EA047547B1 - METHODS TO IDENTIFY PATIENTS WHO MAY BENEFIT FROM TELOMERASE INHIBITOR TREATMENT - Google Patents

Description

Cross-references to related applications

This application claims the benefit of U.S. Provisional Patent Application No. 62/712,841, filed July 31, 2018, and U.S. Provisional Patent Application No. 62/772,849, filed November 29, 2018, the contents of which are incorporated herein by reference.

List of sequences

This application includes a sequence listing that was filed electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy is named Sequence_Listing.txt and is 356 KB in size.

Field of technology

The present invention relates to methods for identifying a patient who is likely to benefit from treatment with a telomerase inhibitor by identifying the patient: who lacks a mutation in each of JAK2, CALR and MPL; and/or who has a high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2. The present invention also relates to methods for treating myelofibrosis in a subject in need thereof (i.e., a patient) with a telomerase inhibitor.

State of the art

Myelofibrosis (MF) is one of the classic BCR-ABL1-negative chronic myeloproliferative neoplasms (MPN), characterized by clonal myeloproliferation deregulated by kinase signaling. Cervantes, Blood, 124(17):2635–2642 (2014). It is also characterized by cytopenias, constitutional symptoms, splenomegaly, and can transform into acute myeloid leukemia. Kuykendall et al., Annals of Hematology, 97: 435–431 (2018). MF is a Philadelphia chromosome-negative myeloproliferative neoplasm with a poor prognosis, for which the currently approved therapy is the JAK1/JAK2 inhibitor ruxolitinib. Ruxolitinib, an inhibitor of the Janus kinases (JAK) JAK-1 and JAK-2, is the first in its class to be licensed in the United States for the treatment of high- and intermediate-risk myelofibrosis (MF). Pardanani, et al. Blood Cancer J.; 4(12): e268 (2014). Several other JAK inhibitors are in development, and some are currently in phase III clinical trials; ibid. Other treatment options for MF include allo-SCT, hydroxyurea, interferon, lenalidomide (Revlimid®), and thalidomide. Clinical trials in MF are ongoing to evaluate selective JAK inhibitors, histone deacetylase/DNA methyltransferase inhibitors, PI3K inhibitors, Hedgehog/mammalian target of rapamycin (MTOR) inhibitors, antifibrotic agents, immunomodulators, monoclonal antibodies, and immune checkpoint inhibitors; Shreenivas, et al., Expert Opin Emerg Drugs, 23(1):37–49 (2018).

Other MPNs include essential thrombocythemia (ET) and polycythemia vera (PV); Cervantes (below). MF may arise de novo (primary MF [PMF]) or after prior ET or PV (post-ET or post-PV MF); ibid. According to Cervantes, MF is a clonal proliferation of pluripotent hematopoietic stem cells in which a population of abnormal cells releases several cytokines and growth factors into the bone marrow that lead to bone marrow fibrosis and stromal changes, and colonizes extramedullary organs such as the spleen and liver; ibid. Myelofibrosis has been associated with mutations in the Janus kinase (JAK) 2 gene (such as the V617F mutation), mutations in the thrombopoietin receptor (MPL) gene, and mutations in the calreticulin (CALR) gene; ibid. It primarily affects older people and, according to Cervantes, there is currently no curative therapy other than allogeneic hematopoietic stem cell transplantation (allo-SCT), which can be used in a small proportion of patients. Ibid.

In fact, according to Langabeer, most patients with the classic myeloproliferative neoplasms (MPNs): polycythemia vera, essential thrombocythemia, and primary myelofibrosis, carry certain disease-causing mutations in the JAK2, CALR, or MPL genes, Langabeer, JAK-STAT, 5: e1248011 (2016). These mutations are called driver mutations. Examples of driver mutations include JAK2 V617F, mutations in exon 12JAK2, exon 10MPL, and exon 9CALR; ibid.

According to Spiegel, in myelofibrosis (MF), driver mutations in JAK2, MPL, or CALR affect survival and progression to the blast cell phase, with the highest risk associated with triple-negative status (i.e. unmutated JAK2, MPL, and CALR). Spiegel et al., Blood Adv., 1(20): 1729–1738 (2017). In fact, the absence of JAK2/MPL/CALR mutations (i.e. triple-negative status) is associated with the poorest outcome. Pardanani, et al. Blood Cancer J.; 4(12): e268 (2014); see also Tefferi et al., Blood, 124(16):2507–13 (2014). In addition, mutations in high molecular risk (HMR) genes such as ASXL1, EZH2, IDH1/2, and SRSF2 were also associated with a worse prognosis. Spiegel et al. The presence of an increasing number of prognostically unfavorable/high molecular risk mutations (i.e., ASXL1, EZH2, SRSF2, and/or IDH-1/2 genes)

- 1 047547 was associated with progressively worse survival outcomes, independent of traditional risk factors. Guglielmelli et al., Leukemia, 28(9): 1804-10 (2014).

Driver mutations in JAK2, MPL, or CALR, alone or in combination with subclonal mutations in genes such as ASXL1, have been associated with differences in overall survival (OB). Spiegel et al. Triple-negative patients who lack canonical mutations in JAK2, MPL, or CALR have an increased risk of leukemic transformation and a shorter OS. Spiegel et al. found that in patients with myelofibrosis who were treated with ruxolitinib or momelotinib (JAK 1/2 inhibitors), these mutations were associated with a shorter time to treatment failure; ibid. Similarly, when comparing clinical characteristics of JAK2-positive, CALR-positive, MPL-positive, and TO-MF patients, patients with CALR mutations had significantly lower hemoglobin (mean 8.6 vs 10.7 g/dL; P 5.001) and white blood cell count (mean 11.0 vs 25 g/dL; P 5.033), trends reported in other MPN cohorts. Patel et al., Blood, 126(6):790–797 (2015). In ruxolitinib-treated patients, carriers of >3 mutations were negatively associated with splenic response and time to treatment discontinuation. Driver mutations or triple negative (JAK2, MPL, CALR) status are found in patients with myelofibrosis who discontinue JAK inhibitors; see, for example, Kuykendall et al.

Brief description of the invention

The present invention provides methods for identifying or selecting a patient who is likely to benefit from treatment with a telomerase inhibitor, such as, for example, imetelstat, by testing the patient for: the absence of a mutation in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes; and/or high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: the extra sex setae homolog 1 (ASXL1) gene, the enhancer of Zeste protein homolog 2 (EZH2) gene, the serine-arginine-rich splicing factor 2 (SRSF2) gene, and the isocitrate dehydrogenase 1/2 (IDH1/2) gene. The patient in need of treatment may have myelofibrosis. The present invention also provides methods for treating myelofibrosis in a patient in need of such treatment, which comprise the step of identifying such a patient.

According to one embodiment of the present invention, there is provided a method for identifying a patient with myelofibrosis who is likely to benefit from treatment with a telomerase inhibitor, comprising: (a) testing the patient for: (i) triple negative status based on the absence of a mutation in each of the JAK2, CALR and MPL genes, and/or (ii) a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2] and (b) selecting said patient if he has: (i) triple negative status based on the absence of a mutation in each of the JAK2, CALR and MPL genes, and/or (ii) high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2, wherein said selected patient is likely to benefit from treatment with a telomerase inhibitor.

According to an alternative embodiment of the present invention, there is provided a method of identifying a patient who is likely to benefit from treatment with a telomerase inhibitor, comprising: (a) testing the patient for: triple negative status based on the absence of a mutation in each of the JAK2, CALR and MPL genes; and (b) selecting said patient if he or she has a triple negative status, wherein said selected patient is likely to benefit from treatment with a telomerase inhibitor. According to an alternative embodiment of the present invention, there is provided a method of identifying a patient who is likely to benefit from treatment with a telomerase inhibitor, comprising: (a) testing said patient for high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2 and (b) selecting a high molecular risk (HMR) patient based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2. The present invention also provides methods for treating myelofibrosis in a patient who has triple negative status and/or HMR with a telomerase inhibitor, such as, for example, imetelstat.

According to another embodiment of the present invention, there is provided a method of identifying a patient having myelofibrosis who is likely to benefit from treatment with a telomerase inhibitor, comprising: (a) obtaining a DNA sample from the patient; (b) testing said DNA sample from such patient for: (i) triple negative status based on the absence of a mutation in each of the JAK2, CALR, and MPL genes; and/or (ii) high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1/2; and (c) selecting said patient if the patient has: (i) triple negative status based on the absence of a mutation in each of the JAK2, CALR, and MPL genes and/or (ii) high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1/2, wherein said selected patient is likely to benefit

- 2 047547 from treatment with a telomerase inhibitor. According to certain embodiments of the said method, a DNA sample is obtained from bone marrow, peripheral blood, or both.

Said DNA sample may be obtained by first obtaining a bone marrow sample, a peripheral blood sample, or both samples, and then isolating DNA from said bone marrow sample, a peripheral blood sample, or both samples. According to one embodiment, the step of obtaining a DNA sample from a patient includes: obtaining a bone marrow sample from the patient, isolating cells from said bone marrow sample, and extracting DNA from the isolated cells. According to another embodiment, the step of obtaining a DNA sample from a patient includes: obtaining a peripheral blood sample from the patient; isolating cells from said peripheral blood sample (e.g., granulocytes); and extracting DNA from the isolated cells.

According to another embodiment of the present invention, there is provided a method for identifying a patient having myelofibrosis who is likely to benefit from treatment with a telomerase inhibitor, comprising testing the patient for: (a) triple negative status based on the absence of any mutation in the JAK2, CALR and MPL genes; (b) high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2; or (c) both; wherein the presence of (a), (b) or (c) indicates a patient who is likely to benefit from treatment with a telomerase inhibitor.

According to any of said methods, said patient may have myelofibrosis. Said myelofibrosis may be: primary myelofibrosis; myelofibrosis that develops after polycythemia vera (post-PV MF); or myelofibrosis that develops after essential thrombocythemia (post-ET MF). In some embodiments, said patient has not previously received therapy with JAK inhibitors. In other embodiments, said patient has previously received therapy with JAK inhibitors and said therapy with JAK inhibitors was ineffective (i.e., the disease was resistant or the patient was refractory to said therapy, or the disease relapsed despite initially responding to treatment). In other embodiments, said patient has received therapy with JAK inhibitors and discontinued therapy with JAK inhibitors due to treatment-related toxicity or intolerance.

The methods may also include the step of administering a telomerase inhibitor after such a patient has been identified. In some embodiments, the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, the imetelstat is imetelstat sodium.

When imetelstat is used to treat patients identified by the above methods, imetelstat is administered in 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each of which comprises: intravenous administration of about 7-10 mg/kg imetelstat once every three weeks; intravenous administration of about 7-10 mg/kg imetelstat once a week for three weeks; intravenous administration of about 2.5-10 mg/kg imetelstat once every three weeks; or intravenous administration of about 0.5-9.4 mg/kg imetelstat once every three weeks. In one embodiment, each dosing cycle comprises intravenous administration of about 7-10 mg/kg imetelstat once every three weeks. In another embodiment, each dosing cycle comprises intravenous administration of about 9.4 mg/kg imetelstat once every three weeks.

When imetelstat sodium is used to treat patients identified by the methods, imetelstat sodium is administered in 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each of which comprises: intravenous administration of about 7-10 mg/kg imetelstat sodium once every three weeks; intravenous administration of about 7-10 mg/kg imetelstat sodium once a week for three weeks; intravenous administration of about 2.5-10 mg/kg imetelstat sodium once every three weeks; or intravenous administration of about 0.5-9.4 mg/kg imetelstat sodium once every three weeks. According to one embodiment, each dosing cycle comprises intravenous administration of about 7-10 mg/kg imetelstat sodium once every three weeks. In another embodiment, each dosing cycle comprises intravenous administration of approximately 9.4 mg/kg imetelstat sodium once every three weeks. In another embodiment, the present invention provides a method of treating a patient having myelofibrosis with a telomerase inhibitor such as imetelstat or imetelstat sodium, comprising: (i) screening said patient to determine whether said patient is: triple negative based on the absence of a mutation in each of JAK2, CALR, and MPL, and/or high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1/2; and (ii) administering said telomerase inhibitor to said patient provided that said patient has a triple negative status based on the absence of a mutation in any of JAK2, CALR, and MPL, and/or is high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1/2. Said myelofibrosis may be: primary myelofibrosis, myelofibrosis that develops after true

- 3 047547 polycythemia vera (post-PV MF), or myelofibrosis that develops after essential thrombocythemia (post-ET MF). According to some embodiments, the patient has not previously received JAK inhibitor therapy. According to another embodiment, the patient has previously received JAK inhibitor therapy and the JAK inhibitor therapy was ineffective, or has previously received JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

According to certain embodiments of the method of treatment, the telomerase inhibitor is imetelstat and is administered in 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each of which comprises intravenous administration of about 7-10 mg/kg imetelstat once every three weeks; intravenous administration of about 7-10 mg/kg imetelstat once a week for three weeks; intravenous administration of about 2.5-10 mg/kg imetelstat once every three weeks; or intravenous administration of about 0.5-9.4 mg/kg imetelstat once every three weeks. According to some embodiments, each dosing cycle comprises intravenous administration of about 7-10 mg/kg imetelstat once every three weeks. According to other embodiments, each dosing cycle comprises intravenous administration of about 9.4 mg/kg imetelstat once every three weeks.

