et al., Current Protocols of Molecular Biology (1997) John Wiley &Sons, and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3 rd ed. (2001) .
Commercially available kits can also be used to isolate RNA, including for example, the NucliSens extraction kit (Biomerieux, Marcy l'Etoile, France) , QIAamp TM mini blood kit, Agencourt Genfind TM, 
Figure PCTCN2021078476-appb-000016
mini columns (Qiagen) , 
Figure PCTCN2021078476-appb-000017
RNA mini kit (Thermo Fisher Scientific) , and Eppendorf Phase Lock Gels  TM. A skilled person can readily extract or isolate RNA or DNA following the manufacturer’s protocol.
ii.  Determination, Measurement and Detection of the Biomarkers
In certain embodiments, the methods provided herein comprise determining in the biological sample if there is deficiency in functional LKB1/STK11. Deficiency in functional LKB1/STK11 can be resulted from, for example, presence of one or more inactivating mutations in LKB1/STK11, presence of negative alteration (e.g. hypermethylation) in the promoter or other cis-regulatory elements of LKB1/STK11, reduced copy number of LKB1/STK11, reduced mRNA level or protein level of LKB1/STK11, and/or reduced protein activity of LKB1/STK11.
In certain embodiments, the deficiency in functional LKB1/STK11 can be indicated by the presence of one or more inactivating mutations in LKB1/STK11. Accordingly, the methods provided herein can comprise the step of detecting the presence of one or more inactivating mutations in LKB1/STK11 in the biological  sample, wherein the presence of the inactivating mutations in STK 11 is indicative of the deficiency in functional STK 11.
The term “inactivating mutation” as used herein with respect to a biomarker, refers to a mutation that results in at least partial (or complete) loss of function or activity of the gene or of the gene product of the biomarker, or results in a nonfunctional gene or gene product of the biomarker, or results in decreased gene expression (e.g. decrease LKB1/STK11 mRNA or protein abundance) . The activity and/or level of the affected gene or gene product of the biomarker would be significantly lower than wild-type counterpart or even be eliminated.
In certain embodiments, the inactivating mutation reduces activity of LKB1/STK11, for example, serine/threonine kinase activity of LKB1/STK11. In certain embodiments, the inactivating mutation reduces LKB1/STK11 mRNA expression or protein expression.
In certain embodiments, an inactivating mutation in LKB1/STK11 can be a deletion, insertion, substitution or any combination thereof. The inactivating mutation can affect, e.g., coding sequence, RNA splicing sites, promoter or other cis-regulatory elements. In some embodiments, the mutation is a large deletion of chromosome 19p13, where LKB1/STK11 resides. These large deletions can span the entire chromosome or an arm of the chromosome. In some embodiments, the mutation can be smaller, e.g., a deletion of less than 1,000 base pairs. For example, the smaller deletion can target one or only a few exons of LKB1/STK11 or can be a deletion in the promoter of LKB1/STK11 that does not target the coding sequence of LKB1/STK11. In addition to larger deletions, the inactivating mutations can be point mutations and small insertion/deletion mutations.
As used herein, a “substitution” is a mutation that exchanges one nucleobase for another in a polynucleotide sequence, or that substitutes one amino acid residue for another in a polypeptide sequence. Substitution in a polynucleotide sequence can: 1) change a codon to one that encodes a different amino acid residue, and therefore will cause change in amino acid sequence in the protein produced ( “missense mutation” ) , or 2) change to a codon that encodes the same amino acid residue thereby causing no change in the protein produced ( “synonymous mutation” ) ; or 3) change an amino-acid- coding codon to a single “stop” codon and cause an incomplete protein (an incomplete protein is usually nonfunctional) ( “nonsense mutation” ) .
As used herein, an “insertion” is a mutation in which one or more extra nucleobase pairs are inserted into a place in a polynucleotide sequence, or in which one or more amino acid residue is inserted into a polypeptide sequence.
As used herein, a “deletion” is a mutation in which one or more nucleobase pairs are lost or deleted from a polynucleotide sequence, or in which one or more amino acid residue are deleted from a polypeptide sequence.
In certain embodiments, insertion or deletion in a polynucleotide sequence may cause frame shift, which changes the reading frame of the codons and results in a completely different translated gene product from the original. This often generates truncated proteins that result in loss of function.
Numerous mutations in LKB1/STK11 have been identified to date. For example, more than 727 mutations in LKB1/STK11 have been identified in various cancer samples, as published in Catalogue of Somatic Mutations in Cancer (COSMIC) database which is available from the following weblink: ( https: //cancer. sanger. ac. uk/cosmic/gene/analysis? ln=STK11) .
Table 1 Mutations in LKB1/STK11
Figure PCTCN2021078476-appb-000018
Figure PCTCN2021078476-appb-000019
It is to be understood that the present disclosure is not limited to any specific LKB1/STK11 mutations. Any inactivating mutations in can be useful in the present disclosure. In some embodiments, inactivating mutations in LKB1/STK11 include, without limitation, one or more mutations in Table 1. In certain embodiments, the inactivating mutation in LKB1/STK11 comprises a mutation selected from the group of mutations as listed in Table 1.
As shown in Table 1, substitution in a polypeptide sequence can be denoted as AnB, where “n” is a number indicating the n th amino acid residue in the polypeptide sequence, “A” is the amino acid residue at the n th residue in the wild-type polypeptide sequence, and “B” is the mutated amino acid residue at the n th residue. When the mutated residue is shown as “*” , it means a mutation leading to a nonsense codon in a nucleotide sequence that results in a truncated, incomplete polypeptide. For instance, “S19P” denotes that the 19 th amino acid residue Serine (S) is changed to a Proline (P) ; “E33*” denotes that the nucleotides encoding amino acid residue 33 (Glutamic acid, E) is changed to a stop codon and the resultant polypeptide is truncated.
Frame shift in a polypeptide is denoted by “AnBfs*m” , indicating a shift in the reading frame starting at the n th amino acid residue and terminating at the m th residues downstream that causes a premature termination of the protein, where “A” and “B” have the same meaning as described above. “I35Lfs*127” denotes a frame shifting starting at the amino acid residue 35 (Isoleucine, I) as the first affected amino acid residue and terminating 127 residues downstream.
Deletion in a polypeptide is denoted by “del” after the amino acid residue number (s) flanking the deletion site. For instance, “E98_G155del” denotes that amino acid residues 98-155 are deleted.
Insertion in a polypeptide sequence can be denoted by as “ins” after the amino acid residue number (s) flanking the insertion site, followed by the amino acid residue (s) inserted.
In some embodiments, the inactivating mutation can be a single-copy or two-copy mutation. Thus, the mutation can be inactivating even if it only causes haploinsufficiency. Accordingly, the inactivating mutation can have a homozygous deletion mutation of LKB1/STK11, a deletion mutation of one allele, a deletion mutation  of one allele and a point mutation of another allele of LKB1/STK11, or heterozygous mutations of LKB1/STK11.
In certain embodiments, the deficiency in functional LKB1/STK11 can be indicated by the presence of negative alteration (e.g. hypermethylation) in the promoter or other cis-regulatory elements of LKB1/STK11 in the biological sample. Accordingly, to determine if there is deficiency in functional LKB1/STK11 in the biological sample, the methods provided herein can comprise the step of determining if promoter or other cis-regulatory elements of LKB1/STK11 is negatively alternated in such a way that would lead to reduced expression of LKB1/STK11. In certain embodiments, the methods of the present disclosure include determining if promoter of LKB1/STK11 is hypermethylated in the biological sample, wherein hypermethylation in the promoter is indicative of the deficiency in functional LKB1/STK11.
In certain embodiments, the deficiency in functional LKB1/STK11 can be indicated by the level of functional LKB1/STK11 in the biological sample. Accordingly, the methods provided herein can comprise the step of determining if level of functional LKB1/STK11 is reduced in the biological sample relative to a reference level, wherein the reduced level of LKB1/STK11 is indicative of the deficiency in functional LKB1/STK11. In certain embodiments, the level of functional LKB1/STK11 includes gene copy number, mRNA expression level, or protein expression level of functional LKB1/STK11. In certain embodiments, functional LKB1/STK11 comprises wild-type LKB1/STK11.
“Copy number” as used herein refers to the number of copies of a particular gene or a particular genomic sequence in the genome of an individual. “Copy number variation (CNV) ” , or “copy number alteration (CNA) ” refers to the variation in the number of copies of a particular gene or a particular DNA sequence from one individual to another individual. For example, although genes are thought to occur in two copies per genome, some genes or genomic sequences are found to be present in one, three, or more than three copies, or even missing (i.e. 0 copy) , in different individuals.
In certain embodiments, the methods of the present disclosure include measuring expression level or gene copy number variation of LKB1/STK11. Without wishing to be bound by any theory, it is found that in some cases, LKB1/STK11  mutations can lead to a reduction or loss of LKB1/STK11 mRNA or protein expression, and in some other cases, hypermethylation of the LKB1/STK11 promoter may also result in decreased mRNA or protein levels.
In certain embodiments, the methods of the present disclosure include measuring biological activity of LKB1/STK11. Methods of assaying, monitoring and modulating activity are described in e.g. US patent application US20050026233A1 or PCT application WO2004113562A1. For example, LKB1/STK11 activity in a biological sample can be assayed by contacting the sample with a substrate kinase under conditions that permit phosphorylation, and monitoring incorporation of phosphate into the substrate kinase, wherein the incorporation of phosphate into the substrate kinase indicates LKB1/STK11 activity. Preferably the substrate kinase is or is derived from AMPK.
In certain embodiments of the methods provided herein, the step of determining further comprises determining in the biological sample if p53 is a functional p53, wherein presence of functional p53 is indicative of likelihood to respond to an MDM2 inhibitor. In certain embodiments, functional p53 comprises wild-type p53.
The term “p53” and “TP53” are used interchangeably herein, and are short for tumor protein p53. Alternative names include, e.g., antigen NY-CO-13, phosphorprotein p53, tumor suppressor p53 and cellular tumor antigen p53. Both TP53 and p53 can refer to the protein or the DNA or RNA sequence of the biomarker p53. Exemplary sequence of human p53 is available in UniProtKB database under the accession number of P04637 (P53-HUMAN) , and in Genbank under the NCBI accession number of AYF55702.1, or AXU92429.1.
p53 is a transcription factor capable of regulating a number of genes that regulate e.g. cell cycle and apoptosis. p53 protein is controlled by MDM2. By binding to p53, MDM2 inhibits p53 transactivation. In addition, MDM2, as E3 ubiquitin ligase, also targets p53 to proteosomal cytosol degradation. Blocking p53-MDM2 interaction therefore can reduce the negative regulation on p53 function, and enable p53 to mediate its downstream functions. Presence of functional p53 is therefore suggested to be beneficial to treatment response to MDM2 inhibitor. p53 used as the biomarker herein can be p53 protein as well as a polynucleotide (e.g. DNA or RNA) encoding the p53  protein. The gene encoding p53 in certain embodiments can be referred to as TP53 in the present disclosure. In certain embodiments, the gene of p53 comprises a gene sequence of SEQ ID NO: 3. In certain embodiments, the protein of p53 comprises an amino acid sequence of SEQ ID NO: 4.
The term “functional p53” , as used herein, refers to wild-type p53 and mutant or allelic variants of p53 that retain at least about 5%of the activity of wild-type p53, e.g., at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of wild-type p53 activity.
In certain embodiments, the methods provided herein further comprise determining in the biological sample presence or absence of a functional p53 (e.g. wild-type p53 protein or TP53 gene) . In certain embodiments, the methods of the present disclosure include determining in the biological sample if p53 is wild-type (e.g. wild-type p53 protein or TP53 gene) .
In some embodiments, the subject to be administered with the MDM2 inhibitor has, or is further determined to have, functional p53 (e.g. wild-type p53 protein or TP53 gene) in the biological sample.
In certain embodiments of the methods provided herein, the step of determining further comprises determining in the biological sample if there is one or more inactivation mutations in KRAS.
“KRAS” as used herein is short for GTPase KRas. The term KRAS is intended to encompass the KRAS gene, as well as the KRAS gene product (e.g. mRNA, protein) . Exemplary sequence of human KRAS is available in UniProtKB database under the accession number of P01116 (RASK-HUMAN) , in the GenBank database under the NCBI accession number of AAM12631.1. KRAS protein converts GTP to GDP in order to modify transductive signals from the cytoplasm to the nucleus and is part of the RAS/MAPK signaling pathway that regulates cells proliferation and differentiation. When KRAS is mutated, GTP is maintained and the RAS/MAPK signaling pathway is persistently in the “active” state, leading to uncontrolled cell proliferation. Despite the well-recognized importance of KRAS mutation in cancer malignancy, continuous efforts in the past decades failed to develop approved therapies for KRAS mutant cancer (Liu P, et al. Acta Pharmaceutica Sinica B (2019) 9: 871) .  KRAS mutation has been considered as an indicator of poor response to cancer therapies, such as treatment with EGFR tyrosine kinase inhibitor in patient with non-small-cell lung cancer (Massarelli E, et al., Clinical Cancer Research (2007) 13: 2890) .
KRAS measured in the methods provided herein can be KRAS protein as well as a polynucleotide (e.g. DNA or RNA) encoding the KRAS protein. In certain embodiments, the gene of KRAS comprises a gene sequence of SEQ ID NO: 5. In certain embodiments, the protein of KRAS comprises an amino acid sequence of SEQ ID NO: 6.
In some embodiments, the determining step further comprises determining in the biological sample presence of one or more mutations in KRAS, wherein the presence of one or more mutations in KRAS is indicative of likelihood to respond to the treatment an MDM2 inhibitor.
In some embodiments, the subject to be administered with an MDM2 inhibitor has, or is further determined to have, one or more mutations in KRAS in the biological sample.
The biomarker LKB1/STK11 as provided herein, and in certain embodiments other biomarkers such as p53 and/or KRAS, are intended to encompass different forms including mRNA, protein and also DNA (e.g. genomic DNA) . Therefore, the level and/or activity of these biomarkers can be measured with RNA (e.g. mRNA) , protein or DNA (e.g. genomic DNA) of the respective biomarker. Similarly, mutation status and/or wild-type status of the biomarkers can also be measured with DNA (e.g. genomic DNA) , RNA (e.g. mRNA) , or protein (for example by measuring for an altered protein product encoded by the mutated gene) .
Mutation status of a biomarker (including LKB1/STK11, p53 and/or KRAS as provided herein) at DNA or RNA level can be measured by any methods known in the art, for example, without limitation, an amplification assay, a hybridization assay, or a sequencing assay. Mutation status at protein level can be measured by any methods known in the art, for example, without limitation, immunoassays.
Expression level of a biomarker (including LKB1/STK11, p53 and/or KRAS as provided herein) at DNA or RNA level can be measured by any methods known in the art, for example, without limitation, an amplification assay, a hybridization assay, or  a sequencing assay. Expression level of a biomarker (including LKB1/STK11, p53 and/or KRAS as provided herein) at protein level can be measured by any methods known in the art, for example, without limitation, immunoassays.
Activity level of a biomarker can be measured by a suitable functional assay known in the art, for example, without limitation, by a phosphorylation assay.
These methods are well-known in the art, and are described in detail below as exemplary illustration.
(a) Amplification assay
A nucleic acid amplification assay involves copying a target nucleic acid (e.g. DNA or RNA) , thereby increasing the number of copies of the amplified nucleic acid sequence. Amplification may be exponential or linear. Exemplary nucleic acid amplification methods include, but are not limited to, amplification using the polymerase chain reaction ( “PCR” , see U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide To Methods And Applications (Innis et al., eds, 1990) ) , reverse transcriptase polymerase chain reaction (RT-PCR) , quantitative real-time PCR (qRT-PCR) ; quantitative PCR, such as
Figure PCTCN2021078476-appb-000020
nested PCR, ligase chain reaction (See Abravaya, K., et al., Nucleic Acids Research, 23: 675-682, (1995) , branched DNA signal amplification (see, Urdea, M.S., et al., AIDS, 7 (suppl 2) : S11-S14, (1993) , amplifiable RNA reporters, Q-beta replication (see Lizardi et al., Biotechnology (1988) 6: 1197) , transcription-based amplification (see, Kwoh et al., Proc. Natl. Acad. Sci. USA (1989) 86: 1173-1177) , boomerang DNA amplification, strand displacement activation, cycling probe technology, self-sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA (1990) 87: 1874-1878) , rolling circle replication (U.S. Patent No. 5,854,033) , isothermal nucleic acid sequence based amplification (NASBA) , and serial analysis of gene expression (SAGE) .
In some embodiments, to measure the mRNA level of the biomarker, the target RNA of the biomarker is reverse transcribed to cDNA before the amplification. Various reverse transcriptases may be used, including, but not limited to, MMLV RT, RNase H mutants of MMLV RT such as Superscript and Superscript II (Life Technologies, GIBCO BRL, Gaithersburg, Md. ) , AMV RT, and thermostable reverse transcriptase from Thermus thermophilus. For example, one method which may be used  to convert RNA to cDNA is the protocol adapted from the Superscript II Preamplification system (Life Technologies, GIBCO BRL, Gaithersburg, Md.; catalog no. 18089-011) , as described by Rashtchian, A., PCR Methods Applic., 4: S83-S91, (1994) .
In certain embodiments, the expression level of RNA (e.g. mRNA) or the copy number variation of DNA of the biomarkers is quantified after the nucleic acid amplification assay. For example, the amplified products can be separated on an agarose gel and stained with ethidium bromide followed by detection and quantification using standard gel electrophoresis methods. Alternatively, the amplified products can be integrally labeled with a suitable detectable label (e.g. a radio-or fluorescence nucleotide) and then visualized using x-ray film or under the appropriate stimulating spectra.
In certain embodiments, the expression level of RNA (e.g. mRNA) or the copy number variation of DNA of the biomarkers is quantified during the nucleic acid amplification assay, which is also known as real-time amplification or quantitative amplification. Methods of quantitative amplification are disclosed in, e.g., U.S. Patent Nos. 6,180,349; 6,033,854; and 5,972,602, as well as in, e.g., Gibson et al., Genome Research (1996) 6: 995-1001; DeGraves, et al., Biotechniques (2003) 34 (1) : 106-10, 112-5; Deiman B, et al., Mol Biotechnol. (2002) 20 (2) : 163-79. Quantification is usually based on the monitoring of the detectable signal representing copies of the template in cycles of an amplification (e.g., PCR) reaction. Detectable signals can be generated by intercalating agents (e.g. SYBR GREEN TM and SYBR GOLD TM) or labeled primer or labeled probes used during the amplification.
In certain embodiments, the labeled primer or labeled probe comprise a detectable label comprising a fluorophore. In certain embodiments, the labeled primer or labeled probe may further comprise a quencher substance. Presence of both a fluorophore and a quencher substance ( “dual labeled” ) in one primer or probe could be helpful to provide for a self-quenching probe such as a TaqMan (U.S. Pat. Nos. 5,210,015 and 5,538,848) or Molecular Beacon probe (U.S. Pat. Nos. 5,118,801 and 5,312,728) , or other stemless or linear beacon probe (Livak et al., 1995, PCR Method Appl., 4: 357-362; Tyagi et al, 1996, Nature Biotechnology, 14: 303-308; Nazarenko et  al., 1997, Nucl. Acids Res., 25: 2516-2521; U.S. Pat. Nos. 5,866,336 and 6,117,635) . In an intact primer or probe, the quencher substance and the fluorophore are in close proximity, such that when the fluorophore is excited by irradiation, it transfers energy to the quencher substance in the same probe via fluorescence resonance energy transfer (FRET) , thereby does not emit a signal.
In a quantitative amplification assay (such as real-time PCR) , the expression level of RNA (e.g. mRNA) or the copy number variation of DNA of the biomarkers can be quantified using methods known in the art. For example, during the amplification, the fluorescence signal can be monitored and calculated during each PCR cycle. The threshold cycle, or Ct value can be further calculated. Ct value is the cycle at which fluorescence intersects a predetermined value. The Ct can be correlated to the initial amount of nucleic acids or number of starting cells using a standard curve. A standard curve is constructed to correlate the differences between the Ct values and the logarithmic level of the measured biomarker.
As a quality control measure, the expression level or copy number variation of an internal control biomarker may be measured. The skilled artisan will understand that an internal control biomarker can be inherently present in the sample and its expression level or copy number variation can be used to normalize the measured expression level or copy number variation of the biomarkers of interest, to offset any difference in the absolute amount of the sample.
(B) Hybridization assay
Nucleic acid hybridization assays use probes to hybridize to the target nucleic acid, thereby allowing detection of the target nucleic acid. Non-limiting examples of hybridization assay include Northern blotting, Southern blotting, in situ hybridization, microarray analysis, and multiplexed hybridization-based assays.
In certain embodiments, the probes for hybridization assay are detectably labeled. In certain embodiments, the nucleic acid-based probes for hybridization assay are unlabeled. Such unlabeled probes can be immobilized on a solid support such as a microarray, and can hybridize to the target nucleic acid molecules which are detectably labeled.
In certain embodiments, hybridization assays can be performed by isolating the nucleic acids (e.g. RNA or DNA) , separating the nucleic acids (e.g. by gel electrophoresis) followed by transfer of the separated nucleic acid on suitable membrane filters (e.g. nitrocellulose filters) , where the probes hybridize to the target nucleic acids and allows detection. See, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7. The hybridization of the probe and the target nucleic acid can be detected or measured by methods known in the art. For example, autoradiographic detection of hybridization can be performed by exposing hybridized filters to photographic film. Densitometric scanning of the photographic films exposed by the hybridized filters provides an accurate measurement of the target nucleic acid levels. Computer imaging systems can also be used to quantify the level of the biomarker.
In some embodiments, hybridization assays can be performed on microarrays. Microarrays provide a method for the simultaneous measurement of the levels of large numbers of target nucleic acid molecules. The target nucleic acids can be RNA, DNA, cDNA reverse transcribed from mRNA, or chromosomal DNA. The target nucleic acids can be allowed to hybridize to a microarray comprising a substrate having multiple immobilized nucleic acid probes arrayed at a density of up to several million probes per square centimeter of the substrate surface. The RNA or DNA in the sample is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative levels of the RNA or DNA. See, U.S. Patent Nos. 6,040,138, 5,800,992 and 6,020,135, 6,033,860, and 6,344,316.
Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Patent No. 5,384,261. Although a planar array surface is often employed the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays may be peptides or nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Patent Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992. Arrays may be packaged in such a manner as to allow for diagnostics or other manipulation of an all-inclusive device. Useful microarrays are also commercially  available, for example, microarrays from Affymetrix, from Nano String Technologies, QuantiGene 2.0 Multiplex Assay from Panomics.
In certain embodiments, hybridization assays can be in situ hybridization assay. In situ hybridization assay is useful to detect the presence of copy number variation (e.g. increase or amplification) at the locus of the biomarker of interest (e.g. LKB1/STK11) . Probes useful for in situ hybridization assay can be locus specific probes, which hybridize to a specific locus on a chromosome to detect the presence or absence of a specific locus of interest (e.g. LKB1/STK11) . Other types of probes may also be useful, for example, chromosome enumeration probes (e.g. hybridizable to a repeat sequence region in a chromosomal of interest to indicate presence or absence of the entire chromosome) , and chromosome arm probes (e.g. hybridizable to a chromosomal region and indicate the presence or absence of an arm of a specific chromosome) . Methods for use of unique sequence probes for in situ hybridization are described in U.S. Pat. No. 5,447,841, incorporated herein by reference. Probes can be viewed with a fluorescence microscope and an appropriate filter for each fluorophore, or by using dual or triple band-pass filter sets to observe multiple fluorophores. See, e.g., U.S. Pat. No. 5,776,688 to Bittner, et al., which is incorporated herein by reference. Any suitable microscopic imaging method can be used to visualize the hybridized probes, including automated digital imaging systems. Alternatively, techniques such as flow cytometry can be used to examine the hybridization pattern of the probes.
(C) Sequencing methods
Sequencing methods allow determination of the nucleic acid sequence of the target nucleic acid, and can also permit enumeration of the sequenced target nucleic acid, thereby measures the level of the target nucleic acid. Examples of sequence methods include, without limitation, RNA sequencing, pyrosequencing, and high throughput sequencing.
High throughput sequencing involves sequencing-by-synthesis, sequencing-by-ligation, and ultra-deep sequencing (such as described in Marguiles et al., Nature 437 (7057) : 376-80 (2005) ) . Sequence-by-synthesis involves synthesizing a complementary strand of the target nucleic acid by incorporating labeled nucleotide or nucleotide analog in a polymerase amplification. Immediately after or upon successful incorporation of a  label nucleotide, a signal of the label is measured and the identity of the nucleotide is recorded. The detectable label on the incorporated nucleotide is removed before the incorporation, detection and identification steps are repeated. Examples of sequence-by-synthesis methods are known in the art, and are described for example in U.S. Pat. No. 7,056,676, U.S. Pat. No. 8,802,368 and U.S. Pat. No. 7,169,560, the contents of which are incorporated herein by reference. Sequencing-by-synthesis may be performed on a solid surface (or a microarray or a chip) using fold-back PCR and anchored primers. Target nucleic acid fragments can be attached to the solid surface by hybridizing to the anchored primers, and bridge amplified. This technology is used, for example, in the 
Figure PCTCN2021078476-appb-000021
sequencing platform.
Pyrosequencing involves hybridizing the target nucleic acid regions to a primer and extending the new strand by sequentially incorporating deoxynucleotide triphosphates corresponding to the bases A, C, G, and T (U) in the presence of a polymerase. Each base incorporation is accompanied by release of pyrophosphate, converted to ATP by sulfurylase, which drives synthesis of oxyluciferin and the release of visible light. Since pyrophosphate release is equimolar with the number of incorporated bases, the light given off is proportional to the number of nucleotides adding in any one step. The process is repeated until the entire sequence is determined.
In certain embodiments, the detection of mutation and/or wild-type status and the measurement of level of biomarkers of interest described herein is by whole transcriptome sequencing, or RNA sequencing (e.g. RNA-Seq) . The method of RNA sequencing has been described (see Wang Z, Gerstein M and Snyder M, Nature Review Genetics (2009) 10: 57-63; Maher CA et al., Nature (2009) 458: 97-101; Kukurba K &Montgomery SB, Cold Spring Harbor Protocols (2015) 2015 (11) : 951-969) . In brief, mRNA extracted from a sample is reverse transcribed into cDNA and sheared into fragments. Fragments within proper length ranges are selected and ligated with sequencing adaptors, followed by amplification, sequencing, and mapping reads to a reference genome.
In certain embodiments, the CNV of a biomarker is determined using whole exome sequencing (WES) . WES involves sequencing DNA exons (i.e. protein encoding regions) using high-throughput sequencing technology. More details of WES can be  found, for example, in Ng SB et al, Nature. 461 (7261) : 272–276 (2009) , and Bao R et al, Cancer Inform. 2014; 13 (Suppl 2) : 67–82, which are incorporated herein to their entirety.
(D) Immunoassays
Immunoassays typically involves using antibodies that specifically bind to the biomarker polypeptide or protein (e.g. the LKB1/STK11, KRAS, and/or p53 protein as provided herein) to detect or measure the presence or level of the target polypeptide or protein. Such antibodies can be obtained using methods known in the art (see, e.g., Huse et al., Science (1989) 246: 1275-1281; Ward et al, Nature (1989) 341 : 544-546) , or can be obtained from commercial sources. Examples of immunoassays include, without limitation, Western blotting, enzyme-linked immunosorbent assay (ELISA) , enzyme immunoassay (EIA) , radioimmunoassay (RIA) , sandwich assays, competitive assays, immunofluorescent staining and imaging, immunohistochemistry (IHC) , and fluorescent activating cell sorting (FACS) . The above methods are all conventional test methods in the field. For a review of immunological and immunoassay procedures, see Basic and Clinical Immunology (Stites &Terr eds., 7 th ed. 1991) . Moreover, the immunoassays can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980) ; and Harlow &Lane, supra. For a review of the general immunoassays, see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993) ; Basic and Clinical Immunology (Stites &Terr, eds., 7 th ed. 1991) .
In certain embodiments, the antibodies are detectably labeled, or alternatively are not labeled but can react with a second molecule which is detectably labeled (e.g. a detectably labeled secondary antibody) . Other detection systems such as time-resolved fluorescence, internal-reflection fluorescence, amplification (e.g., polymerase chain reaction) and Raman spectroscopy are also useful.
In certain embodiments, the antibodies may be immobilized on a solid substrate. The immobilization can be via covalent linking or non-covalent attachment (e.g. coating) . Examples of solid substrate include porous and non-porous materials, latex particles, magnetic particles, microparticles, strips, beads, membranes, microtiter wells and plastic tubes. The choice of solid phase material and method of detectably  labeling the antigen or antibody reagent are determined based upon desired assay format performance characteristics.