According to some embodiments of the methods for identifying or selecting a patient who is likely to benefit from treatment with a telomerase inhibitor, the method further comprises determining the average relative telomere length by analyzing the relative length of telomeric nucleic acids in target cells present in a biological sample from the patient. According to some embodiments of the methods for identifying or selecting a patient who is likely to benefit from treatment with a telomerase inhibitor, the method further comprises selecting a patient identified as a patient in whose biological sample the average relative telomere length in the target cells is determined to be in the 50th percentile or lower of a range of relative telomere lengths determined for one or more known reference standards. According to some embodiments, the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. According to other embodiments, the imetelstat is imetelstat sodium.

According to the present invention, there is provided a method of treating a patient with myelofibrosis with a telomerase inhibitor, comprising: administering said telomerase inhibitor to said patient, provided that the patient has a triple negative status based on the absence of a mutation in each of JAK2, CALR and MPL. In some embodiments, said telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, said imetelstat is imetelstat sodium. According to the present invention, there is provided a method of treating a patient having myelofibrosis with a telomerase inhibitor, comprising: administering said telomerase inhibitor to said patient, provided that the patient has a triple negative status based on the absence of a mutation in each of JAK2, CALR and MPL, and/or has a high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2.

According to the present invention, there is provided a method of treating a patient having myelofibrosis with a telomerase inhibitor, comprising: administering said telomerase inhibitor to said patient if such patient meets one or more of the following characteristics:

(a) the average relative telomere length in the target cells present in the biological sample from the individual is determined to fall within the 50th percentile or lower of the range of relative telomere lengths determined for one or more known reference standards;

(b) triple negative status based on the absence of a mutation in each of JAK2, CALR, and MPL; and (c) high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1/2.

According to some embodiments, said telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. According to other embodiments, said imetelstat is imetelstat sodium. According to the present invention, there is provided a method for identifying a subject having myelofibrosis (MF) for treatment with a telomerase inhibitor, wherein said method comprises: measuring the level of hTERT expression in a biological sample obtained from said patient after administration of a telomerase inhibitor; and comparing the level of hTERT expression in said biological sample with a baseline level of hTERT expression before administration of said telomerase inhibitor; wherein a decrease in the level of hTERT expression in said biological sample identifies a patient who is likely to benefit from treatment with said telomerase inhibitor.

According to the present invention, a method of treating myelofibrosis (MF) is provided, comprising: administering to a subject in need thereof an effective amount of a telomerase inhibitor; and assessing the level of hTERT expression in a biological sample obtained from said patient after administration.

- 4 047547 of said telomerase inhibitor. According to some embodiments, said telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. According to other embodiments, said imetelstat is imetelstat sodium.

According to the present invention, there is provided a method for monitoring therapeutic efficacy in a subject with myelofibrosis (MF), comprising: measuring the level of hTERT expression in a biological sample obtained from said patient after administration of a telomerase inhibitor; and comparing the level of hTERT expression in said biological sample with a baseline level of hTERT expression before administration of said telomerase inhibitor; wherein a decrease in the level of hTERT expression in the biological sample by 50% or more identifies a subject who is likely to benefit from treatment with said telomerase inhibitor. According to some embodiments, said telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. According to other embodiments, said imetelstat is imetelstat sodium.

According to the present invention, there is provided a method for selecting a patient who is likely to benefit from treatment with a telomerase inhibitor, comprising: testing the patient for an average relative telomere length by analyzing the relative length of telomeric nucleic acids in target cells present in a biological sample from the patient; and selecting said patient, provided that the average relative telomere length in the target cells present in the biological sample from the patient is determined to fall within the 50th percentile or lower of a range of relative telomere lengths determined for one or more known standard samples, wherein said selected patient is likely to benefit from treatment with a telomerase inhibitor.

According to the present invention, there is provided a method for identifying a patient who is likely to benefit from treatment with a telomerase inhibitor, comprising: obtaining a biological sample from the patient; determining an average relative telomere length by analyzing the relative length of telomeric nucleic acids in target cells present in the biological sample from the patient; and identifying said patient, provided that the average relative telomere length in the target cells present in the biological sample from the patient is determined to fall within the 50th or lower percentile of a range of relative telomere lengths determined for one or more known standard samples, as a patient who is likely to benefit from treatment with a telomerase inhibitor.

According to the present invention, there is provided a method of treating a patient having myelofibrosis with a telomerase inhibitor, comprising: administering said telomerase inhibitor to said patient, provided that the average relative telomere length in target cells present in a biological sample from said patient is determined to be in the 50th or lower percentile of a range of relative telomere lengths determined for one or more known standard samples. According to some embodiments, said telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. According to other embodiments, said imetelstat is imetelstat sodium.

According to the present invention, there is provided a method for monitoring therapeutic efficacy in a subject with myelofibrosis (MF), comprising measuring the level of hTERT expression in a biological sample obtained from said patient after administration of a telomerase inhibitor; and comparing the level of hTERT expression in said biological sample with a baseline level of hTERT expression before administration of said telomerase inhibitor; wherein a decrease in the level of hTERT expression by 50% or more in said biological sample identifies a subject who is likely to benefit from treatment with said telomerase inhibitor. In some embodiments, the measured or assessed level of hTERT expression is an expression level of hTERT RNA. In some embodiments, said telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. In other embodiments, said imetelstat is imetelstat sodium.

According to the present invention, a method is provided for identifying a patient with myelofibrosis (MF) for treatment with a telomerase inhibitor, wherein said method comprises: measuring the level of hTERT expression in a biological sample obtained from said patient after administration of a telomerase inhibitor; and comparing the level of hTERT expression in said biological sample with a baseline level of hTERT expression before administration of said telomerase inhibitor; wherein a decrease in the level of hTERT expression in said biological sample identifies a patient who is likely to benefit from treatment with said telomerase inhibitor.

According to the present invention, there is provided a method for monitoring therapeutic efficacy in a subject with myelofibrosis (MF), comprising: measuring the level of telomerase activity in a biological sample obtained from said patient after administration of a telomerase inhibitor; and comparing the level of telomerase activity in said biological sample with a baseline level of telomerase activity before administration of said telomerase inhibitor; wherein the level of activity decreases

- 5 047547 telomerase by 50% or more in said biological sample identifies a patient who is likely to benefit from treatment with said telomerase inhibitor. According to some embodiments, said telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof. According to other embodiments, said imetelstat is imetelstat sodium.

Brief description of the drawings

The above brief description, as well as the detailed description of the invention below, can be better understood when studied in conjunction with the accompanying drawings. In order to illustrate the invention, embodiments of the present invention are shown in the drawings. However, it should be understood that the present invention is not limited to the specific circuits, examples and means shown.

Figure 1 shows a cascade plot of spleen volume reduction (SVR) at week 24 for the 4.7 mg/kg and 9.4 mg/kg treatment subgroups from Example 1. SVR is shown as percent change from baseline.

Figure 2 shows a cascade plot of the reduction in total symptom score (TSS) at week 24 for the 4.7 mg/kg and 9.4 mg/kg treatment subgroups from Example 1. TSS is shown as percent change from baseline.

Figure 3 shows the Kaplan-Meier plot of overall survival by JAK2/MPL/CALR mutation status: TO versus non-TO (MUT) for the 4.7 mg/kg subgroup. In particular, Figure 3 shows the survival probability versus time for patients with triple negative status (TO) and patients with at least one mutation (MUT).

Figure 4 shows the Kaplan-Meier overall survival plot stratified by JAK2/MPL/CALR mutation status: TO versus non-TO (MUT) for the 9.4 mg/kg subgroup. In particular, Figure 4 shows the survival probability versus time for patients with triple negative status (TO) and patients with at least one mutation (MUT) for the 9.4 mg/kg subgroup.

Figure 5 shows the Kaplan-Meier plot of overall survival versus time by grouping patients in the 9.4 mg/kg subgroup or the 4.7 mg/kg subgroup. Figure 6 shows the Kaplan-Meier plot of overall survival (OS) by JAK2/MPL/CALR mutation status: TO versus non-TO for the 9.4 mg/kg subgroup. Specifically, Figure 6 shows the survival probability versus time for patients with triple negative status (TO) and patients with at least one mutation (non-TO) for the 9.4 mg/kg subgroup. Figure 7 shows the Kaplan-Meier plot of overall survival (OS) by grouping JAK2/MPL/CALR mutation status: TO versus non-TO for the 4.7 mg/kg subgroup. Specifically, Figure 7 shows the survival probability versus time for triple-negative (TN) patients and patients with at least one mutation (non-TN) for the 4.7 mg/kg subgroup.

Detailed description of the invention

The present application is based on the discovery that patients with myelofibrosis who are triple negative (i.e., lack a mutation in each of JAK2, CALR, and MPL) and/or in the high molecular risk (HMR) category based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1/2, may benefit from treatment with a telomerase inhibitor such as imetelstat or imetelstat sodium. Patients with mutations in the ASXL1, EZH1, IDH1/2, and SRSF2 genes have an increased risk of early death or leukemic transformation. These patients generally do not benefit from treatment with standard therapies such as JAK inhibitors. Gisslinger et al., Blood, 128: 1931 (2016). Accordingly, the fact that these patients benefit from treatment with a telomerase inhibitor is unexpected and surprising.

Accordingly, provided herein are methods for identifying a patient who is likely to benefit from treatment with a telomerase inhibitor, such as imetelstat. The methods comprise testing or identifying the patient to determine whether the patient is triple negative based on the absence of a mutation in each of JAK2, CALR, and MPL; and/or is high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1/2. The present invention also provides methods for treating myelofibrosis with a telomerase inhibitor, such as imetelstat, comprising identifying a patient who is triple negative based on the absence of a mutation in each of JAK2, CALR, and MPL; and/or is high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1/2. Such patients are likely to benefit from treatment with a telomerase inhibitor. Said telomerase inhibitor (e.g., imetelstat) is then administered to said patient. For clarity of understanding of the invention, but not for limitation, the detailed description of the invention is divided into subsections describing or illustrating certain features, embodiments, or uses according to the present invention.

- 6 047547

A. Definitions.

As used herein, a mutation in accessory sex bristle homolog 1 (ASXL1), enhancer of Zeste protein homolog 2 (EZH2), serine-arginine-rich splicing factor 2 (SRSF2), and isocitrate dehydrogenase 1/2 (IDH1/2) includes any mutation in these genes that affects survival and disease progression in a patient with myelofibrosis. Additionally, as used herein, IDH1/2 includes IDH1 and IHD2. Examples of mutations can be found in the following publications, the contents of each of which are incorporated herein for their description of genetic mutations associated with myelofibrosis: Langabeer, JAK-STAT, 5: e1248011 (2016); Cervantes, Blood; 124(17):2635–2642 (2014); Patel et al., Blood; 126(6):790-797 (2015); Spiegel et al., Blood Adv., 1(20): 1729-1738 (2017); Newburry et al., Blood, 130(9):1125–1131 (2017); Kuykendall et al., Annals of Hematology, 97: 435-431 (2018). Examples of sequences are the following: high molecular risk (HMR) can be determined based on the presence of a mutation in at least one of the following genes: the ASXL1 gene, having, for example, the nucleic acid sequence of SEQ ID NO: 5, the EZH2 gene, having, for example, the nucleic acid sequence of SEQ ID NO: 6, the SRSF2 gene, having, for example, the nucleic acid sequence of SEQ ID NO: 7, the IDH1 gene, having, for example, the nucleic acid sequence of SEQ ID NO: 8, the IDH2 gene, having the nucleic acid sequence of SEQ ID NO: 9; and combinations thereof.

In some embodiments, mutations of interest in the ASXL1 gene include mutations in Q575, Q588, Y591, Q592, S604, L614, Q623, A627, E635, T638, A640, G646, G658, R678, C687, D690, R693, Y700, G704, E705, Q708, G710, L721, E727, V751, P763, Q780, W796, V807, T822, K825, S846, D855, C856, L857, L885, L890, S903, S970, Y974, R965, G967, V962, L992, S1028, Q1039, R1073, E1102, H1153, S1209, S1231, A1312, F1305, P1377, R1415 and I1436. In some embodiments, the mutation is a Q575X mutation, a Q588X mutation, a Y591X mutation, a Y591N mutation, a Q592X mutation, a S604F mutation, a L614F mutation, a Q623X mutation, an A627G mutation, an E635R mutation, a T638V mutation, an A640G mutation, a G646W mutation, a G658X mutation, a R678K mutation, a C687R mutation, a C687V mutation, a D690G mutation, a R693X mutation, a Y700X mutation, a G704R mutation, a G704W mutation, an E705X mutation, a Q708X mutation, a G710E mutation, a L721C mutation, an E727X mutation, a V751L mutation, a P763R mutation, mutation Q780X, mutation W796X, mutation W796G, mutation V807F, mutation T822H, mutation K825X, mutation S846Q, mutation D855A, mutation C856X, mutation L857R, mutation L885X, mutation L890F, mutation S903I, mutation S970N, mutation Y974X, mutation R965X, mutation G967del, mutation V962A, mutation L992Q, mutation S1028R, mutation Q1039L, mutation R1073C, mutation E1102D, mutation H1153R, mutation S1209I, mutation S1231F, mutation A1312V, mutation F1305W, mutation P1377S, mutation R1415Q and/or mutation I1436M.

In some embodiments, mutations of interest in the EZH2 gene include mutations in W60, R63, P312, F145, N182, R288, Q328, Q553, R566, T573, R591, R659, D677, V679, R690, A702, V704, E726, D730, and/or Y733. In some embodiments, the mutation is W60X, R63X mutation, P312S mutation, F145S mutation, N182D mutation, R288Q mutation, Q328X mutation, Q553X mutation, R566H mutation, T573I mutation, R591H mutation, R659K mutation, D677H mutation, V679M mutation, R690H mutation, A702V mutation, V704L mutation, E726V mutation, D730X mutation and/or Y733X mutation.

In some embodiments, mutations of interest in the SRSF2 gene include mutations in P95. In some embodiments, said mutation is a P95H mutation, a P95L mutation, or a P95R mutation. In some embodiments, mutations of interest in the IDH1/2 gene include mutations in R132 and/or R140. In some embodiments, said mutation is a R132G mutation, a R132H mutation, or a R140Q mutation.

According to a particular embodiment, mutations of interest include those shown below.

Gene Protein Change ASXL1 r.Tug591* Nonsense mutation ASXL1 p.Gln592* Nonsense mutation ASXL1 r.Pe617* Nonsense mutation ASXL1 p.Glu635Argfs*15 Frame shift ASXL1 p.Gly646Trpfs*12 Frame shift ASXL1 p.Asp667Trpfs*2 Frame shift ASXL1 p.Gln692* Nonsense mutation ASXL1 p.Arg693* Nonsense mutation

- 7 047547

ASXL1 p.Arg693* Nonsense mutation ASXL1 p.Arg693Ter Nonsense mutation ASXL1 p.Tyr700Ilefs*3 Nonsense mutation ASXL1 p.Lys726* Nonsense mutation ASXL1 p.Gln760Hisfs*13 Frame shift ASXL1 p.Leu775* Nonsense mutation ASXL1 p.Trp796* Frame shift ASXL1 p.Pro808fs Frame shift ASXL1 p.Pro808His Missense mutation ASXL1 p.Pro808Leufs*10 Frame shift ASXL1 p.Leu823* Nonsense mutation ASXL1 p.Gly826Glufs*12 Frame shift ASXL1 p.Ala861Aspfs*6 Frame shift ASXL1 p.Pro938* Nonsense mutation ASXL1 p.Asn986Ser Missense mutation ASXL1 p.Glnl234* Nonsense mutation ASXL1 p.Thrl388Serfs*5 Frame shift EZH2 p.Lys400_Glu401 del In-frame deletion EZH2 p.Cys457Tyr Missense mutation EZH2 p.Ser480Argfs*3 Frame shift EZH2 p.Cys552Tyr Missense mutation EZH2 p.Leu56Phefs*2 Frame shift EZH2 p.Ser669Arg Missense mutation EZH2 p. Asp68 l_Lys685del In-frame deletion EZH2 p.Arg684Cys Missense mutation EZH2 p.Tyr733* Nonsense mutation EZH2 p.Glu745Lys Missense mutation EZH2 p.Fe223Phefs*18 Frame shift SRSF2 p.Pro95Ala Missense mutation SRSF2 p.Pro95His Missense mutation SRSF2 p.Pro95Arg Missense mutation SRSF2 p.Pro95Leu Missense mutation SRSF2 p.Prol07His Missense mutation IDHI p.Argl32His Missense mutation IDH2 p.Argl40Gln Missense mutation IDH2 p.Argl40Gln Missense mutation

As used herein, triple negative status, triple negative, or TO refers to patients who lack mutations in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes. Triple negative status can be determined based on the lack of a mutation in each of the JAK2 gene, having, for example, the nucleic acid sequence of SEQ ID NO: 2, the CALR gene, having, for example, the nucleic acid sequence of SEQ ID NO: 3, and the MPL gene, having, for example, the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, triple negative status includes the lack of a mutation in the JAK2 gene, such as a mutation in G335, F556, G571, V617, and/or V625.

- 8 047547

For example, a triple negative status may include the absence of the G335D mutation, the F556V mutation, the G571S mutation, the V617F mutation, and/or the V625S mutation in the JAK2 gene.

In some embodiments, the triple negative status includes the absence of a mutation in the MPL gene, such as a mutation at T119, S204, P222, E230, V285, R321, S505, W515, Y591 and/or R592. For example, the triple negative status can include the absence of the T1191 mutation, the S204F mutation, the S204P mutation, the P222S mutation, the E230G mutation, the V285E mutation, the R321W mutation, the S505N mutation, the W515R mutation, the W515L mutation, the Y591N mutation and/or the R592Q mutation in the MPL gene. In some embodiments, the triple negative status includes the absence of a mutation in the CALR gene, such as a mutation at L367, K368, E381, K385 and/or E396. For example, a triple negative status may include the absence of the L367T mutation, the K368R mutation, the K385N mutation, the E381A mutation, and/or the E396del mutation in the CALR gene.

In some embodiments, mutations of interest include those shown below.

Gene Protein Change JAK2 p.Val617Phe Missense mutation JAK2 p.Val617Phe Missense mutation JAK2 p.Val617Phe Missense mutation MPL p.Ser505Asn Missense mutation MPL p.Trp515Lys Missense mutation MPL p.Trp515Leu Missense mutation CALR p.Leu367Thrfs*46 Frame shift CALR p.Leu367fs Frame shift CALR p.Glu381Ala Missense mutation CALR p.Lys385Asnfs*47 Frame shift CALR p.Glu396del In-frame deletion CALR p.Lys368Argfs*51 Frame shift

In this document, a patient's JAK inhibitor therapy failed if the disease was resistant or the patient was refractory to the therapy or the disease relapsed despite initially responding to the treatment.

As used herein, the term "approximately" in relation to a measurable quantity such as a quantity, a duration of time, etc., encompasses variations within ±20% and ±0.1%, preferably within ±20% or ±10%, more preferably within ±5%, even more preferably within ±1%, and even more preferably within ±0.1% of the stated quantity, if such variations are suitable for implementing the disclosed methods.

The term pharmaceutically acceptable salt means a salt acceptable for administration to a patient, such as a mammal (salts with counterions that have acceptable safety for mammals under a given dosage regimen). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. Pharmaceutically acceptable salt refers to pharmaceutically acceptable salts of a compound that are derived from various organic and inorganic counterions well known in the art and include, for example, sodium, etc.; if the molecule contains a basic functional group, salts of organic or inorganic acids, such as hydrochloride, etc. Pharmaceutically acceptable salts of interest include, but are not limited to, aluminum, ammonium, arginine, barium, benzathine, calcium, cholineate, ethylenediamine, lysine, lithium, magnesium, meglumine, procaine, potassium, sodium, tromethamine, N-methylglucamine, ^N-dibenzylethylenediamine, chloroprocaine, diethanolamine, ethanolamine, piperazine, zinc, diisopropylamine, diisopropylethylamine, triethylamine, and triethanolamine.

The term salt(s) of the above means a compound formed by replacing a proton of an acid with a cation, such as a metal cation or an organic cation, etc. Preferably, the salt is a pharmaceutically acceptable salt. For example, salts of the present compounds include salts characterized in that the compound is protonated with an inorganic or organic acid to form a cation, with the conjugate base of said inorganic or organic acid as the anionic component of the salt. Salts of interest include, but are not limited to, aluminum, ammonium, arginine, barium, benzathine, calcium, cesium, cholineate salts, ethylenediamine, lithium, magnesium, meglumine, procaine, N-methylglucamine, piperazine, potassium,

- 9 047547 tritium, tromethamine, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, ethanolamine, piperazine, diisopropylamine, diisopropylethylamine, triethylamine and triethanolamine. It should be understood that any oligonucleotide structures provided herein that include a backbone of internucleoside linkages may also provide any suitable salt forms. In some embodiments, acid forms of the internucleoside linkages are provided for simplicity. In some cases, a salt of the subject compound is a salt of a monovalent cation. In some cases, a salt of the subject compound is a salt of a divalent cation. In some cases, a salt of the subject compound is a salt of a trivalent cation. A solvate refers to a complex formed by a combination of solvent molecules with solute molecules or ions. The solvent may be an organic compound, an inorganic compound, or a mixture thereof. Some examples of solvents include, but are not limited to, methanol, NH-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, and water. When water is used as a solvent, the resulting solvate is a hydrate. Stereoisomer and stereoisomers refer to compounds that have the same connectivity of atoms, but differ in the arrangement of the atoms in space. Stereoisomers include, for example, cis-trans isomers, E- and Z-isomers, enantiomers, and diastereomers. With respect to any of the groups described herein that contain one or more substituents, it is of course understood that such groups do not contain any substitutions or substitution patterns that would be sterically inadvisable and/or synthetically unrealizable. All stereoisomers are intended to be included within the scope of the present invention.

It will be understood by one of ordinary skill in the art that other tautomeric arrangements of the groups described herein are possible. It should be understood that all tautomeric forms of a subject compound are encompassed by the structure describing one possible tautomeric arrangement of the groups of the said compound, even if this is not specifically stated.

It is intended to include a solvate of a pharmaceutically acceptable salt of a tautomer of a stereoisomer of the subject compound. Such solvates are intended to be included within the scope of the present invention.

Before proceeding to a more detailed description of certain embodiments, it should be noted that the present invention is not limited to the specific embodiments described and therefore may, of course, contain changes. It should also be understood that the terminology in this document is intended solely for the purpose of describing certain embodiments and not for limitation, since the scope of the present invention is limited only by the appended claims.

It should be understood that when a range of values is specified, any intermediate value, up to one-tenth of the lower limit, unless the context clearly indicates otherwise, between the upper and lower limits of the stated range, as well as any other recited or intermediate value in the stated range, is contemplated by the present invention. The upper and lower limits of the stated smaller ranges may independently be included in the stated smaller ranges and are also contemplated by the present invention, taking into account that any limits may be specifically excluded from the stated range. If the stated range includes one or both limits, ranges excluding one or both included limits are also included in the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are described below. All publications and patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are herein incorporated by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is intended to refer to its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antecedent date after such publication by virtue of prior invention. Furthermore, cited publication dates may differ from actual publication dates, which may require independent verification. It is noted that in this document and in the appended claims, the singular forms include the corresponding terms in the plural, unless the context clearly dictates otherwise. It is also noted that the claims may be drafted in such a way as to exclude any optional element. Thus, this statement serves as a prior mention of such excluding terms as exclusively, only, etc., in relation to the listing of elements of the claims, or negative features.

- 10 047547

Each of the individual embodiments described and illustrated herein includes individual components and features that can be easily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any method mentioned can be implemented according to the indicated order of events or in any other logically possible order.

B. Identification of a patient who is most likely to benefit from treatment with a telomerase inhibitor.

According to one aspect of the present invention, methods are provided for identifying or selecting a patient with myelofibrosis who is likely to benefit from treatment with a telomerase inhibitor. The methods are based on identifying triple-negative patients (patients who lack a mutation in each of the JAK2, CALR, and MPL genes) or high molecular risk (HMR) patients based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1/2. Said triple-negative or HMR patients are likely to benefit from treatment with a telomerase inhibitor, such as imetelstat or imetelstat sodium. The myelofibrosis may be primary myelofibrosis, myelofibrosis that develops after polycythemia vera (post-PV MF), or myelofibrosis that develops after essential thrombocythemia (post-ET MF). In some embodiments, the patient has not previously received JAK inhibitor therapy. In other embodiments, the patient has previously received JAK inhibitor therapy and the JAK inhibitor therapy was ineffective (i.e., the disease was resistant or the patient was refractory to the therapy; or the disease relapsed despite initially responding to the therapy). In other embodiments, the patient has previously received JAK inhibitor therapy and has discontinued the JAK inhibitor therapy due to treatment-related toxicity or intolerance. In yet other embodiments, the patient has previously received JAK inhibitor therapy and has discontinued the JAK inhibitor therapy.

In one embodiment, the patient has received JAK inhibitor therapy and the myelofibrosis is refractory to JAK inhibitor therapy. In another embodiment, the patient has received JAK inhibitor therapy and is refractory to JAK inhibitor therapy. In other embodiments, the patient has received JAK inhibitor therapy and is relapsing. In an alternative embodiment, the patient has received JAK inhibitor therapy and has discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. In one embodiment, the present invention provides methods for selecting a patient who is likely to benefit from treatment with a telomerase inhibitor by testing for one or more of: triple negative status based on the absence of a mutation in each of the JAK2, CALR, and MPL genes (i.e., the absence of any mutation). According to this embodiment, the patient may also be tested for high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1/2. According to another embodiment, the present invention provides methods for selecting a patient who is likely to benefit from treatment with a telomerase inhibitor by testing for: triple negative status based on the absence of a mutation in each of the JAK2, CALR, and MPL genes (i.e., the absence of any mutation); and/or high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1/2. According to another embodiment, the present invention provides a method of identifying a patient who is likely to benefit from treatment with a telomerase inhibitor, comprising testing the patient for: (a) triple negative status based on the absence of any mutation in the JAK2, CALR, and MPL genes; (b) high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1/2; or (c) both. In this embodiment, the presence of (a), (b), or (c) indicates a patient who is likely to benefit from treatment with a telomerase inhibitor.