(E) Activity assays
The biological activity of a protein can be measured using a bioassay. For example, LKB1/STK11 activities in a biological sample can be determined by contacting the sample with a substrate kinase (e.g. AMPK) under conditions that permit phosphorylation, and monitoring incorporation of phosphate into the substrate kinase, wherein the incorporation of phosphate into the substrate kinase indicates LKB1/STK11 activity; the activity of p53 can be measured by detecting the phosphorylation of the amino acid residue at position 15 of p53, or by detecting the change in expression level of the downstream target genes of p53. Due to a protein’s ability to exert multiple biological activities, several acceptable bioassays may exist for a particular protein. Exemplary functional assays for measuring the activity of LKB1/STK11 or p53 can be found in Thompson T, et al, Journal Biological Chemistry, 279: 53015-53022 (2004) , US patent application US20050026233A1 or PCT application WO2004113562A1.
iii.  Prediction of Responsiveness to Treatment with MDM2 Inhibitors or Selection  of Patients for Treatment with MDM2 Inhibitors
In certain embodiments, the method further comprises identifying the subject as likely to respond to the treatment with an MDM2 inhibitor based on the deficiency in functional LKB1/STK11 found in the biological sample.
In certain embodiments, detection of one or more inactivating mutation in LKB1/STK11 in the biological sample indicates deficiency of LKB1/STK11. In certain embodiments, the subject is identified as likely to respond to the treatment of MDM2 inhibitor based on having one or more inactivating mutations in LKB1/STK11. In certain embodiments, the one or more inactivating mutations are selected from mutations shown in Table 1.
In certain embodiments, the subject is identified as likely to respond to the treatment of MDM2 inhibitor based on a decrease (e.g. at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%decrease) in expression level or activity level of LKB1/STK11 relative to a reference level of LKB1/STK11 respectively. In certain embodiments, the subject is identified as  likely to respond to the treatment of MDM2 inhibitor based on decrease in copy number of LKB1/STK11 (e.g. 0, 1) relative to a reference copy number of LKB1/STK11.
In certain embodiments, the expression level or activity level or the copy number of LKB1/STK11 can be normalized to an internal control value or to a standard curve. For example, the expression level or activity level or the copy number of LKB1/STK11 can be normalized to a standard level for a standard marker. The standard level of the standard marker can be predetermined, determined concurrently, or determined after a sample is obtained from the subject. The standard marker can be run in the same assay or can be a known standard marker from a previous assay. In the cases when the expression level or activity level or the copy number of the biomarker is determined by sequencing assay (such as RNA sequencing) , the level of the biomarkers can be normalized to the total reads of the sequencing.
The term “reference level” of LKB1/STK11 can be the normal or baseline level (e.g. expression level, activity level or copy number) of LKB1/STK11, for example, a level of LKB1/STK11 in the healthy cell or tissue sample, or an average level of LKB1/STK11 in a general cancer patient population or in a cancer patient population of a particular cancer of interest.
In certain embodiments, the reference level can be a typical level, a measured level, or a range of the level of LKB1/STK11 that would normally be observed in one or more healthy cell or tissue samples, or in one or more control cell or tissue samples. In certain embodiments, the reference level can be an average level of LKB1/STK11 in a healthy subject population, or in a general cancer patient population or in a cancer patient population of a particular cancer of interest. For example, it can be an empirical level of LKB1/STK11 that is considered to be representative of a control sample or a general cancer sample. In certain embodiments, the reference level of LKB1/STK11 is obtained using the same or comparable measurement method or assay as used in the measurement of the level of LKB1/STK11 in the biological sample.
A “general cancer patient population” as used herein, refers to a population of cancer subjects or patients having different kinds of cancers. For example, a general cancer patient population may be a group of at least three (four, five, six, seven, eight, nine, ten, or more) types of cancer patients, with some patients having the first type of  cancer, some having the second type of cancer, some having the third type of cancer, and so on. For example, a general cancer patient population can be a population having all kinds of cancers or a variety of cancer types. In certain embodiments, the reference level can also be an empirical level considered representative of a general cancer patient population.
In certain embodiments, the reference level can be predetermined. For example, the reference level can be calculated or generalized based on measurements of LKB1/STK11 level in a collection of control biological samples (e.g. samples from healthy subjects, or samples from control cancer patients) . For another example, the reference level can be based on statistics of the level of LKB1/STK11 generally observed in healthy subjects, or in general cancer patient population.
In certain embodiments, the method further comprises determining in the biological sample presence or absence of a functional p53. In certain embodiments, the method further comprises determining in the biological sample if p53 is wild-type. In certain embodiments, the method further comprises determining expression level of p53 or activity level of p53.
In certain embodiments, the method further comprises determining in the biological sample presence of one or more mutations KRAS.
III. Treating the Subject Identified as Likely to Respond to the Treatment  with MDM2 inhibitors
In certain embodiments, the methods provided herein further comprises administering the MDM2 inhibitor to the subject identified as likely to respond to the treatment with an MDM2 inhibitor. In certain embodiments, the MDM2 inhibitor is administered at a therapeutically effective amount to the subject.
In another aspect, the present disclosure provides methods of treating a subject with cancer with an MDM2 inhibitor, wherein the subject has been identified as likely to respond to the treatment with MDM2 inhibitors by any of the methods provided herein. In certain embodiments, the step of treating comprising administering a therapeutically effective amount of the MDM2 inhibitor to the subject having been identified as likely to respond to the treatment with the MDM2 inhibitors.
In certain embodiments, the subject to be administered with the MDM2 inhibitor is determined to have, deficiency in functional LKB1/STK11 in the biological sample.
In certain embodiments, the subject to be administered with the MDM2 inhibitor has, or is determined to have, functional p53 in the biological sample.
In certain embodiments, the subject to be administered with the MDM2 inhibitor has, or is determined to have, one or more mutations in KRAS in the biological sample.
In some embodiments, the subject is relapsed from or refractory to an immunotherapy. In some embodiments, the immunotherapy is PD-1/PD-L1 blockade therapy. In some embodiments, the subject has been determined to further have a KRAS mutation.
IV. Methods of inducing ferroptosis
In another aspect, the present disclosure also provides methods of inducing ferroptosis in a subject in need thereof comprising administering to the subject an effective amount of a MDM2 inhibitor provided herein, and optionally, the MDM2 inhibitor has a compound of formula (I) or a pharmaceutically acceptable salt thereof.
In another aspect, the present disclosure also provides methods for treating or ameliorating the effects of a condition that would benefit from ferroptosis in a subject in need thereof comprising administering to the subject a MDM2 inhibitor provided herein in an effective amount to induce ferroptosis, and optionally, the MDM2 inhibitor has a compound of formula (I) or a pharmaceutically acceptable salt thereof.
In another aspect, the present disclosure also provides methods for treating a subject identified as having ferroptosis-sensitive condition or cancer comprising administering to the subject a MDM2 inhibitor provided herein in an effective amount to induce ferroptosis, and optionally, the MDM2 inhibitor has a compound of formula (I) or a pharmaceutically acceptable salt thereof.
As used herein, “ferroptosis” refers to a type of programmed cell death dependent on iron and is genetically and biochemically distinct from other forms of regulated cell death such as apoptosis, necrosis, and autophagy (see e.g. Dixon SJ et al, Cell (2012) 149: 1060-72) . Ferroptosis is characterized by the overwhelming, iron- dependent accumulation of lethal lipid reactive oxygen species (lipid ROS) , a process in which free radicals “steal” electrons from the lipids in cell membranes and promotes their oxidation by oxygen, resulting in cell damage.
In certain embodiments, the subject is identified as having a wild-type p53 or a p53 variant capable of regulating SLC7A11 expression. Without wishing to be bound by any theory, it is believed that MDM2 increases expression of p53, which in turn can regulate expression of SLC7A11. In addition to wild-type p53, some p53 variants or mutants can also regulate SLC7A11, for example, the acetylation-defective mutant p53 3KR (see, e.g. L. Jiang et al., Nature. 520 (7545) : 57-62 (2015) ) , and such p53 variants or mutants are also encompassed in the present disclosure.
In some embodiments, the MDM2 inhibitors provided herein are administered at an effective amount to induce ferroptosis. Ferroptosis can be detected by the measurement of lipid ROS. Induction of ferroptosis is determined by increase in lipid ROS. Any suitable methods for detecting lipid ROS can be used to detect ferroptosis. An exemplary method uses C11-BODIPY TM581/591, which is a fluorescent fatty acid analogue that allows the quantification of lipid peroxidation by indirect measure of ROS production in cells and membranes. Upon free radical-induced oxidation, its fluorescent properties shift from red to green. The measurement of lipid ROS by C11-BODIPY TM581/591 can be carried out following the manufacturer’s instructions. In addition, GSH depletion, glutamate release, and NADPH and cystine uptake assay can also be used as the measurement for ferroptosis (Xie Y, et al, Cell Death Differ., (2016) 23: 369) .
The expression “ferroptosis-sensitive” as used herein means, that the cell or condition that is susceptible to or treatable by ferroptosis-induced cell death. In certain embodiments, the ferroptosis-sensitive condition or cancer is characterized in active or overactive lipid ROS production. By “active lipid ROS production” , it is intended to mean that the biological pathway for lipid ROS production is not significantly inhibited or suppressed, relative to a control (e.g., in a normal cell) . By “overactive lipid ROS production” , it is intended to mean that the lipid ROS production pathway has an elevated activity than a control (e.g. in a normal cell) , for example, due to suppression or inhibition or absence of a negative regulator in the pathway, or due to an increased  expression level or increased activity of a gene or a protein that is not a negative regulator in the pathway.
Various biomarkers indicative for ferroptosis sensitivity are known in the art. For example, NADPH levels in a cancer cell are a biomarker of ferroptosis sensitivity across many cancer cell lines (see, e.g., Shimada et al., Cell Chem Biol.; 23 (2) : 225-235 (2016) ) . Ferroptosis sensitivity is modulated by several pathways and processes, for example glutathione metabolism (e.g. cysteine transportation and biosynthesis of glutathione) , lipid metabolism (e.g. biosynthesis and peroxidation of polyunsaturated fatty acids (PUFAs) ) , iron metabolism (e.g. iron import, export, storage and turnover in cells) , and other metabolic pathways (e.g., production of coenzyme Q10, elimination of lipid hydroperoxides) . Any suitable genes or proteins involved in such pathways or processes could be useful to indicate ferroptosis sensitivity. These metabolism pathways and processes are described in B.R. Stockwell et al., Cell, 171 (2) : 273-285 (2017) , and markers for ferroptosis are also provided.
As ferroptosis is an iron-dependent cell death, without having to be bound by theory, sensitivity to ferroptosis can be at least partly attributable to the amount of iron that is available in cells. Iron can generate reactive oxygen species (ROS) that can damage organelles. Proteins or genes that regulate iron availability by uptake, export, or a shift from storage to the labile iron pool (LIP) can affect the sensitivity of cells to ferroptosis. Such proteins or genes include, without limitation, heme carrier protein 1, integrin, ferroprotin, stimulator of Fe transport (SFT) , iron responsive element binding protein (IRP) , TFRC, transferrin, and DMT1. For example, over-expression or activating mutation of TFRC, DMT1, and/or transferrin can be indicative of sensitivity to ferroptosis.
Additionally or alternatively, proteins or genes that regulate generation of lipid ROS or lipid synthesis or metabolism can also affect the sensitivity of cells to ferroptosis. Such proteins or genes include, without limitation, SLC7A11, GPX4, FSP1/AIFM2, and SAT1. For example, underexpression or inactivating mutation of SLC7A11 or GPX4 can be indicative of sensitivity to ferroptosis (see, e.g. Kim JKM, et al, PNAS (2019) 116: 9433) ) .
In some embodiment, the ferroptosis-sensitive condition or cancer is characterized in having one or more functional or overactive genes or gene products selected from the group consistingf: heme carrier protein 1, integrin, ferroprotin, stimulator of Fe transport (SFT) , iron responsive element binding protein (IRP) , transferrin, DMT1, ACSL4, CARS, ALOXs, ATP5G3, CHAC1, CS, DPP4, GLS2, LPCAT3, NCOA4, RPL8, SAT1, TFRC, and TTC35/EMC2. These markers and their correlation with ferroptosis have been disclosed in detail (see, e.g. B. R. Stockwell et al., Cell, 171 (2) : 273-285 (2017) ) . “Overactive” as used herein refers to that the genes or gene products in the cancer cells or the subject having cancer have higher level or activity than in normal cells or in normal subjects.
In some embodiment, the overactive genes or gene products are over-expressed or having an activating mutation.
In some embodiments, the ferroptosis-sensitive cancer is characterized in having reduced activity in one or more genes or gene products selected from the group consisting of: ZEB2, AKR1C1-3, SLC7A11, SLC3A2, GPX4, CD44v, CBS, CISD1, FANCD2, GCLC/GCLM, GSS, HMGCR, HSPB1/5, NFE2L2, and SQS. These markers and their correlation with ferroptosis have been disclosed in detail (see, e.g. B.R. Stockwell et al., Cell, 171 (2) : 273-285 (2017) ) .
In some embodiments, the ferroptosis-sensitive cancer is characterized in having reduced level or activity in SLC7A11 and/or GPX4. In some embodiments, the genes or gene products having reduced activity are under-expressed or having an inactivating mutation.
The term "underexpression" as used herein with respect to a gene or gene product (e.g. protein) refers to the presence of lower amount of the gene products, usually in a cancer cell, in comparison to a control (e.g. non-cancer cell) . Underexpression of a gene or a gene product may be due to alteration (e.g. decrease) at the level of transcription, post transcriptional processing, translation, post-translational processing, cellular localization protein stability, as compared to a control. Underexpression can be detected using conventional techniques for detecting gene products such as mRNA or proteins. In some embodiments, the underexpression can be  10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or less, or 1-fold, 2-fold, 3-fold, 4-fold or more fold lower as compared to a control.
The term "overexpression" as used herein with respect to a gene or gene product (e.g. protein) refers to the presence of increased amount of the gene product, usually in a cancer cell, in comparison to a control (e.g. non-cancer cell) . Overexpression of a gene or gene product (e.g. protein) may be due to alteration (e.g. increase) at the level of transcription, post transcriptional processing, translation, post-translational processing, cellular localization protein stability, as compared to a control. Overexpression can be detected using conventional techniques for detecting gene products such as mRNA or proteins. In the embodiments, the overexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or more, or 1-fold, 2-fold, 3-fold, 4-fold or more fold higher as compared to a control.
The term “activating mutation” refers to a mutation that results in constitutive activation of a protein, and constitutive activation of a signaling pathway.