According to another embodiment of the present invention, a method for identifying a patient who is likely to benefit from treatment with a telomerase inhibitor is provided, comprising:

a) testing the patient for:

i) triple negative status based on the absence of a mutation in each of the JAK2, CALR and MPL genes, and/or ii) high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2,

b) selection of the specified patient, provided that he has:

i) triple negative status based on the absence of a mutation in each of the JAK2, CALR and MPL genes, and/or ii) high molecular risk (HMR) based on the presence of a mutation in at least

- 11 047547 one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2, and the selected patient is highly likely to benefit from treatment with a telomerase inhibitor.

According to another embodiment of the present invention, there is provided a method for identifying a patient who is likely to benefit from treatment with a telomerase inhibitor, comprising: testing the patient for a triple negative status based on the absence of a mutation in each of the JAK2, CALR and MPL genes; and selecting said patient if he has a triple negative status based on the absence of a mutation in each of the JAK2, CALR and MPL genes, wherein said selected patient is likely to benefit from treatment with a telomerase inhibitor. According to one embodiment, said method also includes testing the patient for high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2; and selecting said patient if he has HMR. According to certain embodiments of any of these methods, patients with a triple negative status lack a mutation in the coding region (exon) of the JAK2, CALR and MPL genes.

In addition, according to other embodiments of any of the specified methods, high molecular risk (HMR) is determined by the presence of a mutation in the coding region (exon) of at least one of the genes ASXL1, EZH2, SRSF2 and IDH1/2.

In a particular embodiment, high molecular risk (HMR) is determined by detecting the presence of a mutation in ASXL1, EZH2, SRSF2, or IDH1/2, or a combination thereof. In some embodiments, the methods comprise detecting the presence of a mutation in ASXL1. In some embodiments, the methods comprise detecting the presence of a mutation in EZH2. In some embodiments, the methods comprise detecting the presence of a mutation in SRSF2. In some embodiments, the methods comprise detecting the presence of a mutation in IDH1/2. In some embodiments, the methods comprise detecting the presence of a mutation in ASXL1 and EZH2. In some embodiments, the methods comprise detecting the presence of a mutation in ASXL1 and SRSF2. In some embodiments, the methods comprise detecting the presence of a mutation in ASXL1 and IDH1/2. In some embodiments, the methods comprise detecting the presence of a mutation in EZH2, SRSF2. In some embodiments, the methods comprise detecting the presence of a mutation in EZH2 and IDH1/2. In some embodiments, the methods comprise detecting the presence of a mutation in SRSF2 and IDH1/2. In some embodiments, the methods comprise detecting the presence of a mutation in ASXL1, EZH2 and SRSF2. In some embodiments, the methods comprise detecting the presence of a mutation in ASXL1, EZH2 and IDH1/2. In some embodiments, the methods comprise detecting the presence of a mutation in EZH2, SRSF2 and IDH1/2. In some embodiments, the methods comprise detecting the presence of a mutation in ASXL1, EZH2, SRSF2 and IDH1/2. In another embodiment, the present invention provides a method for identifying or selecting a patient who is likely to benefit from treatment with a telomerase inhibitor in a patient population. The method comprises screening a patient population for patients with mutations in each of the JAK2, CALR and MPL genes to identify triple negative patients in the population. According to other embodiments, the methods are based on identifying triple negative patients who lack a canonical mutation in each of JAK2, MPL and CALR. According to some embodiments, the methods also include the step of obtaining a DNA sample from the patient. The patient sample may be obtained from a DNA sample collected from bone marrow, peripheral blood or both. Accordingly, according to some embodiments of the present invention, the methods include obtaining a blood sample from the patient and isolating (extracting) DNA from the blood sample from the patient. The methods may also include the step of isolating cells (e.g., granulocytes) from the blood sample from the patient. Similarly, the methods of the present invention include obtaining a bone marrow sample and isolating (extracting) DNA from the bone marrow sample. The method may also include the step of isolating cells from a bone sample from the patient.

The patient's DNA sample is tested for the presence or absence of mutations in each of the JAK2, CALR, and MPL genes using standard techniques. Alternatively, the patient's DNA sample is tested for the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1/2 using standard techniques. In some embodiments, the patient's DNA sample is tested for: (i) the presence or absence of mutations in each of the JAK2, CALR, and MPL genes; and (ii) the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1/2.

In some embodiments, testing of said DNA sample may be performed using next generation sequencing analysis on the Illumina MiSeq platform as described in Patel et al., Blood; 126(6):790-797 (2015), the contents of which, with respect to testing of DNA samples, are incorporated herein.

C. Pharmacodynamics (PD).

The present invention is based in part on the pharmacodynamic effect demonstrated

- 12 047547 association between response to telomerase inhibition therapy in subjects with myelofibrosis and reduction in hTERT telomerase expression levels in subjects relative to baseline levels. In some cases, among subjects achieving a clinical response (spleen or symptom) to telomerase inhibition therapy at week 24, a greater percentage of subjects achieved a 50% or greater reduction in hTERT RNA expression levels than nonresponders.

The present invention provides stratification and selection of patients who may benefit from telomerase inhibition therapy for myelofibrosis and methods for monitoring response, relapse, and prognosis in subjects undergoing treatment.

Aspects of the present invention include methods for selecting subjects with myelofibrosis (MF) for treatment with a telomerase inhibitor, and methods for treating MF. Methods for monitoring therapeutic efficacy in a subject with MF are also provided. In some cases, the pharmacodynamic effect on which an embodiment of the disclosed methods is based is a decrease in hTERT RNA expression by 50% or more, such as 60% or more, 70% or more, 80% or more, or 90% or more.

The telomerase ribonucleoprotein consists of components or subunits, two of which are the telomerase RNA template (hTR) and the telomerase reverse transcriptase protein (hTERT). The assessment, determination and/or measurement of hTERT expression levels can be carried out using any convenient methods. Various methods can be used to amplify, detect and measure mRNA of telomerase components or related proteins in biological fluids. Methods and assays of interest that can be adapted for use in accordance with the disclosed methods include, but are not limited to, quantitative real-time PCR (RT-PCR) assays, such as those based on the TaqMan fluorescence assay, immunohistochemical methods for studying protein expression and the methods described in U.S. Patent No. 6,607,898, Bieche et al., Clin. Cancer Res February 1 2000 (6) (2) 452-459, Terrin et al. (Telomerase expression in B-cell chronic lymphocytic leukemia predicts survival and delineates subgroups of patients with the same igVH mutation status and different outcome. Leukemia 2007; 21: 965-972) and Palma et al. (Telomere length and expression of human telomerase reverse transcriptase splice variants in chronic lymphocytic leukemia. Experimental Hematology 2013; 41: 615-626).

The evaluation or measurement of hTERT expression levels may be performed in any suitable target cells or biological samples. The target cells may be any suitable cells from the patient, including, but not limited to, bone marrow or peripheral blood cells from the patient. In some cases, the target cells are isolated from a bone marrow sample from the patient. In some cases, the target cells are isolated from a peripheral blood sample from the patient. The target cells may be granulocytes.

The evaluation or measurement of hTERT RNA expression levels can be performed in an RNA sample using any convenient methods. The RNA sample can be obtained by first obtaining a bone marrow sample, a peripheral blood sample, or both samples, and then isolating RNA from said bone marrow sample, peripheral blood sample, or both samples. According to one embodiment, the step of obtaining a sample from a patient includes: obtaining a bone marrow sample from said patient, isolating cells from the bone marrow sample, and extracting RNA and/or DNA from the isolated cells. According to another embodiment, the step of obtaining an RNA sample from a patient includes: obtaining a peripheral blood sample from said patient; isolating cells from said peripheral blood sample (e.g., granulocytes); and extracting RNA and/or DNA from the isolated cells.

D. Treatment.

Aspects of the present invention include methods of treating myelofibrosis in a subject in need thereof (i.e., a patient) having: triple negative status based on the absence of any mutation in the JAK2, CALR, and MPL genes (i.e., there is no mutation in said genes, or there are no mutations in said genes); and/or high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2, and IDH1/2. According to one embodiment, the present invention provides a method of treating myelofibrosis in a subject in need thereof (i.e., a patient) having: triple negative status based on the absence of any mutation in the JAK2, CALR, and MPL genes (i.e., there is no mutation in said genes, or there are no mutations in said genes). According to one embodiment, said myelofibrosis is primary myelofibrosis. According to another embodiment, said myelofibrosis is myelofibrosis that develops after polycythemia vera (post-PV MF). According to an alternative embodiment, said myelofibrosis is myelofibrosis that develops after essential thrombocythemia (post-ET MF).

In certain embodiments of the specified treatment methods, the patient has not previously received JAK inhibitor therapy. In other embodiments, the patient has previously received JAK inhibitor therapy and the JAK inhibitor therapy has been ineffective (i.e., the disease was resistant or the patient was refractory to therapy, or the disease was recurrent).

- 13 047547 diverged despite initially responding to treatment). In other embodiments of the treatment methods, the patient was receiving JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. In some embodiments, the treatment methods further comprise premedication with diphenhydramine (25-50 mg) and hydrocortisone (100-200 mg), or equivalent.

The subject is a mammal in need of cancer treatment. Typically, the subject is a human patient. According to some embodiments of the present invention, the subject may be a non-human mammal, such as a non-human primate, animal models (e.g., animals such as mice and rats used for screening, characterization, and evaluation of drugs), and other mammals. As used herein, the terms patient, subject, and individual are used interchangeably.

As used herein and as generally known in the art, treatment is a method for obtaining beneficial or desired results, including clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation or mitigation of one or more symptoms, reduction in the severity of the disease, stabilization (i.e., not worsening) of the disease status, prevention of the spread of the disease, delay or slowing of the progression of the disease, alleviation or temporary relief of the disease state, and remission (partial or complete), detectable or undetectable.

Treatment may also mean a longer survival time than expected without treatment.

E. Telomerase inhibitors.

The methods of the present invention can be used to identify a patient who is likely to benefit from treatment with any suitable telomerase inhibitors. In addition, any suitable telomerase inhibitors can find use in the provided treatment methods. In some embodiments, the telomerase inhibitor is an oligonucleotide with telomerase inhibitory activity, in particular an oligonucleotide as defined in WO 2005/023994 and/or WO 2014/088785, the contents of which are incorporated herein by reference in their entirety. In some cases, one or more telomerase inhibitors (e.g., two or three telomerase inhibitors) can be administered to a mammal to treat a malignant hematological disorder.

I have the State Statistics Service.

In some embodiments, the telomerase inhibitor is imetelstat, including tautomers thereof and salts thereof, such as pharmaceutically acceptable salts. Imetelstat is a novel, first-in-class telomerase inhibitor with clinical activity in hematologic malignancies (Baerlocher et al., NEJM 2015; 373:920928; Tefferi et al., NEJM 2015; 373:908-919) (shown below):

O=P-SH θ [^nps^nps^nps^nps^npsAnps^npsAnps^npsAnps] “1 A

NH ₂ where nps stands for the thiophosphoramidate linkage -NH-P(=O)(SH)-O-, which connects the 3' carbon atom of one nucleoside to the 5' carbon atom of the adjacent nucleoside.

In some cases, the telomerase inhibitor in question is imetelstat sodium, including its tautomers. Imetelstat sodium is the sodium salt of imetelstat, which is a synthetic lipid-conjugated 13-mer oligonucleotide N3'^P5'thiophosphoramidate. Imetelstat sodium is a telomerase inhibitor that is a covalently lipidated 13-mer oligonucleotide (shown below) complementary to the human telomerase RNA template region (hTR). The chemical name of imetelstat sodium is DNA, d(3'amino-3'-deoxy-P-thio) (T-A-G-G-G-T-T-A-G-A-C-A-A), 5'-[O-[2-hydroxy-3-(hexadecanoylamino)propyl]phosphorothioate], sodium salt (1:13) (SEQ ID NO: 1). Imetelstat sodium is not used for

- 14 047547 functioning antisense mechanism and, accordingly, it does not have side effects often observed with such therapy.

Imetelstat sodium

Unless otherwise stated or obvious from the context, in this document, when referring to imetelstat, its tautomers and its salts, such as pharmaceutically acceptable salts, are also meant. As mentioned, imetelstat sodium is in particular represented by the sodium salt of imetelstat. Unless otherwise stated or obvious from the context, in this document, when referring to imetelstat, all its tautomers are also meant.

Imetelstat and imetelstat sodium may be manufactured, prepared or produced according to existing descriptions (see, for example, Asai et al., Cancer Res., 63:3931- 3939 (2003), Herbert et al., Oncogene, 24:5262-5268 (2005), and Gryaznov, Chem. Biodivers., 7:477-493 (2010)). Unless otherwise stated or obvious from the context, references herein to imetelstat also include its salts. As mentioned, imetelstat sodium is in particular the sodium salt of imetelstat.

Imetelstat targets telomerase template RNA and inhibits telomerase activity and cell proliferation in a variety of cancer cell lines and tumor xenografts in mice. Phase I studies in patients with breast cancer, non-small cell lung cancer and other solid tumors, multiple myeloma, or chronic lymphocytic leukemia provided information on the pharmacokinetics and pharmacodynamics of the drug. A subsequent phase II study in patients with essential thrombocythemia demonstrated platelet-lowering activity accompanied by significant reductions in JAK2 V617F and CALR mutant allele load. Imetelstat sodium is typically administered intravenously; when implementing the proposed methods, it is also possible to administer by other methods, such as intrathecal administration, intratumoral injection, oral administration, etc. Imetelstat sodium can be administered in doses comparable to conventional doses for clinical use. According to some embodiments, imetelstat sodium is administered as described in the text of this document.

According to a specific embodiment, corresponding to any other embodiment, imetelstat is limited to imetelstat sodium.

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F. Pharmaceutical compositions.

To facilitate administration, the telomerase inhibitor (e.g., as described herein) can be formulated into various pharmaceutical forms for administration. In some cases, the telomerase inhibitor is administered as a pharmaceutical composition. The carrier or diluent of the pharmaceutical composition must be acceptable in the sense of being compatible with the other ingredients of the composition and not harmful to recipients thereof.

The pharmaceutical composition can be enclosed in a unit dosage form suitable, in particular, for administration orally, rectally, transdermally, by parenteral injection or by inhalation. In some cases, the administration can be carried out by intravenous injection. For example, when preparing the composition in the form of a dosage form for oral use, any usual pharmaceutical media can be used, such as, for example, water, glycols, oils, alcohols, etc. in the case of liquid preparations for oral use, such as suspensions, syrups, elixirs, emulsions and solutions; or solid carriers, such as starches, sugars, kaolin, diluents, lubricants, binders, disintegrating agents, etc., in the case of powders, pills, capsules and tablets. Because of their ease in administration, tablets and capsules represent the most useful oral dosage unit forms and solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will usually comprise sterile water, at least in large part, though other ingredients, for example, to aid solubility, may be included. Injectable solutions, for example, may be prepared in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable solutions may be prepared, for example, in which the carrier comprises saline solution, glucose solution or a mixture of saline and glucose solution. Injectable solutions containing the telomerase inhibitor described herein may be formulated with an oil for prolonged action. Suitable oils for the said purpose are, for example, peanut oil, sesame oil, cottonseed oil, corn oil, soybean oil, synthetic glycerol esters of long-chain fatty acids, and mixtures of these and other oils. Injectable suspensions can also be prepared, in which case suitable liquid carriers, suspending agents, etc., can be used. Also provided are solid form compositions which are intended to be converted, shortly before use, into liquid form compositions. In compositions suitable for transdermal administration, the carrier optionally contains a penetration enhancing agent and/or a suitable wetting agent, optionally in combination with suitable additives of any nature in minor amounts which do not have a significant deleterious effect on the skin. The said additives can facilitate application to the skin and/or can be useful for obtaining the desired composition. The composition may be administered in various ways, for example, as a transdermal patch, as a spot-on, as an ointment. It is particularly advantageous to formulate the above pharmaceutical compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as dosage units, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect associated with the required pharmaceutical carrier. Examples of such unit dosage forms are tablets (including scored or coated tablets), capsules, pills, packeted powders, wafers, suppositories, injectable solutions or suspensions and the like, and segregated multiples thereof.

To improve the solubility and/or stability in pharmaceutical compositions of the drug described herein, it may be useful to use α-, β- or γ-cyclodextrins or derivatives thereof, in particular hydroxyalkyl-substituted cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin or sulfobutyl-β-cyclodextrin. Cosolvents such as alcohols may also improve the solubility and/or stability of the said telomerase inhibitor in pharmaceutical compositions.

Depending on the route of administration, said pharmaceutical composition preferably comprises 0.05-99% by weight, more preferably from 0.1 to 70% by weight, even more preferably from 0.1 to 50% by weight of the telomerase inhibitor described herein, and from 1 to 99.95% by weight, more preferably from 30 to 99.9% by weight, even more preferably from 50 to 99.9% by weight of a pharmaceutically acceptable carrier; all percentages are based on the total weight of the composition.

G. Administration and modes of administration.

The frequency of administration may be any frequency that reduces the severity of the myelofibrosis symptom without causing significant toxicity to the subject. For example, a frequency of administration ranging from approximately once every two months to approximately once a week, or from approximately once a month to approximately once a week, may be used.

- 16 047547 about twice a month, alternatively about once every six weeks, about once every 5 weeks, alternatively about once every 4 weeks, alternatively about once every 3 weeks, alternatively about once every 2 weeks, or alternatively about once a week. The frequency of administration may remain constant or may vary throughout the treatment. A course of treatment with a composition comprising one or more telomerase inhibitors may include rest periods. For example, a composition comprising a telomerase inhibitor may be administered weekly for a three-week period followed by a two-week rest period, and such a regimen may be repeated multiple times. As with the effective amount, the actual frequency of administration used for a particular application may be influenced by various factors. For example, the effective amount, the duration of treatment, the use of multiple agents for treatment, the route of administration, and the severity of myelofibrosis and associated symptoms may require an increase or decrease in the frequency of administration.

An effective duration of administration of a composition comprising a telomerase inhibitor (e.g., imetelstat or imetelstat sodium) may be any duration that reduces the severity of a symptom of myelofibrosis (e.g., as described herein) without causing significant toxicity to the subject. Accordingly, an effective duration may range from one month to several months or years (e.g., one month to two years, one month to one year, three months to two years, three months to ten months, or three months to 18 months). In general, an effective duration for the treatment of myelofibrosis may range from two months to twenty months. In some cases, an effective duration may be the entire period during which an individual subject remains alive. Several factors may influence the actual effective duration for a particular treatment. For example, an effective duration may vary depending on the frequency of administration, the effective amount, the use of multiple agents for treatment, the route of administration, and the severity of myelofibrosis and associated symptoms.

In some cases, the course of treatment and the severity of one or more symptoms associated with myelofibrosis may be monitored. Any method is suitable for determining whether or not the severity of a myelofibrosis symptom is improving. For example, assessment of the severity of a myelofibrosis symptom (e.g., as described herein) may be performed using biopsy techniques.

The telomerase inhibitors according to the disclosed methods can be administered at any dose that is therapeutically effective, for example, at doses comparable to those commonly used in the clinic. Specific dosing regimens for known and approved anticancer agents (e.g., the recommended effective dose) are known to treating physicians and are provided, for example, in product descriptions in the sources: Physicians' desk reference, 2003, 57th Ed., Medical Economics Company, Inc., Oradell, N.J.; Goodman & Gilman's The pharmacological basis of therapeutics, 2001, 10th Edition, McGrawHill, New York; and/or provided by the Federal Drug Administration and/or described in the medical literature.

In some aspects, the dose of the telomerase inhibitor, imetelstat sodium, administered to the subject is from about 1.0 mg/kg to about 13.0 mg/kg. In other aspects, the dose of the telomerase inhibitor is from about 4.5 mg/kg to about 11.7 mg/kg, or from about 6.0 mg/kg to about 11.7 mg/kg, or from about 6.5 mg/kg to about 11.7 mg/kg. In some embodiments, the dose of the telomerase inhibitor comprises at least about 4.5 mg/kg, or 4.6 mg/kg, 4.7 mg/kg, 4.8 mg/kg, 4.9 mg/kg, 5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.1 mg/kg, 6.2 mg/kg, 6.3 mg/kg, 6.4 mg/kg, 6.5 mg/kg, 6.6 mg/kg, 6.7 mg/kg, 6.8 mg/kg, 6.9 mg/kg, 7 mg/kg, 7.1 mg/kg, 7.2 mg/kg, 7.3 mg/kg, 7.4 mg/kg, 7.5 mg/kg, 7.6 mg/kg, 7.7 mg/kg, 7.8 mg/kg, 7.9 mg/kg, 8 mg/kg, 8.1 mg/kg, 8.2 mg/kg, 8.3 mg/kg, 8.4 mg/kg, 8.6 mg/kg, 8.7 mg/kg, 8.8 mg/kg, 8.9 mg/kg, 9 mg/kg, 9.1 mg/kg, 9.2 mg/kg, 9.3 mg/kg, 9.4 mg/kg, 5 mg/kg, 9.6 mg/kg, 9.7 mg/kg, 9.8 mg/kg, 9.9 mg/kg, 10 mg/kg, 10.1 mg/kg, 10.2 mg/kg, 10.3 mg/kg, 10.4 mg/kg, 10.5 mg/kg, 10.6 mg/kg, 10.7 mg/kg, 8 mg/kg, 10.9 mg/kg, 11 mg/kg, 11.1 mg/kg, 11.2 mg/kg, 11.3 mg/kg, 11.4 mg/kg, 11.5 mg/kg, 11.7 mg/kg, 11.8 mg/kg, 11.9 mg/kg, 12 mg/kg, 12.1 mg/kg, 12.2 mg/kg, 12.3 mg/kg, 12.4 mg/kg, 12.5 mg/kg, 12.6 mg/kg, 12.7 mg/kg, 12.8 mg/kg, 12.9 mg/kg, or 13 mg/kg. In some embodiments, the effective amount of a telomerase inhibitor administered to the individual comprises at least about 1 mg/kg, or 2.5 mg/kg, 3.5 mg/kg, 4.7 mg/kg, 5 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.4 mg/kg, 10 mg/kg, 15 mg/kg, or 20 mg/kg. In some embodiments, the effective amount of a telomerase inhibitor administered to an individual is about 1 mg/kg, or 2.5 mg/kg, 3.5 mg/kg, 4.7 mg/kg, 5 mg/kg, 6.5 mg/kg, 7.5 mg/kg, 9.4 mg/kg, 10 mg/kg, 15 mg/kg, or 20 mg/kg. In various embodiments, the effective amount of a telomerase inhibitor administered to an individual comprises less than about 350 mg/kg, 300 mg/kg, 250 mg/kg, 200 mg/kg, 150 mg/kg, 100 mg/kg, 50 mg/kg, 30 mg/kg, 25 mg/kg, 20 mg/kg, 10 mg/kg, 7.5 mg/kg, 6.5 mg/kg, 5 mg/kg, 3.5 mg/kg, 2.5 mg/kg, 1 mg/kg, or 0.5 mg/kg of a telomerase inhibitor.

Examples of dosage frequency of a pharmaceutical composition comprising a telomere inhibitor

- 17 047547 zy, include, but are not limited to, daily dosing; dosing every other day; twice a week; three times a week; weekly without a break; weekly for three weeks out of four weeks; once every three weeks; once every two weeks; weekly for two weeks out of three weeks. According to some embodiments, the specified pharmaceutical composition is administered about once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks. According to some embodiments, the specified composition is administered at least about: 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, or 7 times (i.e., daily) per week, or three times daily, twice daily. In some embodiments, the intervals between all administrations are less than about: 6 months, 3 months, 1 month, 20 days, 15 days, 12 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the intervals between all administrations are more than about: 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, or 12 months. In some embodiments, there are no breaks in the dosing regimen. In some embodiments, the interval between all administrations is no more than about a week.

Telomerase inhibitors such as imetelstat (e.g., imetelstat sodium) can be administered using any suitable route. For example, telomerase inhibitors such as imetelstat (e.g., imetelstat sodium) can be administered intravenously once every 4 weeks for a period of time (e.g., one, two, three, four, or five hours). In some embodiments, imetelstat is administered intravenously once a week for a period of about 2 hours at a dose of 7-10 mg/kg. In some embodiments, imetelstat is administered intravenously once every 3 weeks for a period of about 2 hours at a dose of about 0.5-9.4 mg/kg. In an embodiment, imetelstat is administered intravenously over a period of about 2 hours once every 4 weeks at a dose of 0.5-5 mg/kg. In an embodiment, imetelstat is administered intravenously once every 3 weeks over a period of about 2 hours at a dose of about 2.5-10 mg/kg. Alternatively, imetelstat is administered intravenously over a period of about 2 hours once every 4 weeks at a dose of about 0.5-9.4 mg/kg.

According to certain embodiments of the method, imetelstat is administered in 1, 2, 3, 4, 5, 6, 7, 8, or more than 8 dosing cycles, each of which comprises: intravenous administration of about 7-10 mg/kg imetelstat once every three weeks, intravenous administration of about 7-10 mg/kg imetelstat once a week for three weeks, intravenous administration of about 2.5-10 mg/kg imetelstat once every three weeks, or intravenous administration of about 0.5-9.4 mg/kg imetelstat once every three weeks. In certain cases, each dosing cycle comprises intravenous administration of about 7-10 mg/kg imetelstat once every three weeks. In some cases, each dosing cycle comprises intravenous administration of about 9.4 mg/kg imetelstat about once every three weeks.

In one embodiment of the present invention, imetelstat is administered intravenously at a dosage of about 7-10 mg/kg imetelstat once every three weeks after premedication with an antihistamine, a corticosteroid, or both. In other embodiments, imetelstat is administered intravenously at a dosage of about 9.4 mg/kg, optionally about 7.0 mg/kg to about 9.8 mg/kg imetelstat once every three weeks after premedication with an antihistamine, a corticosteroid, or both. In some embodiments, imetelstat is administered at a dosage of about 7.5 mg/kg, optionally about 7.0 mg/kg to about 7.7 mg/kg, once every three weeks for at least three cycles, after which the dosage is increased. In some embodiments, the dosage of imetelstat may be increased to about 9.4 mg/kg, optionally to about 8.8 mg/kg to about 9.6 mg/kg, provided that the maximum reduction in ANC and platelet count does not reach a level of from about 1.5 x 10 ⁹ /L to about 75 x 10 ⁹ /L, respectively, and there is no non-hematological toxicity of > grade 3.