In addition, some cancer biomarkers are also identified as potentially indicative of sensitivity to ferroptosis. Such cancer biomarkers include, without limitation, RAS (e.g. HRAS and/or KRAS) , NRF2, MRP1 (multidrug resistance protein 1) . For example, over-expression or activating mutation of RAS (e.g. HRAS and/or KRAS) , NRF2, and/or MRP1 can be indicative of sensitivity to ferroptosis (see, e.g., C. M .Bebber et al, Cancers (2020) : 12, 164) . Cancer cells harboring oncogenic Ras are reported to be more sensitive to ferroptosis induction. (see e.g., Bebber CM et al, Cancers (2020) 12, 164, Yang WS, et al, Cell (2014) 156: 317-331) .
In some embodiments, cancers having a high-mesenchymal cell state are also ferroptosis-sensitive. High-mesenchymal cell state as used herein is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids, as disclosed by V.S. Viswanathan et al. in Nature 547 (7664) , 453-457 (2017) . Cancers having a high-mesenchymal cell state are dependent on GPX4 for survival. In some embodiments, cancers having a high-mesenchymal cell state are characterized in upregulation of one or more genes (e.g. stemness markers or mesenchymal markers) selected from the group consisting of: CD133, CD44, VIM, FN1, ZEB1, TWIST1, SNAI2, and CDH2. These markers have been disclosed in detail in V. S. Viswanathan et al., Nature 547 (7664) ,  453-457 (2017) , and M.J. Hangauer et al., Nature 551, 247-250 (2017) , which are incorporated herein to its entirety. Without being limited to any theories, it is believed that the subjects are particularly sensitive or vulnerable to ferroptosis, thereby are likely to respond to the MDM2 inhibitors provided herein.
In some embodiment, the subject is identified as having wild-type 53 or a p53 variant capable of regulating SLC7A11 expression, and optionally one or more mutations in KRAS in the biological sample. In some embodiment, the subject is identified as having wild-type 53 or a p53 variant capable of regulating SLC7A11 expression, and optionally as having high-mesenchymal cell state. In some embodiment, the high-mesenchymal cell state is characterized in upregulation of one or more genes selected from the group consisting of: CD133, CD44, VIM, FN1, ZEB1, TWIST1, SNAI2, and CDH2. Without being limited to any theories, it is believed that such subjects are particularly sensitive or vulnerable to ferroptosis, thereby are likely to respond to the MDM2 inhibitors provided herein.
In some embodiment, a ferroptosis-sensitive condition can be any condition that can be treated by inducing ferroptosis in the subject. For example, suitable conditions may involve elimination or growth arrest of unwanted cells (such as cancer) .
In some embodiment, ferroptosis-sensitive conditions or cancers are characteristic of having over-expression or activating mutation of RAS (e.g. KRAS and/or HRAS) . In some embodiment, ferroptosis-sensitive conditions or cancers are characteristic of having functional p53 (e.g. wild-type 53) and/or one or more mutations in KRAS.
In some embodiment, ferroptosis-sensitive conditions or cancers are further characterized in having deficiency in functional LKB1/STK11.
V. MDM2 inhibitors
The MDM2 inhibitors disclosed in the present invention inhibit the interaction between p53 or p53-related proteins and MDM2 or MDM2-related proteins. By inhibiting the negative effect of MDM2 or MDM2-related proteins on p53 or p53-related proteins, the MDM2 inhibitors of the present invention sensitize cells to inducers of apoptosis and/or cell cycle arrest, and/or ferroptosis. In one embodiment, the MDM2  inhibitors of the present invention induce apoptosis and/or cell cycle arrest, and/or ferroptosis.
Activities of MDM2 inhibitors can be determined by fluorescence-polarization MDM2 binding assay, a competitive binding assay between MDM2 inhibitors and a p53-based peptidomimetic compound competing for binding to a MDM2 protein as described in US patent 9,745,314B2.
Fluorescence polarization measurement of competitive binding works by titrating a mixture of a protein of interest and a fluorescently labeled probe with an unlabeled competitor and demonstrating that the fluorescence polarization decreases to the value observed with the free fluorescently labeled probe (Moerke N, Current Protocols in Chemical Biology, (2009) 1: 1) . In the fluorescence-polarization MDM2 binding assay, a recombinant human His-tagged MDM2 protein (residue 1-118) and a fluorescently tagged p53-based peptide called PMDM6-F (Garcia-Echeverria et al., J. Med. Chem. 43: 3205-3208 (2000) ) are used, and the Kd value of PMDM6-F with the recombinant MDM2 protein is determined. A dose-dependent, competitive binding experiments are then performed with serial dilutions of a tested MDM2 inhibitor in the presence of pre-incubated MDM2 protein and PMDM6-F peptide. The polarization values are measured and the IC50 values are determined from a plot using nonlinear least-squares analysis.
In some embodiments, the MDM2 inhibitors has an IC50 of no more than 1μM, e.g. no more than 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 150 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, in inhibiting the binding of MDM2 to P53 as determined by the fluorescence-polarization MDM2 binding assay.
In some embodiments, MDM2 inhibitor is selected from idasanutlin (RG7388) , RG7112 (PubChem Compound CID: 57406853) , HDM201 (PubChem Compound CID: 71678098) , KRT-232 (also known as AMG232, PubChem Compound CID: 58573469) , AMG 232 (PubChem Compound CID: 58573469) , BI907828 (accessible in NCI Thesaurus (version: 19.10d) under code C156709) , SAR-405838 (also known as MI-77301, PubChem Compound CID: 53476877) , MK-8242 (also  known as SCH 900242, accessible in NCI Thesaurus (version: 19.10d) under code C116867) , DS3032-b (PubChem Compound CID: 9051550) , ALRN-6924 (PubChem Compound CID: 381833444) and CGM097 ( (PubChem Compound CID: 53240420) ; or a pharmaceutically acceptable salt of any of the foregoing.
In some embodiments, the MDM2 inhibitor comprises a compound represented by formula (I) :
Figure PCTCN2021078476-appb-000022
or a pharmaceutically acceptable salt thereof, wherein
Figure PCTCN2021078476-appb-000023
is selected from the group consisting of
Figure PCTCN2021078476-appb-000024
B is a C 4-7 carbocyclic ring;
R 1 is H, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, OR a, or NR aR b;
n is 0, 1, or 2;
R 2, R 3, R 4, R 5, R 7, R 8, R 9, and R 10, independently, are selected from the group consisting of H, F, Cl, CH 3, and CF 3;
R 6 is
Figure PCTCN2021078476-appb-000025
R a is hydrogen or substituted or unsubstituted C 1-4 alkyl;
R b is hydrogen or substituted or unsubstituted C 1-4 alkyl;
R c and R d are substituents on one carbon atom of ring B, wherein
R c is H, C 1-3 alkyl, C 1-3 alkylene-OR a, OR a, or halo;
R d is H, C 1-3 alkyl, C 1-3 alkylene-OR a, OR a, or halo; or
R c and R d are taken together with the carbon to which they are attached to form a 4 to 6-membered Spiro substituent, optionally containing an oxygen atom; and
R e is -C (=O) OR a, -C (=O) NR aR b, or -C (=O) NHSO 2CH 3.
In some embodiments, 
Figure PCTCN2021078476-appb-000026
is
Figure PCTCN2021078476-appb-000027
In some embodiments, B is
Figure PCTCN2021078476-appb-000028
In some embodiments, n is 0 or 1 and R 1 is H or CH 3.
In some embodiments, 
Figure PCTCN2021078476-appb-000029
is H, CH 3, or CH 2CH 3.
In some embodiments, R 2 is H. In other embodiments, R 3 is halo, and preferably chloro. In still another embodiments, R 4 is H, R 5 is H, or both R 4 and R 5 are H.
In some embodiments, R 7 is halo, and more preferably is fluoro.
In some embodiments, each of R 8, R 9, and R 10 are H.
In some embodiments, R a and R b, individually, are H, CH 3, or CH 2CH 3.
In some embodiments, R c and R d, individually, are H, halo, OH, CH 3, CH 2CH 3, or CH 2OH. In some embodiments, R c and R d are F and F, H and H, OH and CH 3, OH and H, CH 3 and CH 3, CH 3 and OH, H and OH, CH 2CH 3 and CH 2CH 3, and CH 2OH and CH 2OH.
In some embodiments, R e is -C (=O) OH, -C (=O) NH 2, or -C (=O) NHSO 2CH 3.
In one embodiment, the MDM2 inhibitor is a compound selected from
Figure PCTCN2021078476-appb-000030
Figure PCTCN2021078476-appb-000031
Figure PCTCN2021078476-appb-000032
or a pharmaceutically acceptable salt thereof.
In one embodiment, the MDM2 inhibitor is a compound having the following structure
Figure PCTCN2021078476-appb-000033
or a pharmaceutically acceptable salt thereof.
In one embodiment, the MDM2 inhibitor is a compound having the following structure (also known as Compound C)
Figure PCTCN2021078476-appb-000034
or a pharmaceutically acceptable salt thereof.
More MDM2 inhibitors and the synthesis of the MDM2 inhibitors that can be used in the present application are further disclosed in U.S. Patent No. 9,745,314, which is incorporated herein by reference.
The MDM2 inhibitors provided herein can exist as salts. Pharmaceutically acceptable salts of the MDM2 inhibitors provided herein often are preferred in the methods of the invention. As used herein, the term “pharmaceutically acceptable salts” refers to salts or zwitterionic forms of the compounds of structural formula (I) . Salts of compounds of formula (I) can be prepared during the final isolation and purification of the compounds or separately by reacting the compound with an acid having a suitable cation, such as, but not limited to, alkali and alkaline earth metal ions, e.g., Na +, K +, Ca 2+, and Mg 2+ well as organic cations such as, but not limited to, ammonium and substituted ammonium ions, e.g., NH 4 +, NHMe 3 +, NH 2Me 2 +, NHMe 3 + and NMe 4 +. Examples of monovalent and divalent pharmaceutically acceptable cations are discussed, e.g., in Berge et al. J. Pharm. Sci., 66: 1-19 (1997) .
The pharmaceutically acceptable salts of compounds of structural formula (I) can be acid addition salts formed with pharmaceutically acceptable acids. Examples of acids which can be employed to form pharmaceutically acceptable salts include inorganic acids such as nitric, boric, hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric. Nonlimiting examples of salts of compounds of the invention include, but are not limited to, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2-hydroxyethansulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerolphsphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate,  salicylate, methanesulfonate, mesitylenesulfonate, naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, picrate, pivalate, propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate, tartrate, gluconate, methanesulfonate, ethanedisulfonate, benzene sulphonate, and p-toluenesulfonate salts. In addition, available amino groups present in the compounds of the invention can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. In light of the foregoing, any reference to compounds of the present invention appearing herein is intended to include compounds of structural formula (I) as well as pharmaceutically acceptable salts thereof.
Compounds having one or more chiral centers can exist in various stereoisomeric forms. Stereoisomers are compounds that differ only in their spatial arrangement. Stereoisomers include all diastereomeric, enantiomeric, and epimeric forms as well as racemates and mixtures thereof.
The term “geometric isomer” refers to cyclic compounds having at least two substituents, wherein the two substituents are both on the same side of the ring (cis) or wherein the substituents are each on opposite sides of the ring (trans) . When a disclosed compound is named or depicted by structure without indicating stereochemistry, it is understood that the name or the structure encompasses one or more of the possible stereoisomers, or geometric isomers, or a mixture of the encompassed stereoisomers or geometric isomers.
When a geometric isomer is depicted by name or structure, it is to be understood that the named or depicted isomer exists to a greater degree than another isomer, that is that the geometric isomeric purity of the named or depicted geometric isomer is greater than 50%, such as at least 60%, 70%, 80%, 90%, 99%, or 99.9%pure by weight. Geometric isomeric purity is determined by dividing the weight of the named or depicted geometric isomer in the mixture by the total weight of all of the geomeric isomers in the mixture.
Racemic mixture means 50%of one enantiomer and 50%of is corresponding enantiomer. When a compound with one chiral center is named or depicted without indicating the stereochemistry of the chiral center, it is understood that the name or structure encompasses both possible enantiomeric forms (e.g., both enantiomerically-pure, enantiomerically-enriched or racemic) of the compound. When a compound with two or more chiral centers is named or depicted without indicating the stereochemistry of the chiral centers, it is understood that the name or structure encompasses all possible diasteriomeric forms (e.g., diastereomerically pure, diastereomerically enriched and equimolar mixtures of one or more diastereomers (e.g., racemic mixtures) ) of the compound.
Enantiomeric and diastereomeric mixtures can be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Enantiomers and diastereomers also can be obtained from diastereomerically-or enantiomerically-pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.
When a compound is designated by a name or structure that indicates a single enantiomer, unless indicated otherwise, the compound is at least 60%, 70%, 80%, 90%, 99%or 99.9%optically pure (also referred to as “enantiomerically pure” ) . Optical purity is the weight in the mixture of the named or depicted enantiomer divided by the total weight in the mixture of both enantiomers.
When the stereochemistry of a disclosed compound is named or depicted by structure, and the named or depicted structure encompasses more than one stereoisomer (e.g., as in a diastereomeric pair) , it is to be understood that one of the encompassed stereoisomers or any mixture of the encompassed stereoisomers is included. It is to be further understood that the stereoisomeric purity of the named or depicted stereoisomers at least 60%, 70%, 80%, 90%, 99%or 99.9%by weight. The stereoisomeric purity in this case is determined by dividing the total weight in the mixture of the stereoisomers encompassed by the name or structure by the total weight in the mixture of all of the stereoisomers.
In certain embodiments, the MDM2 inhibitor is administered as a pharmaceutical composition. The pharmaceutical composition can comprise an MDM2 inhibitor or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.
“Pharmaceutically acceptable carrier” and “pharmaceutically acceptable diluent” refer to a substance that aids the formulation and/or administration of an active agent to and/or absorption by a subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the subject. Non-limiting examples of pharmaceutically acceptable carriers and/or diluents include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer’s solution) , alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, hydroxymethycellulose, fatty acid esters, polyvinyl pyrrolidine, and colors, and the like. The carriers, diluents and/or excipients are “acceptable” in the sense of being compatible with the other ingredients of the pharmaceutical composition and not deleterious to the recipient thereof. One skilled in the art would be able, by routine experimentation, to determine what an effective, non-toxic amount of an MDM2 inhibitor (e.g., Compound C) would be for the purpose of treating cancers. For example, a therapeutically active amount of MDM2 inhibitor (e.g., Compound C) may vary according to factors such as the disease stage (e.g., stage I versus stage IV) , age, sex, medical complications (e.g., immunosuppressed conditions or diseases) and weight of the subject, and the ability of the MDM2 inhibitor (e.g., Compound C) to elicit a desired response in the subject. In certain embodiments, a therapeutically active amount is a safe amount of MDM2 inhibitor which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this disclosure.
The dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or administered by continuous infusion or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. In certain embodiments, an MDM2 inhibitor  (e.g., Compound C) is administered in an amount that would be therapeutically effective if delivered alone, i.e., MDM2 inhibitor (e.g., Compound C) is administered and/or acts as a therapeutic anti-cancer agent, and not predominantly as an agent to ameliorate side effects of other chemotherapy or other cancer treatments.
In certain embodiments, an MDM2 inhibitor (e.g., Compound C) is administered in an amount that would be effective to improve or augment the immune response to the tumor. The dosages provided below may be used for any mode of administration of MDM2 inhibitor (e.g., Compound C) , including topical administration, administration by inhalation, and intravenous administration (e.g. continuous infusion) .
In some embodiments, the pharmaceutical composition comprises Compound C or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical composition is in a solid dose form. In some embodiments, the solid dose form is capsules. In some embodiments, the solid dose form is dry-filled capsules. In some embodiments, the solid dose form is dry-filled size 1 gelatin capsules. In some embodiments, the capsule comprises from about 10-500 mg of an MDM2 inhibitor, such as Compound C. In some embodiments, the pharmaceutical composition or capsule comprises silicified microcrystalline cellulose.
In certain embodiments, the MDM2 inhibitor (e.g. Compound C) or a pharmaceutically acceptable salt thereof is administered in the range of about 0.5mg/kg to about 10000 mg/kg. In certain embodiments, the MDM2 inhibitor (e.g. Compound C) or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 mg/kg/day (24 hours) to about 150 mg/kg/day (24 hours) . In certain embodiments, the MDM2 inhibitor (e.g. Compound C) or a pharmaceutically acceptable salt thereof is administered at a dose of about 10 mg/kg/week to about 700 mg/kg/week. In certain embodiments, the MDM2 inhibitor (e.g. Compound C) or a pharmaceutically acceptable salt thereof is administered orally every other day (QOD)
Other embodiments of pharmaceutical compositions or dosage regimens of MDM2 inhibitors contemplated for the treatment have been described previously (See, e.g, U.S. Patent No. 9,745,314, the entire contents of which are incorporated herein by reference) ..
VI. Combination Therapy
In some embodiments, the MDM2 inhibitor may also be administered in combination with one or more additional therapy to the subject identified as likely to respond to the treatment with an MDM2 inhibitor by any of the methods provided herein.
In some embodiments, the method of treating a subject with cancer comprises to administering to the subject with a therapeutically effective amount of an MDM2 inhibitor in combination with one or more additional therapy, wherein the subject has been determined to have deficiency in functional LKB1/STK11 in a biological sample from the subject.
In some embodiments, the one or more additional therapy comprises a radiotherapy, a chemotherapy, a targeted cancer therapy, or a therapy with a modulator of an immune checkpoint molecule. In some embodiments, the one or more additional therapy comprise administering an anti-PD-1 antibody, a Bcl-2 inhibitor, a FAK inhibitor, a MEK inhibitor, or a MET inhibitor. It is noted that the additional therapy may comprise administering traditional small organic chemical molecules or macromolecules such as a proteins, antibodies, peptibodies, DNA, RNA or fragments of such macromolecules.
In some embodiments, the Bcl-2 inhibitor is compound D,
Figure PCTCN2021078476-appb-000035
or a pharmaceutically acceptable salt thereof.
Compound D: (R) -1 - (3 - (4 - (4 - (4 - (2 - (4-chlorophenyl) -1-isopropyl-5-methyl-4 - (methylsulfonyl) -1h-pyrrole-3-yl) -5-fluorophenyl) piperazine-1-yl) phenyl) -aminosulfonyl) -2 - (trifluoromethylsulfonyl) phenylamino) -4 - (phenylthio) butyl) piperidine-4-carboxylic acid. The synthesis method of compound D can be prepared according to the description of WO2014/113413A1.
In some embodiments, the FAK inhibitor is Compound E,
Figure PCTCN2021078476-appb-000036
or a pharmaceutically acceptable salt thereof.
Compound E : (5-chloro-N 2- (2-isopropoxy-5-methyl-4- (1- (tetrahydro-2H-pyran-4-yl) -1, 2, 3, 6-tetrahydropyridin-4-yl) phenyl) -N 4- (2- (isopropylsulfonyl) phenyl) pyrimidine-2, 4-diamine) are synthesized according to the production methods described in WO 2018/044767, which is incorporated herein by reference in its entirety and for all purposes, or a method analogous thereto.
In some embodiments, the MEK inhibitor is trametinib.
As used herein, the term “radiotherapy” refers to the treatment of cancers with ionizing radiation. The term "chemotherapy" refers to the treatment of cancers using specific chemical agents. The term “targeted cancer therapy” refers to the treatment of cancers with agents (chemical compounds or macromolecules) that selectively interact with a chosen biomolecule.
As used herein, an “immune checkpoint” or “immune checkpoint molecule” is a molecule in the immune system that modulates a signal. An immune checkpoint molecule can be a co-stimulatory checkpoint molecule, i.e., turn up a signal, or an inhibitory checkpoint molecule, i.e., turn down a signal. As used herein, a “modulator of an immune checkpoint molecule” is an agent capable of altering the activity of an immune checkpoint in a subject.
In some embodiment, methods as provided herein for inducing ferroptosis in a subject in need thereof further comprises administering to the subject an additional therapy. In some embodiments, the method as provided herein for treating or ameliorating the effects of a condition that would benefit from ferroptosis in a subject in  need thereof further comprises administering to the subject an additional therapy. In some embodiments, the method as provided herein for treating a subject identified as having ferroptosis-sensitive condition or cancer further comprises administering to the subject an additional therapy.
In some embodiment, the additional therapy comprises an anti-cancer agent that does not induce ferroptosis. Without being bound to any theory, it is believed that by combining the MDM2 inhibitors provided herein which can induce ferroptosis, to an anti-cancer agents that do not induce ferroptosis, the MDM2 inhibitors can enhance the anti-cancer effects of such anti-cancer agents by providing a different mechanism to kill the cancer cells. Examples of such anti-cancer agents that do not induce ferroptosis include, for example, DNA damaging agents such as etoposide and doxorubicin. DNA damaging agents are believed to be unable to induce ferroptosis (see, e.g. L. Jiang et al, Nature. 2015 April 2; 520 (7545) : 57-62. )
In some embodiment, the additional therapy comprises ferroptosis inducing agent. As used herein, the “ferroptosis inducing agent” refers to agents that can either directly induce ferroptosis in cells or increase the sensitivity of cells to ferroptosis. Without being bound to any theory, it is believed that combination of the MDM2 inhibitors provided herein and additional ferroptosis inducing agent can provide additive or synergistic ferroptosis activity. Ferroptosis inducing agents can act on multiple factors that are involved in the regulation of ferroptosis, for example, (i) RSL3 and/or other compounds which inhibit GPX4, (ii) erastin (e.g., and/or another compound which inhibits amino acid transporters system xc-) , and (iii) buthionine sulfoximine (BSO) which inhibits gamma-glutamylcysteine synthetase and production of glutathione. Examples of ferroptosis inducing agents include RSL3, altretamine, artesunate, buthioninesulfoximine, BAY 87-2243, cyct (e) inase, DP17 erastin, FIN56, lanperisone, piperazine-coupled erastin, imidazole-ketone erastin, statins, sulfasalazine, sorafenib and withaferin A (Bebber CM et al, Cancers (2020) 12, 164, Xie Y, et al, Cell Death Differ., (2016) 23: 369) .
In some embodiments, the ferroptosis inducing agent is lenvatinib, the statins is Atorvastatin.
VII. Cancers
In certain embodiments, the cancer is solid tumor or hematologic malignancy. In various embodiments, the cancer is selected from the group consisting of leukemia, a lymphoma, a melanoma, a carcinoma, and a sarcoma. In certain embodiments, the cancer is selected from the group consisting of adrenal cortical cancer, advanced cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain/CNS tumors in adults, brain/CNS tumors in children, breast cancer, breast cancer in men, cancer in children, cancer of unknown primary, Castleman disease, cervical cancer, colon/rectum cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor (GIST) , gestational trophoblastic disease, head and neck cancer, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia-acute lymphocytic (ALL) in adults, leukemia-acute myeloid (AML) , leuhepatomakemia-chronic lymphocytic (CLL) , leukemia-chronic myeloid (CML) , leukemia-chronic myelomonocytic (CMML) , leukemia in children, liver cancer, lung cancer-non-small cell, lung cancer-small cell, lung carcinoid tumor, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-Hodgkin lymphoma in children, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma-adult soft tissue cancer, skin cancer-basal and squamous cell, skin cancer-melanoma, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms Tumor. In one embodiment, the cancer is selected from the group consisting of melanoma, Hodgkin lymphoma, lung cancer, kidney cancer, bladder cancer, head and neck cancer, Merkel cell carcinoma, urothelial carcinoma, solid tumors that are microsatellite instability-high or mismatch repair-deficient, sarcoma, colon cancer, prostate cancer, choriocarcinoma, breast cancer, retinoblastoma, stomach carcinoma, acute myeloid leukemia, lymphoma, multiple myeloma, and leukemia.
In certain embodiments, the cancer is selected from the group consisting of gastric cancer (e.g. stomach cancer) , cholangiocarcinoma, lung cancer, melanoma, breast cancer (e.g. invasive breast carcinoma) , colon cancer, ovian cancer, prostate cancer, liver cancer (e.g. hepatocellular carcinoma) , bladder cancer, pancreatic cancer, renal cancer, esophageal cancer, head and neck cancer, thyroid cancer, cutaneous squamous cell carcinoma, glioblastoma. neuroblastoma, urinary bladder cancer, hysterocarcinoma, melanoma, osteosarcoma, lymphoma (e.g., mantel cell lymphoma, diffuse large B cell lymphoma) , leukemia (e.g., T-cell prolymphocytic leukemia, chronic lymphocytic leukemia, or acute myeloid leukemia) , multiple myeloma, ulterine cancel, colorectal cancer, lung adenocarcinoma, uterine carcinosarcoma, lung squamous cell carcinoma, cervical cancer, esophagus cancer, sarcoma, chromophobe, renal cell carcinoma (RCC) , clear cell RCC, papillary RCC, uveal melanoma, testicular germ cell tumor, low grade glioma (LGG) , mesothelioma, pheochromocytoma and paraganglioma (PCPG) , thymoma and adenoid cystic carcinoma (ACC) .
In some embodiments, the cancer is selected from small cell lung carcinoma and non-small cell lung carcinoma (e.g. lung adenocarcinoma, lung squamous cell carcinoma, lung large cell carcinoma) .
In certain embodiments, the cancer is locally advanced or metastatic solid tumor or lymphoma. In certain embodiments, the subject is treatment-experienced and shows disease progression. “Treatment-experienced” as used herein means that the subject has been treated with an anti-cancer therapy. Disease progression can be characterized by a sign of reduced responsiveness to the previous treatment, for example, increase in tumor size, increase in tumor cell number, or tumor growth.
In some embodiment, the cancer is a ferroptosis-sensitive cancer comprising lung cancer, neuroblastoma, pancreatic cancer, acute myeloid leukemia, hepatocellular carcinoma, rhadomyoscarcoma, diffuse large B-cell lymphoma, renal cell carcinoma, prostate cancer, melanoma, fibrosarcoma, ovarian cancer, brain cancer or breast cancer.
In some embodiment, ferroptosis-sensitive cancers can have a high-mesenchymal cell state. In some embodiments, the high-mesenchymal cell state is characterized in upregulation of one or more genes selected from the group consisting of: CD133, CD44, VIM, FN1, ZEB1, TWIST1, SNAI2, and CDH2.
In one embodiment, administration of an MDM2 inhibitor (e.g., Compound C) as described herein results in one or more of, reducing tumor size, weight or volume, increasing time to progression, inhibiting tumor growth and/or prolonging the survival time of a subject having cancer.
VIII. Kits
In another aspect, the present disclosure further provides a kit for predicting responsiveness to treatment with MDM2 inhibitor, comprising one or more reagents for detecting deficiency in functional LKB1/STK11. In some embodiments, the kit comprises reagents for detecting presence of one or more inactivating mutation in LKB1/STK11, measuring expression or activity level of LKB1/STK11, measuring copy number of LKB1/STK11, or determining methylation status of the promoter of LKB1/STK11. In some embodiments, the kit further comprises one or more reagents for detecting presence or absence of functional p53 (e.g. wild-type p53) . In some embodiment, the kit further comprises one or more reagents for detecting the presence of one or more mutations in KRAS.
In some embodiments, the kit for predicting responsiveness to treatment with MDM2 inhibitor comprises one or more reagents for detecting the function for production of lipid ROS.
In some embodiments, the kit further comprises one or more reagents for detecting the over expression or activation mutation in the genes or gene products selected from the group consisting of: heme carrier protein 1, integrin, ferroprotin, stimulator of Fe transport (SFT) , iron responsive element binding protein (IRP) , transferrin, DMT1, ACSL4, CARS, ALOXs, ATP5G3, CHAC1, CS, DPP4, GLS2, LPCAT3, NCOA4, RPL8, SAT1, TFRC, and TTC35/EMC2.
In some embodiments, the kit further comprises one or more reagents for detecting the underexpression or inactivating mutation in the genes or gene products selected from the group consisting of: ZEB2, AKR1C1-3, SLC7A11, SLC3A2, GPX4, CD44v, CBS, CISD1, FANCD2, GCLC/GCLM, GSS, HMGCR, HSPB1/5, NFE2L2, and SQS.
In some embodiments, the kit further comprises one or more reagents for detecting the upregulation of genes or gene products selected from the group consisting of: CD133, CD44, VIM, FN1, ZEB1, TWIST1, SNAI2, and CDH2.
In some embodiments, the kit further comprises one or more reagents for detecting the overexpression or activating mutation of RAS (e.g. KRAS and/or HRAS) , TFRC or MRP1.