It should be understood that cancer treatment sometimes involves multiple rounds or cycles of drug administration, where each cycle involves administering said drug one or more times according to a set schedule (e.g., every three weeks for three consecutive days; once a week; etc.). For example, anticancer drugs may be administered over 1-8 cycles, or over a longer period. When more than one drug (e.g., two drugs) is administered to a subject, each drug may be administered according to its own schedule (e.g., weekly; once every three weeks; etc.). It is understood that administration of drugs, even those administered at different intervals, may be coordinated such that at least sometimes both drugs are administered on the same day, or, alternatively, such that at least sometimes the drugs are administered on consecutive days.

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In some embodiments, imetelstat may be administered according to a regimen that includes dose reduction. In one embodiment, said patient is initially administered about 9.4 mg/kg every three weeks, then the dose is adjusted to about 7.5 mg/kg every three weeks, after which the dose is adjusted to about 6.0 mg/kg every three weeks.

As is known in the art, treatment with anticancer therapeutic drugs may be temporarily suspended if toxicity is observed or for the convenience of the patient, without departing from the scope of the present invention, after which treatment is resumed.

Aspects of the disclosed methods include identifying or selecting a patient who is likely to benefit from treatment based on the relative telomere length of target cells (e.g., as described herein) of said patient. The target cells may be any suitable cells of the patient, including, but not limited to, bone marrow or peripheral blood cells of the patient. In some cases, said target cells are isolated from a bone marrow sample of the patient. In some cases, said target cells are isolated from a peripheral blood sample of the patient. Said target cells may be granulocytes. In some cases, the patient lacks a mutation in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes; and said patient, in particular, has a short telomere length in the target cells. As used herein, a short telomere length is a length less than or equal to the median or average telomere length when compared to a suitable control, such as one or more known standards as described herein. Accordingly, the disclosed methods may also include determining the relative telomere length by analyzing the relative length of telomeric nucleic acids in target cells present in a biological sample from an individual; selecting an individual who will benefit from treatment with a telomerase inhibitor, provided that the average relative telomere length in target cells present in a biological sample from said individual is determined to fall within the 50th or lower percentile of a range of relative telomere lengths determined for one or more known reference standards, such as determined to fall within the 45th or lower percentile, the 40th or lower percentile, the 35th or lower percentile, the 30th or lower percentile, the 25th or lower percentile, the 20th or lower percentile, or even a lower percentile of a range of relative telomere lengths determined for one or more known reference standards.

In some cases, according to said method, one or more known reference standards are represented by a range of telomere lengths established in a population of naturally occurring target cells (e.g., as described herein) from a population of individuals diagnosed with a disease. In certain cases, according to said method, one or more known reference standards are represented by characterized cell lines. Characterized cell lines mean that the corresponding telomeric nucleic acids of said cells are known and relatively constant. According to some embodiments, the telomere length in cancer cells present in said biological sample is determined to be equal to or less than the median or average telomere length. According to some embodiments, the telomere length in cancer cells present in said biological sample is determined to fall within the 50th or lower percentile, the 40th or lower percentile, the 35th or lower percentile, the 30th or lower percentile, the 25th or lower percentile, the 20th or lower percentile, the 15th or lower percentile, the 10th or lower percentile, or the 5th or lower percentile of a range of relative telomere lengths determined for one or more known standard samples.

Telomere length in a target cell may be determined using any convenient assay method, including but not limited to qPCR, telo-FISH (telomere fluorescence in situ hybridization) or Southern blotting as described by Bassett et al. in U.S. Patent No. 9,200,327. In one aspect, telomere length may be determined by measuring the average terminal restriction fragment (TRF) length. TRF is defined as the length, typically the average length, of fragments resulting from complete digestion of genomic DNA with a restriction enzyme that does not cleave nucleic acid at the telomeric sequence. In some cases, DNA is digested with restriction enzymes that often cleave genomic DNA but do not cleave telomeric sequences. In some cases, these restriction enzymes contain a 4-base recognition sequence (e.g., AluI, HinfI, RsaI, and Sau3A1) and are used singly or in combination. The resulting terminal restriction fragment contains both telomeric repeats and subtelomeric DNA. Subtelomeric DNA is DNA sequences adjacent to tandem repeats of telomeric sequences and contains telomeric repeat sequences interspersed with variable telomeric-like sequences. The digested DNA is separated by electrophoresis and blots are prepared on a support, such as a membrane. Fragments containing telomeric sequences are detected by hybridization of the probe, i.e., labeled repeat sequences, to the membrane. After visualization of telomere-containing fragments, average lengths of terminal restriction fragments can be calculated (Harley, C.B. et al., Nature. 345(6274):458-60 (1990), incorporated herein by reference). Estimation of TRF using Southern blot analysis yields the distribution of telomere lengths in cells or tissue, and, accordingly, the median and average telomere lengths in all cells.

In another aspect, telomere length can be measured by flow cytometry (Hultdin, M. et al., Nucleic Acids Res. 26: 3651-3656 (1998); Rufer, N. et al., Nat. Biotechnol. 16:743-747 (1998); incorporated herein by reference). Flow cytometric methods are variants of FISH techniques. In case the starting material is tissue, a cell suspension is prepared, usually by mechanical separation and/or treatment with proteases. The cells are fixed with a fixative and hybridized with a probe specific for telomeric sequences, preferably a fluorescently labeled PNA probe. After hybridization, the cells are washed and then analyzed using FACS. The fluorescent signal is measured for cells in the Go/G1 phases after appropriate subtraction of background fluorescence. This method is suitable for rapid calculation of telomere length in a large number of samples. As in the case of TRF, telomere length is represented by the average telomere length in the cell.

In other aspects, the median or average telomere length from cells in a biological sample is determined by quantitative PCR (qPCR) or telomere fluorescence in situ hybridization (telo-FISH). In qPCR, a DNA binding dye binds to the entire double-stranded DNA, causing the dye to fluoresce. An increase in the amount of DNA product during the PCR reaction results in an increase in fluorescence intensity and is measured at each cycle of the PCR reaction. This allows for a quantitative determination of the DNA concentration. The relative concentration of DNA present in the exponential phase of the reaction is determined by plotting the fluorescence level versus the PCR cycle number on a semi-logarithmic scale. A threshold for detecting fluorescence above background is determined. The cycle during which the fluorescence in a given sample exceeds the threshold is called a threshold cycle (Ct). Since the amount of DNA theoretically doubles in each cycle during the exponential phase, relative amounts of DNA can be calculated. The baseline corresponds to the initial PCR cycles, in which the fluorescent signal changes little. In some aspects, telomere length is determined using telo-FISH. In this method, cells are fixed and hybridized with a probe conjugated with a fluorescent label, such as Cy-3, fluorescein, rhodamine, etc. The probes for this method are oligonucleotides designed to specifically hybridize to telomere sequences. Typically, the probes are 8 or more nucleotides long, such as 12-20 or more nucleotides. In one aspect, the probes are oligonucleotides comprising naturally occurring nucleotides. In one aspect, the probe is a peptide nucleic acid whose Tm is higher than similar naturally occurring sequences and, accordingly, allows for more stringent hybridization conditions. The cells can be treated with an agent such as colcemid to induce cell cycle arrest in metaphase and obtain metaphase chromosomes for hybridization and analysis.

In some embodiments, the cellular DNA may also be stained with the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI).

Digital images of intact metaphase chromosomes are acquired and the fluorescence intensity of probes hybridized to the elomeres is quantified. This allows the telomere length of individual chromosomes, as well as the average or median telomere length in a cell, to be measured, thereby avoiding problems associated with the presence of subtelomeric DNA (Zjilmans, J. M. et al., Proc. Natl. Acad Sci. USA 94:7423–7428 (1997); Blasco, M. A. et al., Cell 91:25–34 (1997); incorporated by reference). The intensity of the fluorescent signal correlates with telomere length, with a brighter fluorescent signal indicating a longer telomere.

Some embodiments of the present invention relate to a telomerase inhibitor for use in a method of treating myelofibrosis, said method comprising:

identifying a patient who is likely to benefit from treatment with a telomerase inhibitor, including testing the patient for:

(a) triple negative status based on the absence of any mutation in the JAK2, CALR and MPL genes;

(b) high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2 or (c) both;

wherein the presence of (a), (b) or (c) indicates a patient who is likely to benefit from treatment with a telomerase inhibitor and administering to said patient an effective amount of a telomerase inhibitor. Some embodiments of the present invention relate to a telomerase inhibitor for use in a method as defined in any other embodiments.

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According to another embodiment of the present invention there is provided a telomerase inhibitor for use in the treatment of myelofibrosis, comprising: (a) screening a patient to determine whether said patient is: triple negative based on the absence of a mutation in each of JAK2, CALR and MPL and/or has a high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2; and; (b) administering said telomerase inhibitor to said patient, provided that said patient is triple negative based on the absence of a mutation in each of JAK2, CALR and MPL, and/or has a high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2. According to one embodiment, said using comprises screening said patient for triple negative based on the absence of a mutation in each of JAK2, CALR and MPL. According to another embodiment of the present invention there is provided a telomerase inhibitor for use in the treatment of myelofibrosis, comprising: (a) screening a patient to determine whether the patient is triple negative based on the absence of a mutation in each of JAK2, CALR and MPL; and (b) administering said telomerase inhibitor to said patient, given that the patient is triple negative. According to another embodiment of the present invention there is provided a use of a telomerase inhibitor for the treatment of myelofibrosis, comprising: (a) screening a patient to determine whether the patient is: triple negative based on the absence of a mutation in each of JAK2, CALR and MPL, and/or high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2; and; (b) administering said telomerase inhibitor to said patient, provided that said patient is triple negative based on the absence of a mutation in each of JAK2, CALR and MPL, and/or has a high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2. According to one embodiment, said use comprises screening said patient for triple negative based on the absence of a mutation in each of JAK2, CALR and MPL. According to another embodiment of the present invention there is provided a use of a telomerase inhibitor for the treatment of myelofibrosis, comprising: (a) screening a patient to determine whether said patient is: triple negative based on the absence of a mutation in each of JAK2, CALR and MPL; and (b) administering said telomerase inhibitor to said patient, provided that said patient is triple negative.

According to some embodiments of the present invention, triple negative status can be determined based on the absence of a mutation in each of the following genes: the JAK2 gene having the nucleic acid sequence of SEQ ID NO: 2, the CALR gene having the nucleic acid sequence of SEQ ID NO: 3, and the MPL gene having the nucleic acid sequence of SEQ ID NO: 4. According to other embodiments, triple negative status can be determined based on the absence of a mutation in each of SEQ ID NO: 2; CALR and MPL. According to an alternative embodiment, triple negative status can be determined based on the absence of a mutation in each of the sequences: JAK2, SEQ ID NO: 3 and MPL. According to an alternative embodiment, a triple negative status may be determined based on the absence of a mutation in each of the sequences: JAK2, CALR, and SEQ ID NO: 4. According to other embodiments of the present invention, a high molecular risk (HMR) may be determined based on the presence of a mutation in at least one of the following genes: the ASXL1 gene having the nucleic acid sequence of SEQ ID NO: 5, the EZH2 gene having the nucleic acid sequence of SEQ ID NO: 6, the SRSF2 gene having the nucleic acid sequence of SEQ ID NO: 7, the IDH1 gene having the nucleic acid sequence of SEQ ID NO: 8, the IDH2 gene having the nucleic acid sequence of SEQ ID NO: 9, and combinations thereof.

According to other embodiments of the present invention, telomerase activity and hTERT expression levels can be determined in biological samples obtained from patients to assess pharmacodynamic effects and/or monitor patients treated with telomerase inhibition. Telomerase activity can be measured using the Telomerase Repeat Amplification Protocol (TRAP) telomerase activity assay. The hTERT expression level can be determined by measuring the hTERT RNA expression level in cells in a biological sample using Northern blots or serial analysis of gene expression (SAGE), or other methods. Some embodiments of the present invention relate to a telomerase inhibitor for use in the treatment of myelofibrosis as defined in any of the other embodiments.

Some embodiments of the present invention relate to the use of a telomerase inhibitor for the treatment of myelofibrosis as defined in any other embodiments.

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Additional implementation options

Additional implementation options of interest are presented in the paragraphs below.

Item 1. A method for identifying a patient who is likely to benefit from treatment with a telomerase inhibitor, comprising:

(a) testing a patient for triple negative status based on the absence of a mutation in each of the Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL) genes; and (b) selecting said patient if said patient has triple negative status based on the absence of a mutation in each of the JAK2, CALR, and MPL genes, where said selected patient is likely to benefit from treatment with a telomerase inhibitor.

Item 2. A method for identifying a patient who is likely to benefit from treatment with a telomerase inhibitor, comprising:

(c) testing the patient for:

i) triple negative status based on the absence of a mutation in each of the JAK2, CALR and MPL genes, and/or ii) high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2, (d) patient selection based on the presence of:

i) triple negative status based on the absence of a mutation in each of the JAK2, CALR and MPL genes, and/or ii) high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2, wherein said selected patient is highly likely to benefit from treatment with a telomerase inhibitor.

Item 3. A method for identifying a patient who is likely to benefit from treatment with a telomerase inhibitor, comprising:

(e) obtaining a DNA sample from the patient;

(f) testing said DNA sample from the patient for triple negative status based on the absence of a mutation in each of the JAK2, CALR, and MPL genes; and (g) selecting said patient on the condition that he or she has a triple negative status based on the absence of a mutation in each of the JAK2, CALR, and MPL genes;

however, the selected patient is highly likely to benefit from treatment with a telomerase inhibitor.

Item 4. A method for identifying a patient who is likely to benefit from treatment with a telomerase inhibitor, comprising: (h) obtaining a DNA sample from the patient; (i) testing said DNA sample from the patient for:

i) triple negative status based on the absence of a mutation in each of the JAK2, CALR and MPL genes; and/or ii. high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2; and (j) selection of the specified patient subject to the presence of:

i. triple negative status based on the absence of a mutation in each of the JAK2, CALR and MPL genes; and/or ii) high molecular risk (HMR) based on the presence of a mutation in at least one of the following genes: ASXL1, EZH2, SRSF2 and IDH1/2, wherein said selected patient is highly likely to benefit from treatment with a telomerase inhibitor.

Item 5. Use of a telomerase inhibitor in the treatment of a patient with myelofibrosis, wherein said patient is defined as having a triple negative status, where said triple negative status is characterized by the absence of a mutation in each of the genes: Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL).

Item 6. Use of a telomerase inhibitor in the treatment of a patient with myelofibrosis, characterized in that said patient is determined to have a high molecular risk (HMR), wherein the presence of HMR includes the presence of a mutation in at least one gene selected from the group consisting of the extra sex bristle homolog 1 gene (ASXL1), the enhancer of Zeste protein homolog 2 gene (EZH2), the serine-arginine-rich splicing factor 2 gene (SRSF2), and the isocitrate dehydrogenase 1/2 gene (IDH1/2).

Item 7. Use of a telomerase inhibitor in the treatment of a patient who has myelofibrosis, wherein it has been determined that cells present in a biological sample from said patient have a mean relative telomere length that is defined as falling within the 50th percentile or lower of a range of relative telomere lengths determined for one or more known reference standards.

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Item 8. Use of a telomerase inhibitor in obtaining a medicament for the treatment of a patient with myelofibrosis, wherein said patient is defined as having a triple negative status, characterized by the absence of a mutation in each of the genes: Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL).

Item 9. Use of a telomerase inhibitor in the preparation of a medicament for the treatment of a patient with myelofibrosis, wherein said patient is determined to have a high molecular risk (HMR), wherein the presence of HMR comprises the presence of a mutation in at least one gene selected from the group consisting of the extra sex setae homologue 1 gene (ASXL1), the enhancer of Zeste protein homologue 2 gene (EZH2), the serine-arginine-rich splicing factor 2 gene (SRSF2), and the isocitrate dehydrogenase 1/2 gene (IDH1/2).

Item 10. Use of a telomerase inhibitor in the preparation of a medicament for the treatment of a patient with myelofibrosis, wherein it has been determined that cells present in a biological sample from the patient have a mean relative telomere length that is determined to fall within the 50th percentile or lower of a range of relative telomere lengths determined for one or more known reference standards.

The examples below are provided for illustrative purposes and not to be limiting.

Examples

Example 1: Imetelstat sodium is an effective treatment for patients with intermediate-risk 2 (Int-2) or high-risk myelofibrosis (MF) that is relapsed or refractory to Janus kinase (JAK) inhibitor therapy.

Introduction.

Imetelstat, a 13-mer oligonucleotide that specifically targets the human telomerase template RNA, is a potent competitive inhibitor of telomerase enzymatic activity (Asai et al., Cancer Res 2003; Herbert, Oncogene 2005). Clinical activity and an acceptable safety profile have been reported in intermediate-risk 2 (int-2) or high-risk myelofibrosis (MF) in a 33-patient pilot study, where 48% of patients had previously received a Janus kinase inhibitor (JAKi) (Tefferi, N Engl J Med 2015). This example presents results from a phase II clinical trial of imetelstat sodium at two dose levels in patients with MF.

Methods.

A randomized, multicenter, phase II study of two doses of imetelstat sodium (9.4 mg/kg or 4.7 mg/kg IV every 3 weeks) was conducted in adults with MF with a Dynamic International Prognosis Score (DIPSS) int-2 risk score; or high-risk MF that was relapsed/refractory to prior JAKi therapy (i.e., no improvement in splenomegaly at 12 weeks or worsening splenomegaly at any time after initiation of JAK inhibitor (JAKi) therapy). Eligibility criteria included a diagnosis of primary MF, MF after essential thrombocythemia, or MF after polycythemia vera; other inclusion criteria included measurable splenomegaly (by magnetic resonance imaging [MRI]), systemic symptoms associated with active MF, and a platelet count >75 x 10 ⁹ /L. The primary endpoints included: spleen response rate (achieving >35% % spleen volume reduction [SVR] by MRI at week 24); and symptom response rate (achieving >50% % reduction in total symptom score [TSS] by Myelofibrosis Symptom Assessment Form (MFSAF) v2 at week 24). Key secondary endpoints included safety, overall survival (OS), treatment response, molecular response, and pharmacokinetic-pharmacodynamic interactions.

Results.

A total of 107 patients from 55 institutions were included (48 on 4.7 mg/kg; 59 on 9.4 mg/kg). The main characteristics are summarized in Table 1 below. In addition, the median duration of the period on JAKi was 23 (0.9-89.7) months and the median platelet count was 147 x 10 ⁹ /L. Triple negative status (TN; ie, no JAK2, MPL, or CALR mutations) was observed in 24.8% of patients, and 67.6% of patients were considered to have high molecular risk (HMR; ie, > 1 mutation in ASXL1, EZH2, SRSF2, or IDH1/2).

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Table 1

Basic characteristics

4.7 mg/kg (n = 48) 9.4 mg/kg (n = 59) Median age (range; y) 68.5 (44, 84) 67.0(31, 86) Male gender 32 (66.7%) 35 (59.3%) Myelofibrosis Subtype Primary Post-ET Post-IP 27 (56.3%) 9(18.8%) 12 (25.0%) 36 (61.0%) 10 (16.9%) 13 (22.0%) DIPSS Risk Status Intermediate Risk Level 1 Intermediate Risk Level 2 High Risk 1 ^a (2.1%) 28 (58.3%) 19 (39.6%) 0 34 (57.6%) 25 (42.4%) Spleen length (by palpation) - median value (range; cm) > 15 cm 18.0(4.0, 35.0) 31 (64.6%) 18.0(3.0, 32.0) 35 (59.3%) Spleen volume (MRI) - Median value ^Ь (range; cm ³ ) 3352.8 (726; 7243) 2997.5 (890; 7607) Total Symptom Score - Median (Range; TSS) 22.29 (6.7, 58.4) 23.86 (3.1, 57.2) ^Red blood cell transfusion dependence 14 (29.2%) 12 (20.3%) Platelet count - Median value (range; *10 ⁹ /L) 153 (74, 1097) 146 (65, 798) Time since initial diagnosis - Median (range; 48.90 (2.0; 227.3) 42.84 (6.6; 200.7) months) Time since last JAKi therapy - Median (range; months) 1.41 (0.5, 31.2) 1.71 (0.5, 37.8) Duration of prior JAKi therapy - Median (range; months) 22 (3, 90) 25 (1, 73)

^a Protocol option.

^b As assessed by an independent review committee.

^c Received 6 units of PRBC 12 weeks prior to inclusion.

At the primary data cutoff date, the median duration of the study period was 22.6 months (range, 0.2-27.4 months); the median duration of the treatment period was 6.2 months (range, 0.0-27.2 months). Six patients (10.2%) in the 9.4 mg/kg subgroup had an independent review committee (IRC)-confirmed splenic response based on MRI.

At the clinical data cutoff date, patients had been followed for a median of 22.6 (0.2-27.4) months, including a median treatment duration of 6.2 (0.0-27.2) months. The median treatment duration was longer in the 9.4 mg/kg subgroup (7.7 months) compared with the 4.7 mg/kg subgroup. Six (10.2%) patients in the 9.4 mg/kg subgroup had a splenic MRI response, whereas no responses were observed in the 4.7 mg/kg subgroup (see Figure 1). Nineteen (32%) patients in the 9.4 mg/kg subgroup and three (6%) patients in the 4.7 mg/kg subgroup had a symptomatic response (> 50% reduction in TSS) (see Figure 2). The median OS in the 9.4 mg/kg subgroup was not

- 24,047,547 was reached at the first clinical data cutoff date, whereas in the 4.7 mg/kg subgroup, the median OS was 19.9 months. The 18-month survival rates were 76.7% and 62.9% for the 9.4 mg/kg subgroup and 4.7 mg/kg subgroup, respectively. Sensitivity analyses censoring patients at dose escalation for subsequent JAKi therapy or stem cell transplantation yielded similar results. In the 9.4 mg/kg subgroup, an association was observed between patients with TO and OS (median OS was not reached in patients with TO and was 23.6 months for patients without TO). Splenic response rates were higher in patients with 1 HMR mutation (ASXL1, EZH2, SRSF2, or IDH1/2).

The most common treatment-related adverse events (all grades) in the 9.4 mg/kg subgroup were thrombocytopenia (49%), anemia (44%), neutropenia (36%), and nausea (34%); and in the 4.7 mg/kg subgroup, diarrhea (38%), nausea (31%), anemia (31%), and thrombocytopenia (23%). Grade 10 neutropenia and thrombocytopenia were more common in the 9.4 mg/kg subgroup (34% and 42%, respectively) compared with the 4.7 mg/kg subgroup (13% and 29%, respectively); most cytopenias resolved within 4 weeks. Grade 10 elevations in FFP were observed in 7 patients during the study period. No imetelstat-related hepatotoxicity was observed as assessed by an independent liver review committee.

At the second data cutoff date, patients had been followed for a median of 27.4 (0.2-33.0) months, including a median treatment duration of 26.9 (0.1-118.1) weeks. The median treatment duration was longer in the 9.4 mg/kg subgroup (33.3 weeks) compared with the 4.7 mg/kg subgroup (23.9 weeks). The 4.7 mg/kg subgroup was terminated early, impacting treatment duration. The median OS with 95% CI in the 9.4 mg/kg subgroup was 29.9 months (22.8, NE) (NE = not evaluable) in the 9.4 mg/kg subgroup and was reached at the second data cutoff date.

Triple negative status and OV.

Subjects were grouped by JAK2/MPL/CALR mutation status, triple negative (TNE, no mutation in any of the JAK2/MPL/CALR genes), and no TNE (no mutation in any of the JAK2/MPL/CALR genes). Median OS was not estimable (NE) for subjects with TNE with 95% CI (23.2, NE) and was 23.6 months for subjects without TNE with 95% CI (20.7, NE) in the 9.4 mg/kg subgroup, while in the 4.7 mg/kg subgroup, median OS with 95% CI was 22.3 (17.0, NE) and 20.3 (18.3, NE) for subjects with TNE and subjects without TNE, respectively. In the 9.4 mg/kg subgroup, lower mortality was observed in the triple negative (TN) group compared to the non-TN group (see Table 2, Figs. 3 and 4).

Table 2

Overall survival grouped by JAK2/MPL/CALR gene mutation status

Dose (mg/kg) Mutation status Mortality rate % Median survival, months (95% CI) HR (95% CI) P-value (log-rank test) 4.7 THAT 40 22.3 (17, N O.) Without maintenance 36.8 20.3 (18.3, NO.) 0.98 (0.32, 2.98) 0.9719 9.4 THAT 18.8 N O. (23.2, NO.) Without maintenance 41,5 23.6 (20.7, NO.) 2.76 (0.81, 9.46) 0,0910

At the second clinical data cutoff date, a lower mortality rate was observed in the 9.4 mg/kg subgroup in the triple negative (TN) group compared with the non-TN group (see Table 3, Figs. 6 and 7).

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Table 3

Overall survival grouped by JAK2/MPL/CALR gene mutation status

Imetelstat dose (mg/kg) Mutation status Percentage of subjects who died Median survival (months) (95% CI) HR* (95% D.i.) P-value (Log-rank test) 4.7 Triple negative (TN) 6/10 (60.0%) 23.1 (4.2, NO.) Without maintenance (MUT) 21/38 (55.3%) 19.5 (16.7, 31.6) 1.16 (0.46, 2.87) 0,7562 9.4 Triple negative (TN) 4/16 (25.0%) N O. (23.2, NO.) Without maintenance (MUT) 21/41 (51.2%) 24.6 (19.7, NO.) 2.60 (0.89, 7.59) 0,0700

*HR = Hazard Ratio.

Triple negative status and response at 24 weeks.

For the 9.4 mg/kg subgroup, a higher response rate (SVR or TSS) was observed in the TO group compared to the no-TO group (see Table 4 below).