The measurement or detection can be at RNA level, DNA level and/or protein level. Suitable reagents for detecting target RNA, target DNA or target proteins can be used. In certain embodiments, the detection reagents comprise primers or probes that can hybridize to the polynucleotide of the gene of interest (e.g., LKB1/STK11, p53 or KRAS) . In certain embodiments, the detection reagents comprise antibodies that can specifically bind to the protein of interest (e.g., LKB1/STK11, p53 or KRAS) . In some embodiments, the primers, the probes, and/or the antibodies may or may not be detectably labeled. In certain embodiments, the kits may further comprise other reagents to perform the methods described herein. In such applications the kits may include any or all of the following: suitable buffers, reagents for isolating nucleic acid, reagents for amplifying the nucleic acid (e.g. polymerase, dNTP mix) , reagents for hybridizing the nucleic acid, reagents for sequencing the nucleic acid, reagents for quantifying the nucleic acid (e.g. intercalating agents, detection probes) , reagents for isolating the protein, and reagents for detecting the protein (e.g. secondary antibody) . Typically, the reagents useful in any of the methods provided herein are contained in a carrier or compartmentalized container. The carrier can be a container or support, in the form of, e.g., bag, box, tube, rack, and is optionally compartmentalized.
The term “primer” as used herein refers to oligonucleotides that can specifically hybridize to a target polynucleotide sequence, due to the sequence complementarity of at least part of the primer within a sequence of the target polynucleotide sequence. A primer can have a length of at least 8 nucleotides, typically 8 to 70 nucleotides, usually of 18 to 26 nucleotides. For proper hybridization to the target sequence, a primer can have at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%sequence complementarity to the hybridized portion of the target polynucleotide sequence. Oligonucleotides useful as primers may be chemically  synthesized according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts. (1981) 22: 1859-1862, using an automated synthesizer, as described in Needham-Van Devanter et al, Nucleic Acids Res. (1984) 12: 6159-6168.
Primers are useful in nucleic acid amplification reactions in which the primer is extended to produce a new strand of the polynucleotide. Primers can be readily designed by a skilled artisan using common knowledge known in the art, such that they can specifically anneal to the nucleotide sequence of the target nucleotide sequence of the at least one biomarker provided herein. Usually, the 3' nucleotide of the primer is designed to be complementary to the target sequence at the corresponding nucleotide position, to provide optimal primer extension by a polymerase.
The term “probe” as used herein refers to oligonucleotides or analogs thereof that can specifically hybridize to a target polynucleotide sequence, due to the sequence complementarity of at least part of the probe within a sequence of the target polynucleotide sequence. Exemplary probes can be, for example DNA probes, RNA probes, or protein nucleic acid (PNA) probes. A probe can have a length of at least 8 nucleotides, typically 8 to 70 nucleotides, usually of 18 to 26 nucleotides. For proper hybridization to the target sequence, a probe can have at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%sequence complementarity to hybridized portion of the target polynucleotide sequence. Probes and also be chemically synthesized according to the solid phase phosphoramidite triester method as described above. Methods for preparation of DNA and RNA probes, and the conditions for hybridization thereof to target nucleotide sequences, are described in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition. Cold Spring Harbor Laboratory Press, 1989,  Chapters  10 and 11.
The term “antibody” as used herein refers to an immunoglobulin or an antigen-binding fragment thereof, which can specifically bind to a target protein antigen. Antibodies can be identified and prepared by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing animals such as rabbits or mice (see, e.g., Huse et al., Science (1989) 246: 1275-1281; Ward et al, Nature (1989) 341 : 544-546) .
In certain embodiments, the primes or probes provided herein comprise a polynucleotide sequence hybridizable to a portion within the sequence of SEQ ID NO: 1, 3 or 5. In certain embodiments, the primes or probes provided herein comprise a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%or 100%complementarity to a portion within the sequence of SEQ ID N.: 1, 3 or 5. In certain embodiments, the antibodies provided herein comprise an antigen-binding region capable of specifically binding to an epitope within the protein or polypeptide having the sequence of SEQ ID NO: 2, 4 or 6.
In certain embodiments, the primers, the probes and the antibodies provided herein are detectably labeled. Examples of the detectable label suitable for labeling primers, probes and antibodies include, for example, chromophores, radioisotopes, fluorophores, chemiluminescent moieties, particles (visible or fluorescent) , nucleic acids, ligand, or catalysts such as enzymes.
Examples of radioisotopes include, without limitation,  123I,  124I,  125I,  131I,  35S,  3H,  111In,  112In,  14C,  64Cu,  67Cu,  86Y,  88Y,  90Y,  177Lu,  211At,  186Re,  188Re,  153Sm,  212Bi, and  32P.
Examples of fluorophores include, without limitation, Acridine, 7-amino-4-methylcoumarin-3-acetic acid (AMCA) , BODIPY, Cascade Blue, Cy2, Cy3, Cy5, Cy7, Edans, Eosin, Erythrosin, Fluorescein, 6-FAM, TET, JOC, HEX, Oregon Green, Rhodamine, Rhodol Green, Tamra. Rox, and Texas Red TM (Molecular Probes, Inc., Eugene, Oreg. ) .
Examples of enzymes include, without limitation, alkaline phosphatase, acid phosphatase, horseradish peroxidase, beta-galactosidase, and ribonuclease.
Examples of ligands include, without limitation, biotin, avidin, an antibody or an antigen.
It should be understood that it is not necessary for a detectable label to produce a detectable signal, for example, in some embodiments, it may can react with a detectable partner or react with one or more additional compounds to generate a detectable signal. For example, the detectable label can be a ligand capable of functioning as a specific binding pair member for a labeled ligand (e.g. a secondary labeled antibody) . For another example, enzymes are useful a detectable labels due to  their catalytic activity to catalyze chromo-, fluoro-, or lumo-genic substrate which results in generation of a detectable signal.
In certain embodiments, the detectably labeled primers, probes or antibodies as provided herein can further comprise a quencher substance. A quencher substance refers to a substance which, when present in sufficiently close proximity to a fluorescent substance, can quench the fluorescence emitted by the fluorescent substance as a result of, for example, fluorescence resonance energy transfer (FRET) .
Examples of a quencher substance include, without limitation, Tamra, Dabcyl, or Black Hole Quencher (BHQ, Biosearch Technologies) , DDQ (Eurogentec) , Iowa Black FQ (Integrated DNA Technologies) , QSY-7 (Molecular Probes) , and Eclipse quenchers (Epoch Biosciences) .
Primer and probes can be labeled to high specific activity by either the nick translation method or by the random priming method. Useful probe labeling techniques are described in the literature (Fan, Y-S, Molecular cytogenetics: protocols and applications, Humana Press, Totowa, N.J. xiv, 411 (2002) ) .
In addition, the kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods provided herein. While the instructional materials typically comprise written or printed materials they are not limited to such.
In certain embodiments, the kits can further comprise a computer program product stored on a computer readable medium. When computer program product is executed by a computer, it performs the step of identifying the subject as likely to respond to the treatment with an MDM2 inhibitor based on the deficiency in functional LKB1/STK11 found in the biological sample. Any medium capable of storing such computer executable instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips) , optical media (e.g., CD ROM) , and the like. Such media may include addresses to internet sites that provide such instructional materials.
The computer programs may also be encoded and transmitted using carrier signals adapted for transmission via wired, optical, and/or wireless networks conforming to a variety of protocols, including the Internet. As such, a computer readable medium  according to an embodiment of the present invention may be created using a data signal encoded with such programs. Computer readable media encoded with the program code may be packaged with a compatible device or provided separately from other devices (e.g., via Internet download) . Any such computer readable medium may reside on or within a single computer product (e.g. a hard drive, a CD, or an entire computer system) , and may be present on or within different computer products within a system or network.
In some embodiments, the present disclosure provides oligonucleotide probes attached to a solid support, such as an array slide or chip, e.g., as described in Eds., Bowtell and Sambrook DNA Microarrays: A Molecular Cloning Manual (2003) Cold Spring Harbor Laboratory Press. Construction of such devices are well known in the art, for example as described in US Patents and Patent Publications U.S. Patent No. 5,837,832; PCT application WO95/11995; U.S. Patent No. 5,807,522; US Patent Nos. 7,157,229, 7,083,975, 6,444,175, 6,375,903, 6,315,958, 6,295,153, and 5,143,854, 2007/0037274, 2007/0140906, 2004/0126757, 2004/0110212, 2004/0110211, 2003/0143550, 2003/0003032, and 2002/0041420. Nucleic acid arrays are also reviewed in the following references: Biotechnol Annu Rev (2002) 8: 85-101; Sosnowski et al. Psychiatr Genet (2002) 12 (4) : 181-92; Heller, Annu Rev Biomed Eng (2002) 4: 129-53; Kolchinsky et al., Hum. Mutat (2002) 19 (4) : 343-60; and McGail et al., Adv Biochem Eng Biotechnol (2002) 77: 21-42. The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.
EXEMPLIFICATION
While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this disclosure. Therefore, it will be appreciated that the scope of this disclosure is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example.
The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties by reference. Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one with ordinary skill in the art. Most of the compounds of the invention can be purchased commercially, such as from selleck.
Example 1. LKB1/STK11 mutations in cancers.
Mutations in LKB1/STK11 have been identified in various neoplasms, such as malignant melanoma (Guldberg, P. et al., (1999) 18: 1777) , breast cancer (Stephens P. et al., Nature (2005) 37: 590) , lung cancer (Shah U., et al, Cancer Res (2008) 68: 3562) and pancreatic cancer (Goggins, M. et al., (1999) 10: Suppl 4) . In particular, LKB1/STK11 has been suggested to be relevant to resistance to immune checkpoint inhibitor therapy in patients with lung adenocarcinoma, especially when mutation (s) in KRAS are also present (Skoulidis F, et al., J Clin Oncol. 2017; 35 (suppl_15_abstr 9016) ) . Patients with metastatic NSCLC characterized LKB1/STK11 mutations have also be found to be less likely to benefit from first-line chemotherapy (Shire N, et al. IASLC 2019 World Conference on Lung Cancer, September 7-10, 2019; Barcelona, Spain. Abstract OA07.02) .
LKB1/SKT11 mutations in various cancers were analyzed using data available from NIH Genomic Data Commons under TCGA dataset (John N et al. The Cancer Genome Atlas Pan-Cancer Analysis Project. Nat Genet. 2013 October ; 45 (10) : 1113–1120. doi: 10.1038/ng. 2764) ..
The data shown in Figure 1A demonstrates that LKB1/SKT11 mutations (including copy number variation (CNA) ) are most frequent in lung adenocarcinoma, occurring at a rate as high as 14%. Additionally, LKB1/STK11 mutations in lung adenocarcinoma may be accompanied with TP53 mutation, at a frequency of 36% (Figure 1B) . In other words, the patients with LKB1/STK11 mutant and TP53 wild-type make up about 8.7%of the population of lung adenocarcinoma.
Example 2 Binding affinity of MDM2 inhibitors to MDM2 protein
The experiment is to determining the binding affinity of Compound C to MDM2 protein by a fluorescence polarization-based (FP-based) binding assay using a recombinant human His-tagged MDM2 protein (residues 1-118) and a fluorescently tagged p53-based peptide.
The design of the fluorescence probe was based upon a previously reported high-affinity p53-based peptidomimetic compound called PMDM6-F (García-Echeverría et al., J. Med. Chem. 43: 3205-3208 (2000) ) . The Kd value of PMDM6-F with the recombinant MDM2 protein was determined from the saturation curve. MDM2 protein was serially double diluted in a Dynex 96-well, black, round-bottom plate, and the PMDM6-F peptide was added at 1 nM concentration. The assay was performed in the buffer: 100 mM potassium phosphate, pH 7.5; 100 μg/mL bovine gamma globulin; 0.02%sodium azide, 0.01%Triton X-100) and the polarization values were measured after 3 h of incubation using an ULTRA READER (Tecan U.S. Inc., Research Triangle Park, NC) . The IC50 value was obtained by fitting the mP values in a sigmoidal dose-response curve (variable slope) with a non-linear regression, and was determined to be 1.40 nM±0.25. The Kd value was calculated using the equation: Kd value=IC50-L0/2. L0/2 is the concentration of the free ligand (PMDM6-F) . Since PMDM6-F was used at a final concentration of 1 nM, L0/2 was 0.5 nM.
Dose-dependent, competitive binding experiments were performed with serial dilutions of Compound C in DMSO. A 5 μL sample of Compound C and pre-incubated MDM2 protein (10 nM) and PMDM6-F peptide (1 nM) in the assay buffer (100 mM potassium phosphate, pH 7.5; 100 μg/mL bovine gamma globulin; 0.02%sodium azide, 0.01%Triton X-100) , were added in a Dynex 96-well, black, round- bottom plate to produce a final volume of 125 μL. For each assay, the controls included the MDM2 protein and PMDM6-F (equivalent to 0%inhibition) , PMDM6-F peptide alone (equivalent to 100%inhibition) . The polarization values were measured after 3 h of incubation. The IC50 values, i.e., Compound C concentration at which 50%of bound peptide is displaced, were determined from a plot using nonlinear least-squares analysis. Curve fitting was performed using GRAPHPAD PRISM software (GraphPad Software, Inc., San Diego, Calif. ) .
The IC50 of Compound C was determined to be 3.8 nM.
Example 3. Development of predictive biomarker for Compound C
The study was to evaluate the antiproliferative activity of Compound C as a single agent in the NSCLC lines.
Methods
Cell
Figure PCTCN2021078476-appb-000037
luminescence cell viability assay kit (Promega, Cat. #G7571) was used to quantify the anti-proliferative effect of Compound C.
Briefly, cells at the logarithmic growth phase were collected, centrifuged, counted and diluted to the desired concentration. 90 μL of cell suspension containing 8000 cells were seeded to each well in an opaque 96-well plate (8000 cells/well) and incubated overnight. 3 wells containing only medium (100 μL/well) were introduced to obtain background luminescence signals (control wells) . Cell lines that were tested were shown in Table 2.
Compound C (10 μM) was serially diluted in a 1: 3 ratios to obtain 5-7 series concentration. 10 μL/well of diluted Compound C solution was added into a 96-well plate while 10 μL/well of medium was added to control wells. Plates were incubated at 37℃ in a 5%CO 2 incubator for 72 hours. Cell growth was observed daily under an inverted microscope.
At the end of the treatment, 96-well plate was removed from the incubator and equilibrated to room temperature for 30 minutes before adding 30 μL Cell Titer-
Figure PCTCN2021078476-appb-000038
reagent (protected from light) to each well. Solutions were mixed thoroughly in each well to generate cell lysis and the 96-well plate was kept at room temperature for another 10 minutes to stabilize the luminescent signal. Luminescence signal was then  detected using a Biotek synergy H1 microplate reader. Cell viability (%) was calculated using the mean luminescence (LN) value from control replicates (blank) using the following equation:
Percentage of cell viability (%) = (test cell fluorescence signal value -negative control cell fluorescence signal value) / (control cell fluorescence signal value -negative control cell fluorescence signal value) × 100%
Cell proliferative (i.e., viability) curves were plotted using Graphpad Prism 6.0 software (Golden software, Golden, Colorado, USA) .
The genetic status of LKB1/STK11, KRAS and TP53 of these cell lines were available from COSMIC database.
Results:
As shown in Table 2 below and Figure 2, MDM2 inhibitor Compound C showed anti-cancer activity in all the cells having putative inactivating mutation in LKB1/STK11 and putative functional TP53. KRAS is co-mutated in the cells having putative inactivating mutation in LKB1/STK11.
Table 2
Figure PCTCN2021078476-appb-000039
Example 4. Association of treatment with Compound C and increased lipid ROS level
Methods
A549, NCI-H460, NCI-H292, NCI-H1944, NCI-H1666, DV90 and MOLM-13 cells were cultured and prepared for the treatment with Compound C as described in  Example 2. Compound C of different concentration and a vehicle control (DMSO) as indicated were tested. The cells were harvested at the indicated times for the analysis.
Lipid ROS was detected by using BODIPY TM 581/591 C11 kit (Invitrogen, cat#D3861) according to the manufacturer’s instruction. For BODIPY 581/591 C11 staining, the signals from both non-oxidized C11 (PE channel) and oxidized C11 (FITC channel) were monitored. The ratio of MFI of FITC to MFI of PE was calculated for each sample.
The expression level of SCL7A11, p53 or p21 in was detected by western blot. Briefly, after the treatment with Compound C, cells were collected and washed with pre-cooled PBS. The cell pellets were lysed using RIPA lysis (Beyotime Biotechnology, Cat. #P0013B) containing 1%PMSF (Yeasen Biotechnology, Cat. #20104ES08) and 1%protease inhibitor. The protein concentration was measured using BCA protein assay kit (Beyotime Biotechnology, Cat. #P0012) . Cell lysates (20-50 μg) were subject to 4-20%SDS-PAGE to separate proteins. The separated proteins were transferred to a PVDF membrane (Millipore, Cat. #IPVH00010) . The PVDF membrane was then blocked with 1%BSA buffer for 1 hour at room temperature, and then it was incubated with the primary antibody anti-SLC7A11 (abcam, Cat. ab37185) , anti-p53 (CST, Cat. #2524S) or anti-p21 (CST, Cat. #2947S) diluted in 1%BSA TBST on a 4 ℃ shaker overnight. The membrane was washed for 10 minutes in 1 x TBST for 3 times. Then the PVDF membrane was incubated for 1 hour at room temperature with HRP (horse radish peroxidase) labeled secondary antibody (Yeasen, Cat. #33101ES60; Yeasen, Cat. #33201ES60) , which was prepared following the supplier's instruction. The membrane was washed for 10 minutes in 1 x TBST for 3 times again. HRP substrate was applied to the PVDF membrane. Signals were detected using ECL hypersensitive reagent.
Results
The results demonstrated that the treatment with Compound C increased the lipid ROS levels in a dose and/or time dependent manner (Figures 3A-3E) . The effects of Compound C on lipid ROS level is more significant in A549 and NCI-H460 cells that have LKB1/SKT11 mutation (Figures 3A, 3B) . The treatment with Compound C also increased the expression of p53 and p21 (the presence of p21 is an indicator of p53  activity) , and suppressed the expression of SCL7A11 (Figures 3F-3H) . These results suggested that Compound C can promote ferroptosis in human cancer cells.
Example 5. Responsiveness to the treatment with Compound C in LKB1/STK11 mutant PDX models.
The object of the study is to evaluate the anti-tumor activity of MDM2 inhibitor Compound C in LKB1/STK11 lung cancer PDX models.
A set of BALB/c nude mice (female, 6-8W, 18-20g) are inoculated subcutaneously at the right flank region with LKB1/STK11 mutant and TP53 wild-type A549 lung cancer cells (5 × 10 6) in Matrigel for tumor development. An independent set of mice are inoculated H460 lung cancer cells. The treatments are initiated when the tumors reach suitable mean size. Both mice bearing A549 lung cancer cells and mice bearing H460 lung cancer cells are administered with vehicle or Compound C according to the experimental design shown in Table 3. After the treatment, fresh tumor tissues are collected for pharmacodynamic studies and serum samples are collected for pharmacokinetic studies.
Table 3.
Figure PCTCN2021078476-appb-000040
Note: “PO” indicates oral administration, “QD” indicates once daily, “D” indicates doses.
Efficacy of the treatment with Compound C is further evaluated in additional LKB1/STK11 mutant and TP53 wild-type lung cancer PDX models available from Crowbio and Wuxi Biologics.
It is expected that Compound C exerts effective anti-proliferative activity in LKB1/STK11 mutant and TP53 wild-type lung cancer PDX models.
Example 6. Compound C significantly induces apoptosis in STK11 mutant A549 and NCI-H460 cell lines
Methods: Apoptosis was detected using an Annexin V-PI (propidium iodide) staining kit. Briefly, cells were harvested 48 hours after the treatment and washed with PBS. Cells were then stained with Annexin-V and PI, analyzed by an Attune NxT flow cytometer following manufacturer’s instruction. Apoptosis data were obtained by analyzing 20,000 cells from each experimental condition. The data were analyzed by the FlowJo software.
STK11 mutant A549 and NCI-H460 cell lines are sourced from ATCC.
Results: As shown in Figure 4 and Figure 5 Compound C significantly induces apoptosis in STK11 mutant A549 and NCI-H460 cell lines after 48 hours treatment.
Example 7. Over-expression of STK11 in A549 cell reduced COMPOUND C sensitivity
Methods: A Western blotting assay B CTG assay C Flow cytometry assay D Lipid ROS were assessed using the flow cytometer Attune NxT Flow Cytometer.
Figure 6C detailed methods: for apoptosis analysis, STK11 null or overexpressed lung cancer cell lines were treated with COMPOUND C alone for 48 hours, then subjected to flow cytometry analysis using an Annexin V/fluorescein isothiocyanate (FITC) /propidium iodide (PI; Annexin V-FITC-PI) Apoptosis Detection Kit (BD Biosciences Cat#556547) . In brief, cells were washed once with cold PBS and incubated for 30 minutes on ice with Annexin V-FITC-PI in binding buffer. Cells were then analyzed on an Attune NxT Flow Cytometer (Life Technologies, Thermo Fisher, Carlsbad, CA USA) using FlowJo software. Results were expressed as percentages of Annexin V + cells. Experiments were performed in triplicate in two independent experiments.
Figure 6 D detailed methods: Cells were seeded into 24-well plates and incubated overnight. Cells were treated with COMPOUND C for the indicated time, harvested by trypsinization and resuspended in 200 μL RPMI-1640 containing 5 μM C11-BODIPY 581/591 (Invitrogen, Cat#D3861, for lipid peroxidation detection) or 10  μM CM-H2DCFDA (Beyotime, Cat#S0033, for ROS detection) . Cells were incubated for 30 min at 37 ℃ in an incubator. Lipid peroxidation or ROS were assessed using the flow cytometer Attune NxT Flow Cytometer. A minimum of 3,0000 single cells were analyzed per well.
Results and Conclusion:
As shown in Figure 6 -8, in Figure 6A, A549-STK11 overexpression was confirmed by WB.
And as shown in Figure 6 B, Over-expression of STK11 in A549 cell reduced COMPOUND C sensitivity. As shown in Figure 6C, Cell apoptosis induced by COMPOUND C was reduced when Over-expression of STK11 in A549 cell, and as shown in shown in Figure 6 D, lipid ROS were assessed using the flow cytometer Attune NxT Flow Cytometer. No significantly change of lipid ROS when Over-expression of STK11 in A549 cell.
A549, NCI-H460 cell sourced from ATCC.
Example 8. Synergistic antiproliferative activity of Compound C plus Trametinib in STK11 and KRAS co-mutated NSCLC cell lines
Methods: A549, NCI-H460, NCI-H2122 cell sourced from ATCC.
Table 4
Cell line STK11 KRAS TP53 status
A549 p. Q37* p. G12S Wild type
NCI-H2122 p. G279fs p. G12C Q16L, C176F
NCI-H460 p. Q37* p. Q61H Wild type
Results: as shown in the table 4 and Figure 9-11. Combination treatment showed lower cell viability compared to single agents. COMPOUND C plus trametinib  (selleck) enhanced cell viability inhibition in A549, NCI-H460, NCI-H2122 STK11 and KRAS co-mutated NSCLC cells after 72h combination treatment.
Example 9. Synergistic antiproliferative activity of Compound C plus Compound D in STK11 and KRAS co-mutated NSCLC cell lines
Method Cell viability CTG assay.
Results: As shown in table 5 and Figure12-14, Combination treatment showed lower cell viability compared to single agents.
Compound C plus Compound D enhanced cell viability inhibition in A549, NCI-H1944, NCI-H2122 STK11 and KRAS co-mutated NSCLC cells after 72h combination treatment.
Table 5
Figure PCTCN2021078476-appb-000041
Example 10. Antitumor activity of COMPOUND C single agent in STK11mut LUAD PDX model
Methods: STK11mut LUAD PDX model is sourced from Crownbio.
Result: As shown in the figure 15, COMPOUND C single agent showed antitumor activity in LU5209 STK11mut LUAD PDX , T/C (%) value was 54%on Day 26.
Example 11. Combination treatment with COMPOUND C and other therapeutic agents in TP53 wt, STK11 mut LU5209 lung PDX model
Methods: the TP53 wt, STK11 mut LU5209 lung PDX is sourced from crownbio.
Table 6
Figure PCTCN2021078476-appb-000042
Results:
As shown in table 6 and FIG 16, COMPOUND C single agent showed minor antitumor activity, atorvastatin (selleck) single agent showed no antitumor activity, lenvatinib (selleck) single agent showed significantly antitumor activity. COMPOUND C plus atorvastatin combination treatment achieved a synergistic antitumor effect. COMPOUND C plus Lenvatinib combination treatment achieved an enhanced antitumor effect.
As shown in the table 6, T/C (%) value of COMPOUND C and atorvastatin combination group was 41.32%on Day 42 compared to 74.04%or 92.86%from single agents groups, the synergy score was 1.66, indicating synergistic effects. T/C (%) value of COMPOUND C and lenvastain combination group was 26.21%on Day 42 compared to 74.04%or 35.43%from single agents groups, the synergy score was 1.00.
Combination of COMPOUND C plus atorvastatin achieved synergistic antitumor effect in s.c. LU5209 STK11mut LUAD PDX .
Combination of COMPOUND C plus lenvastain achieved enhanced antitumor effect in s.c. LU5209 STK11mut LUAD PDX.
Example 12. Treatment with COMPOUND C single agent or combined with Compound E in subcutaneous A549 lung cancer
Methods: A549 sourced from ATCC.
Table 7
Figure PCTCN2021078476-appb-000043
Results: as shown in table7 and figure 18 COMPOUND C and COMPOUND E single agents showed minor antitumor activity. Compound C plus compound E combination treatment achieved an enhanced antitumor effect.
As shown in the table, T/C (%) value of COMPOUND C and COMPOUND E combination group was 43.85%on Day 53 compared to 78.91%or 71.66%from single agents groups, the synergy score was 1.29, indicating synergistic effects.
Conclusion:
Combination of COMPOUND C plus COMPOUND E achieved synergistic antitumor effect in s.c. A549 lung cancer.
Example 13. Combination treatment with COMPOUND C and Compound E/RSL3/Atorvastatin in subcutaneous TP53 wt , KRAS  mut, STK11 mut A549 lung cancer
Methods: A549 sourced from ATCC, animal model are sourced form Crownbio. RSL3 100mg/kg intratumorally injected to the model BIW (twice per week) . Atorvastain 100mg/kg, PO TIW (three times per week) .
Table 8
Figure PCTCN2021078476-appb-000044
As shown in the Figure 19, Figure 20, COMPOUND C single agent showed antitumor activity, RSL3 (selleck) and atorvastatin (selleck) single agents showed minor antitumor activity. COMPOUND C plus RSL3 or atorvastatin combination treatment achieved synergistic antitumor effect.
As shown in the table, T/C (%) value of COMPOUND C and RSL3 combination group was 26.85%on Day 63 compared to 38.20%or 77.37%from single agents groups, the synergy score was 1.10, indicating synergistic effects. T/C (%) value of COMPOUND C and atorvastatin combination group was 25.05%on Day 63  compared to 38.20%or 75.76%from single agents groups, the synergy score was 1.16, indicating synergistic effects.
Conclusion: Combination of COMPOUND C plus RSL3 or atorvastatin achieved synergistic antitumor effect in s.c. A549 lung cancer.

Claims (63)

  1. A method of identifying a subject with cancer as likely to respond to treatment with an MDM2 inhibitor, the method comprising:
    a) providing a biological sample from the subject;
    b) determining in the biological sample if there is deficiency in functional LKB1/STK11; and
    c) identifying the subject as likely to respond to the treatment with an MDM2 inhibitor based on the deficiency in functional LKB1/STK11 found in the biological sample.
  2. The method of claim 1, wherein the method further comprising:
    d) administering the MDM2 inhibitor in a therapeutically effective amount to the subject identified as likely to respond to the treatment with an MDM2 inhibitor.
  3. A method of treating a subject with cancer, the method comprising:
    a) determining in a biological sample from the subject if there is deficiency in functional LKB1/STK11; and
    b) administering the subject with a therapeutically effective amount of an MDM2 inhibitor based on the deficiency in functional LKB1/STK11 found in the biological sample.
  4. A method of treating a subject with cancer, the method comprising administering to the subject with a therapeutically effective amount of an MDM2 inhibitor, wherein the subject has been determined to have deficiency in functional LKB1/STK11 in a biological sample from the subject.
  5. The method of any one of the preceding claims, wherein said determining comprises detecting presence of one or more inactivating mutations in LKB1/STK11 in the biological sample, wherein the presence of one or more inactivating mutations in LKB1/STK11 is indicative of the deficiency in functional LKB1/STK11.
  6. The method of claim 5, wherein the one or more inactivating mutations in LKB1/STK11 comprises deletion, insertion, substitution or any combination thereof that reduces serine/threonine kinase activity of LKB1/STK11.
  7. The method of claim 6, wherein the one or more inactivating mutations in LKB1/STK11 comprises a mutation selected from the group of mutations as listed in Table 1.
  8. The method of any one of claims 1-4, wherein said determining comprises determining if level of LKB1/STK11 is reduced in the biological sample relative to a reference level, wherein the reduced level of LKB1/STK11 is indicative of the deficiency in functional LKB1/STK11.
  9. The method of any one of claims 1-4, wherein said determining comprises determining if promoter of LKB1/STK11 is hypermethylated in the biological sample, wherein the hypermethylated LKB1/STK11 promoter is indicative of the deficiency in functional LKB1/STK11.
  10. The method of any of claims 1-3 and 5-9, wherein said determining further comprises:
    determining in the biological sample presence or absence of functional p53 (e.g. wild-type p53) , wherein the presence of functional p53 (e.g. wild-type p53) is indicative of likelihood to respond to the treatment an MDM2 inhibitor.
  11. The method of claim 4, wherein the subject to be administered with the MDM2 inhibitor has, or is further determined to have, functional p53 (e.g. wild-type 53) in the biological sample.
  12. The method of claim 4 or 11, wherein the subject to be administered with the MDM2 inhibitor is relapsed from or refractory to an immunotherapy or a chemotherapy.
  13. The method of claim 4, 11 or 12, wherein the immunotherapy is PD-1/PD-L1 blockade therapy.
  14. The method of any of claims 1-3 and 5-10, wherein said determining further comprises: determining in the biological sample presence of one or more mutations in KRAS, wherein the presence of one or more mutations in KRAS is indicative of likelihood to respond to the treatment an MDM2 inhibitor.
  15. The method of any one of claims 4 and 11-13 , wherein the subject to be administered with an MDM2 inhibitor has, or is further determined to have, one or more mutations in KRAS in the biological sample.
  16. The method of any of the preceding claims, wherein i) the deficiency in functional LKB1/STK11, ii) the presence or absence of functional p53, and/or iii) the one or more mutations in KRAS, is measured by an amplification assay, a hybridization assay, a sequencing assay, or an immunoassay.
  17. The method of any one of the preceding claims, wherein the biological sample comprises a cancer cell or a non-cancer cell.
  18. The method of any one of the preceding claims, wherein the cancer is solid tumor or hematologic malignancy.
  19. The method of any one of the preceding claims, wherein the cancer is gastric cancer (e.g. stomach cancer) , cholangiocarcinoma, lung cancer, melanoma, breast cancer (e.g. invasive breast carcinoma) , colon cancer, ovarian cancer, prostate cancer, liver cancer (e.g. hepatocellular carcinoma) , bladder cancer, pancreatic cancer, renal cancer, esophageal cancer, head and neck cancer, thyroid cancer, cutaneous squamous cell carcinoma, glioblastoma. neuroblastoma, urinary bladder cancer, hysterocarcinoma, melanoma, osteosarcoma, lymphoma (e.g., mantel cell lymphoma, diffuse large B cell lymphoma) , leukemia (e.g., T-cell prolymphocytic leukemia, chronic lymphocytic leukemia, or acute myeloid leukemia) , multiple myeloma, uterine cancer, colorectal cancer, lung adenocarcinoma, uterine carcinosarcoma, lung squamous cell carcinoma, cervical cancer, esophagus cancer, sarcoma, chromophobe, renal cell carcinoma (RCC) , clear cell RCC, papillary RCC, uveal melanoma, testicular germ cell tumor, low grade glioma (LGG) , mesothelioma, pheochromocytoma and paraganglioma (PCPG) , thymoma, or adenoid cystic carcinoma (ACC) .
  20. The method of claim 19, wherein the cancer is selected from small cell lung carcinoma and non-small cell lung carcinoma (e.g. lung adenocarcinoma, lung squamous cell carcinoma, or lung large cell carcinoma) .
  21. The method of any one of the preceding claims, wherein the MDM2 inhibitor has an IC50 of no more than 1 μM (e.g. no more than 500 nM, 400 nM, 300 nM, 200 nM, 150 nM, 100 nM , 50 nM, 20 nM, 10 nM or 5 nM) in inhibiting the binding of MDM2 to p53 as determined by a fluorescence-polarization MDM2 binding assay.
  22. The method of any one of the preceding claims, wherein the MDM2 inhibitor comprises a compound of formula (I) :
    Figure PCTCN2021078476-appb-100001
    or a pharmaceutically acceptable salt thereof, wherein
    Figure PCTCN2021078476-appb-100002
    is selected from the group consisting of
    Figure PCTCN2021078476-appb-100003
    B is a C 4-7 carbocyclic ring;
    R 1 is H, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, OR a, or NR aR b;
    n is 0, 1, or 2;
    R 2, R 3, R 4, R 5, R 7, R 8, R 9, and R 10, independently, are selected from the group consisting of H, F, Cl, CH 3, and CF 3;
    R 6 is
    Figure PCTCN2021078476-appb-100004
    R a is hydrogen or substituted or unsubstituted C 1-4 alkyl;
    R b is hydrogen or substituted or unsubstituted C 1-4 alkyl;
    R c and R d are substituents on one carbon atom of ring B, wherein
    R c is H, C 1-3 alkyl, C 1-3 alkylene-OR a, OR a, or halo;
    R d is H, C 1-3 alkyl, C 1-3 alkylene-OR a, OR a, or halo; or
    R c and R d are taken together with the carbon to which they are attached to form a 4 to 6-membered Spiro substituent, optionally containing an oxygen atom; and R e is -C (=O) OR a, -C (=O) NR aR b, or -C (=O) NHSO 2CH 3.
  23. The method of claim 22, wherein
    Figure PCTCN2021078476-appb-100005
    is
    Figure PCTCN2021078476-appb-100006
    B is
    Figure PCTCN2021078476-appb-100007
    R c and R d are F and F, H and H, OH and CH 3, OH and H, CH 3 and CH 3, CH 3 and OH, H and OH, CH 2CH 3 and CH 2CH 3, or CH 2OH and CH 2OH.
  24. The method of claim 22 or 23, wherein
    Figure PCTCN2021078476-appb-100008
    is H, CH 3, or CH 2CH 3.
  25. The method of any one of claims 22-24, wherein R 2 is H; R 3 is halo; R 4 and R 5 are H.
  26. The method of any one of claims 22-25, wherein R 7 is fluoro; each of R 8, R 9, and R 10 is H; and R e is -C (=O) OH, -C (=O) NH 2, or -C (=O) NHSO 2CH 3.
  27. The method of claim 20, wherein the MDM2 inhibitor is a compound selected from:
    Figure PCTCN2021078476-appb-100009
    Figure PCTCN2021078476-appb-100010
    Figure PCTCN2021078476-appb-100011
    or a pharmaceutically acceptable salt thereof.
  28. The method of claim 22 wherein the MDM2 inhibitor is
    Figure PCTCN2021078476-appb-100012
    or a pharmaceutically acceptable salt thereof.
  29. The method of any one of claims 1-21, wherein the MDM2 inhibitor is selected from the group consisting of idasanutlin, RG7112, HDM201, KRT-232, AMG 232, BI907828, SAR-405838, MK-8242, DS3032-b, ALRN-6924 and CGM097; or a pharmaceutically acceptable salt of any of the foregoing.
  30. The method of any one of claims 2-29, wherein the method further comprises further administering an effective amount of one or more additional therapies.
  31. The method of claim 30, wherein the one or more additional therapies comprise a radiotherapy, a chemotherapy, a targeted cancer therapy, or a therapy with a modulator of an immune checkpoint molecule.
  32. The method of claim 31, wherein the one or more additional therapies comprise administering an anti-PD-1 antibody, a Bcl-2 inhibitor, a FAK inhibitor, a MEK inhibitor, or a MET inhibitor.
  33. A kit for predicting responsiveness of a subject with cancer to treatment with an MDM2 inhibitor, comprising
    a) one or more reagents for detecting deficiency in functional LKB1/STK11.
  34. The kit of claim 33, further comprising:
    b) one or more reagents for detecting presence or absence of functional p53 (e.g. wild-type p53) .
  35. The kit of claim 33 or 34, further comprising:
    c) one or more reagents for detecting one or more mutations in KRAS.
  36. A method of inducing ferroptosis in a subject in need thereof comprising administering to the subject an effective amount of a MDM2 inhibitor.
  37. A method for treating or ameliorating the effects of a condition that would benefit from ferroptosis in a subject in need thereof comprising administering to the subject a MDM2 inhibitor in an effective amount to induce ferroptosis.
  38. A method of treating a subject identified as having ferroptosis-sensitive condition or cancer , comprising administering to the subject a MDM2 inhibitor in an effective amount to induce ferroptosis.
  39. The method of any of claims 36-38, wherein the MDM2 inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof,
    Figure PCTCN2021078476-appb-100013
    wherein
    Figure PCTCN2021078476-appb-100014
    is selected from the group consisting of
    Figure PCTCN2021078476-appb-100015
    B is a C 4-7 carbocyclic ring;
    R 1 is H, substituted or unsubstituted C 1-4 alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, OR a, or NR aR b;
    n is 0, 1, or 2;
    R 2, R 3, R 4, R 5, R 7, R 8, R 9, and R 10, independently, are selected from the group consisting of H, F, Cl, CH 3, and CF 3;
    R 6 is
    Figure PCTCN2021078476-appb-100016
    R a is hydrogen or substituted or unsubstituted C 1-4 alkyl;
    R b is hydrogen or substituted or unsubstituted C 1-4 alkyl;
    R c and R d are substituents on one carbon atom of ring B, wherein
    R c is H, C 1-3 alkyl, C 1-3 alkylene-OR a, OR a, or halo;
    R d is H, C 1-3 alkyl, C 1-3 alkylene-OR a, OR a, or halo; or
    R c and R d are taken together with the carbon to which they are attached to form a 4 to 6-membered Spiro substituent, optionally containing an oxygen atom; and
    R e is -C (=O) OR a, -C (=O) NR aR b, or -C (=O) NHSO 2CH 3.
  40. The method of claim 39, wherein
    Figure PCTCN2021078476-appb-100017
    is
    Figure PCTCN2021078476-appb-100018
    B is
    Figure PCTCN2021078476-appb-100019
    R c and R d are F and F, H and H, OH and CH 3, OH and H, CH 3 and CH 3, CH 3 and OH, H and OH, CH 2CH 3 and CH 2CH 3, or CH 2OH and CH 2OH.
  41. The method of claim 39 or 40, wherein
    Figure PCTCN2021078476-appb-100020
    is H, CH 3, or CH 2CH 3.
  42. The method of any one of claims 39-41, wherein R 2 is H; R 3 is halo; R 4 and R 5 are H.
  43. The method of any one of claims 39-42, wherein R 7 is fluoro; each of R 8, R 9, and R 10 is H; and R e is -C (=O) OH, -C (=O) NH 2, or -C (=O) NHSO 2CH 3.
  44. The method of claim 39, wherein the MDM2 inhibitor is a compound selected from
    Figure PCTCN2021078476-appb-100021
    Figure PCTCN2021078476-appb-100022
    Figure PCTCN2021078476-appb-100023
    or a pharmaceutically acceptable salt thereof.
  45. The method of any one of claims 36-44, wherein the induction of ferroptosis is determined by increase in lipid reactive oxygen species (ROS) .
  46. The method of claim 36-45, wherein the subject is identified as having a wild-type p53 or a p53 variant capable of regulating SLC7A11 expression.
  47. The method of any of claims 38-46, wherein the ferroptosis-sensitive condition or cancer is characterized in active or overactive lipid ROS production.
  48. The method of claim 47, wherein the ferroptosis-sensitive condition or cancer is characterized in having one or more functional or overactive genes or gene products selected from the group consisting of: heme carrier protein 1, integrin, ferroprotin, stimulator of Fe transport (SFT) , iron responsive element binding protein (IRP) ,  transferrin, DMT1, ACSL4, CARS, ALOXs, ATP5G3, CHAC1, CS, DPP4, GLS2, LPCAT3, NCOA4, RPL8, SAT1, TFRC, and TTC35/EMC2.
  49. The method of claim 48, wherein the overactive genes or gene products are over-expressed or having an activating mutation.
  50. The method of any of claims 47-49, wherein the ferroptosis-sensitive condition or cancer is characterized in having reduced activity in one or more genes or gene products selected from the group consisting of: ZEB2, AKR1C1-3, SLC7A11, SLC3A2, GPX4, CD44v, CBS, CISD1, FANCD2, GCLC/GCLM, GSS, HMGCR, HSPB1/5, NFE2L2, and SQS.
  51. The method of any of claims 47-50, wherein the ferroptosis-sensitive cancer is characterized in having reduced activity in SLC7A11 and/or GPX4.
  52. The method of claim 50 or 51, wherein the genes or gene products having reduced activity are under-expressed or having an inactivating mutation.
  53. The method of any of claims 38-52, wherein the ferroptosis-sensitive condition or cancer is characterized in a high-mesenchymal cell state.
  54. The method of claim 53, wherein the high-mesenchymal cell state is characterized in upregulation of one or more genes selected from the group consisting of: CD133, CD44, VIM, FN1, ZEB1, TWIST1, SNAI2, and CDH2.
  55. The method of any one of claims 38-54, wherein the ferroptosis-sensitive condition or cancer is characterized in one or more of the following: over-expression or activating mutation of RAS (e.g. KRAS and/or HRAS) , TFRC or MRP1.
  56. The method of any one of claims 38-55, wherein the subject is further identified as having functional p53 (e.g. wild-type 53) and/or one or more mutations in KRAS.
  57. The method of any one of claims 38-56, wherein the subject is identified as having deficiency in functional LKB1/STK11.
  58. The method of any one of claims 38-57, wherein the ferroptosis-sensitive condition or cancer comprises lung cancer, neuroblastoma, pancreatic cancer, acute myeloid  leukemia, hepatocellular carcinoma, rhadomyoscarcoma, diffuse large B-cell lymphoma, renal cell carcinoma, prostate cancer, melanoma, fibrosarcoma, ovarian cancer, brain cancer or breast cancer.
  59. The method of any one of claims 36-58, further comprising administering to the subject an additional therapy.
  60. The method of claim 59, wherein the additional therapy comprises a radiotherapy, a chemotherapy, a targeted cancer therapy (e.g. a therapy with a Bcl-2 inhibitor, a FAK inhibitor, a MEK inhibitor, or a MET inhibitor) , a therapy with a modulator of an immune checkpoint molecule (e.g. an anti-PD-1 antibody) .
  61. The method of claim 59, wherein the additional therapy comprises an anti-cancer agent that does not induce ferroptosis.
  62. The method of claim 59, therein the additional therapy comprises a ferroptosis inducing agent.
  63. The method of claim 62, wherein the ferroptosis inducing agent comprises RSL3, altretamine, artesunate, buthioninesulfoximine, BAY 87-2243, cyct (e) inase, DP17 erastin, FIN56, lanperisone, piperazine-coupled erastin, imidazole-ketone erastin, statins, sulfasalazine, sorafenib or withaferin A.
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