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Table 4

Baseline JAK2/CALR/MPL mutation status and clinical response

SVR > 20% at 24 weeks according to IRC Mutational status of JAK2/CALR/MPL Total, N=105 4.7 mg/kg, N=48 9.4 mg/kg, N=57 Total number Present, N=2 (4.2%) Absent, N=46 (95.8%) Total number Present, N=12(21.1%) Absent, N=45 (78.9%) THAT 26(24.8%) 10 0 (0%) 10 (100%) 16 5 (31.25%) And (68.75)) Without maintenance 79 (75.2%) 38 2 (5.3%) 36 (94.7%) 41 7(17.1%) 34 (82.9%) SVR > 35% at 24 weeks according to IRC Mutational status of JAK2/CALR/MPL Total, N=105 4.7 mg/kg, N=48 9.4 mg/kg, N=57 Total number Present, N=0 (0%) Number, N=48 (100%) Total number Present, N=6 (10.5%) Absent, N=51 (89.5%) THAT 26(24.8%) 10 0 (0%) 10 (100%) 16 3 (18.75%) 13 (81.25%) Without maintenance 79 (75.2%) 38 0 (0%) 38 (100%) 41 3 (7.3%) 38 (92.7%) TSS > 50% reduction at 24 weeks Mutational status of JAK2/CALR/MPL Total, N=105 4.7 mg/kg, N=48 9.4 mg/kg, N=57 Total number Present, N=3 (6.3%) Absent, N=45 (93.8%) Total number Present, N=18 (31.6%) Absent, N=39 (68.4%) THAT 26(24.8%) 10 1 (10.0%) 9 (90.0%) 16 8 (50%) 8 (50%) Without maintenance 79 38 2 (5.3%) 36 (94.7%) 41 10 (24.4%) 31 (75.6%) (75.2%)

Molecular risk and responses at 24 weeks.

In the 9.4 mg/kg subgroup, a higher response rate (SVR or TSS) was observed in the low molecular risk (LMR) group or the high molecular risk (HMR) group with a single mutation (MUT) compared with the HMR group with more than 1 mutation (see Table 5).

- 27 04Ί54Ί

Table 5

Molecular risk and responses at 24 weeks

SVR > 20% at 24 weeks Molecular risk Total, N=105 4.7 mg/kg, N=48 9.4 mg/kg, N=57 Total number Present, N=1 (2.1%) Absent, N=47(97.8%) Total number Present, N=12 (21.1%) Absent, N=45 (78.9%) LMR, No mutations 34 (32.4%) 12 1 (8.3%) 11 (91.7%) 22 5 (22.7%) 17 (77.3%) HMRc 1 mutation 48 (45.7%) 27 0 (0%) 27(100%) 21 6 (28.6%) 15 (71.4%) HMR with more than 1 mutation 23 (21.9%) 9 0 (0%) 9 (100%) 14 1 (7.1%) 13 (92.9%) SVR > 35% at 24 weeks according to IRC Molecular risk Total, N=105 4.7 mg/kg, N=48 9.4 mg/kg, N=57 Total number Present, N=0 (0%) None, N=48 (100%) Total number Present, N=6 (10.5%) Absent, N=51 (89.5%)

- 28 04Ί54Ί

LMR, No mutations 34 (32.4%) 12 0 (0%) 12 (100%) 22 2(9.1%) 20 (90.9%) HMRc 1 mutation 48 (45.7%) 27 0 (0%) 27 (100%) 21 4 (19%) 17 (81%) HMR with more than 1 mutation 23 (21.9%) 9 0 (0%) 9 (100%) 14 0 (0%) 14 (100% TSS > 50% reduction at 24 weeks Molecular risk Total, N=105 4.7 mg/kg, N=48 9.4 mg/kg, N=57 Total number Present, N=3 (6.3%) Absent, N=45 (93.8%) Total number Present, N=18 (31.6%) Absent, N=39 (68.4%) LMR, No mutations 34 (32.4%) 12 3 (25%) 9 (75%) 22 8 (36.4%) 14 (63.6%) HMRc 1 mutation 48 (45.7%) 27 0 (0%) 27 (100%) 21 7 (33.3%) 14 (66.7%) HMR with more than 1 mutation 23 (21.9%) 9 0 (0%) 9 (100%) 14 3 (21.4%) 11 (78.6%)

For subjects receiving 9.4 mg/kg, associations were observed between the following factors and clinical responses or OS.

Triple negative status (TN): response (SVR or TSS) was more common in TN subjects. Median OS was not estimable for TN subjects, non-TN subjects = 23.6 months; and

Molecular risk: Response (SVR or TSS) was more common in subjects with HMR with only one mutation, and responses were observed in subjects with HMR with more than one mutation who received 9.4 mg/kg imetelstat treatment.

Example 2. Baseline telomere length (TL) and overall survival (OS).

Subjects were grouped by median baseline DT. In the 9.4 mg/kg subgroup, the median OS was not estimable (NE) at the first clinical data cutoff date with 95% CI (23.2, NE) for subjects with lower baseline DT (< median) and was 22.8 months (16.2, NE) for subjects with higher DT (> median), respectively (Table 6). In the 4.7 mg/kg subgroup, the median OS with 95% CI was 20.3 (17.2, NE) months and 22.3 (16.6, NE) months for subjects with lower baseline DT and subjects with higher DT, respectively (Table 6).

In the 9.4 mg/kg subgroup, a trend towards better OS was observed for subjects with lower baseline DT, i.e., with baseline DT less than or equal to the median DT.

- 29 047547

Table 6

Overall survival as a function of baseline telomere length (TL) grouped by median value

4.7 mg/kg 9.4 mg/kg ^Median >Median ^Median >Median

N value value N value value

Subjects with a basic meaning 40 18 22 53 29 24 Number of subjects being assessed 40 18 (45.0%) 22(55.0%) 53 29(54.7%) 24(45.3%) Number of subjects with censored data (%) 20 8 (20.0%) 12(30.0%) 35 21(39.6%) 14(26.4%) Number of events (%) 20 10 (25.0%) 10(25.0%) 18 8(15.1%) 10(18.9%) Overall survival (months) Median (95% D.I.) 22.3 (18.2, NO.) 20.3 (17.2, NO.) 22.3 (16.6, NO.) NO. (22.8, NO.) N O. (23.2, NO.) 22.8 (16.2, NO.) Survival rate at 18 months, % (95% CI) 68.2 (50.7, 80.6) 76.7 (49.2, 90.6) 61.0 (36.7, 78.3) 75.9 (61.5, 85.6) 81.2 (60.5, 91.7) 69.8 (46.9, 84.3) Survival rate at 24 months, % (95% CI) 39.6 (21.1, 57.6) 34.1 (10.9, 59.3) 46.6 (21.8, 68.2) 54.2 (35.1, 69.9) 59.3 (30.9, 79.3) 47.6(22.1, 69.4)

Basic DT and answers at 24 weeks.

Baseline Telomere Length (TL): SVR or TSS response at week 24 was more common in subjects with shorter baseline TL (< median). 17.3% (5/29) of subjects with shorter baseline TL and 4.2% (1/24) of subjects with longer baseline TL had a splenic response, respectively. 34.5% (10/29) of subjects with shorter baseline TL and 25% (6/24) of subjects with longer baseline TL had a TSS response, respectively. In the 9.4 mg/kg subgroup at week 24, a higher response rate (SVR or TSS) was observed more often in subjects with shorter baseline TL compared to subjects with longer TL (Table 7).

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Table 7

Review of the relationship between clinical response and baseline telomere length (TL)

Basic DT SVR > 35% at 24 weeks according to IRC Total, N=93 With this and 4.7 mg/kg, N=40 9.4 mg/kg, N=53 Total number Present N=0 (0%) None 2 N=40 (100%) Total number Present 2 N=6 (10.5%) None 2 N=47 (89.5%) Lower DT « Median value 47 18 0 (0%) 10 (100%) 29 5 (17.25%) 24 (82.75%) Large DT (>Median value) 46 22 0 (0%) 38 (100%) 24 1 (4.2%) 23 (92.7%) Basic DT TSS > 50% reduction at 24 weeks Total, N = 93 with the given and 4.7 mg/kg, N=40 9.4 mg/kg, N=53 Total number Present 2 N = 2 (5%) None 2 N = 38 (95%) Total number Present 2 N= 16 (30.2%) None 2 N = 37 (69.8%) Lower DT « Median value 47 18 1 (5.6%) 17 (94.4%) 29 10 (34.5%) 19 (65.6%) Big DT 46 22 1 (4.5%) 21 (95.5%) 24 6 (25%) 18 (75%) (>Median value)

Example 3. Dose-dependent pharmacodynamic (PD) effect.

Telomerase and hTERT activities were analyzed to evaluate the pharmacodynamic effects of imetelstat. Of the subjects with available baseline and post-treatment data, 23 (51.1%) subjects in the 9.4 mg/kg subgroup and 10 (29.4%) subjects in the 4.7 mg/kg subgroup achieved a >50% reduction in telomerase activity from baseline; this PD effect correlated with antitumor activity in preclinical in vivo xenograft models. Additionally, 35 (61.4%) subjects in the 9.4 mg/kg subgroup and 20 (47.7%) subjects in the 4.7 mg/kg subgroup achieved a >50% reduction in hTERT RNA from baseline, respectively (Table 8). Thus, a dose-dependent PD effect was demonstrated, indicating on-target binding.

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Table 8

Subjects achieved reductions in telomerase (TA) (30%, 50%) or hTERT (50%) activity relative to baseline at all time points

4.7 MG/KG 9.4 MG/KG Subjects with baseline (hTERT) 48 57 P With a measure of >5 zenka (>50% with (table 9 hTERT >50% decrease Subjects with baseline (TA) TA >30% decrease TA >50% decrease Example 4. Relationship between PD effect among those who had a response sel 0% decrease in hT expression levels 55.6%); among those who had a decrease in hTER expression levels ). hTERT RNA expression levels from 20 (41.7%) 34 14 (41.2%) 10 (29.4%) I and response at 24 weeks. spleen, in a higher % of subjects ERT RNA compared to those TSS response, in a higher % of sub RNA compared to those were measured in whole-cell samples 35 (61.4%) 45 28 (62.2%) 23 (51.1%) of those (83.3%) were achieved by m and those who did not have responses were achieved by less than those who did not have TS of blood taken from patients before to a lesser extent S-response and after

treatment.

Table 9

Association between hTERT reduction (50%) from baseline and SVR or TSS response at week 24

Spleen response at 24 weeks according to IRC hTERT > 50% reduction Present 5/6 (83.3%) Absent 55/99(55.6%) TSS response at 24 weeks Present 15/24 (62.5%) Absent 45/81 (55.6%)

Compared with subjects who did not have a spleen or TSS response, at least >30% or >50% reduction in telomerase activity was achieved in a higher percentage of subjects who had a spleen or TSS response (Table 10).

Table 10

Association between telomerase activity reduction (30%, 50%) relative to baseline and SVR or TSS response at week 24

Telomerase activity Spleen response at 24 weeks according to IRC >50% reduction >30% reduction Present 2/4 (50%) 3/4 (75%) Absent 34/70 (48.6%) 41/70 (58.6%) TSS response at 24 weeks Present 12/18 (66.7%) 15/18 (83.3%) Absent 24/56 (42.9%) 29/56 (51.8%)

The aspects, including embodiments of the subject matter, described herein may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the present disclosure, certain non-limiting aspects of the present invention are set forth below. As will be apparent to those skilled in the art upon reading this document, each of the individually numbered aspects may be applied to or combined with any preceding or following individually numbered aspects. Thus, all such combinations are contemplated.

- 32,047,547 aspects, without limitation to the combinations of aspects directly presented below.

1. Use of a telomerase inhibitor in the treatment of a patient with myelofibrosis who is defined as having a triple-negative status, where triple-negative status is characterized by the absence of a mutation in each of the genes: Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL).

2. The use according to claim 1, characterized in that said myelofibrosis is primary myelofibrosis.

3. The use according to paragraph 2, characterized in that said myelofibrosis is myelofibrosis that develops after true polycythemia (post-PV MF).

4. The use according to paragraph 2, characterized in that said myelofibrosis is myelofibrosis that develops after essential thrombocythemia (post-ET MF).

5. Use according to any one of paragraphs 1-4, characterized in that said patient has not previously received therapy with JAK inhibitors.

6. The use according to any of 1-4, characterized in that said patient has received JAK inhibitor therapy and was refractory to JAK inhibitor therapy.

7. The use according to any of 1-4, characterized in that said patient has received therapy with JAK inhibitors and experiences a relapse.

8. The use according to any of 1-4, characterized in that said patient received therapy with JAK inhibitors and discontinued therapy with JAK inhibitors due to treatment-related toxicity or intolerance.

9. The use according to any of 1-8, characterized in that said telomerase inhibitor is imetelstat.

10. The use according to claim 9, characterized in that said imetelstat is sodium imetelstat.

11. The use according to claim 10, characterized in that said telomerase inhibitor is imetelstat and is administered during 1, 2, 3, 4, 5, 6, 7, 8 or more than 8 dosing cycles, each of which comprises intravenous administration of approximately 7-10 mg/kg imetelstat once every three weeks;

intravenous administration of approximately 7-10 mg/kg imetelstat once a week for three weeks;

intravenous administration of approximately 2.5-10 mg/kg imetelstat once every three weeks or intravenous administration of approximately 0.5-9.4 mg/kg imetelstat once every three weeks.

12. The use according to claim 11, characterized in that each dosing cycle comprises intravenous administration of approximately 7-10 mg/kg imetelstat once every three weeks.

13. The use according to claim 12, characterized in that each dosing cycle comprises intravenous administration of approximately 9.4 mg/kg imetelstat once every three weeks.

14. The use according to any one of claims 1 to 13, characterized in that the average relative telomere length is determined by analyzing the relative length of telomeric nucleic acids in target cells present in a biological sample from a patient.

15. The use according to any one of claims 1 to 14, further comprising selecting a patient identified as a patient in whose biological sample the average relative telomere length in the target cells is determined to fall within the 50th or lower percentile of a range of relative telomere lengths determined for one or more known standard samples.

16. The use according to any one of claims 1 to 15, further comprising screening a patient to determine whether said patient has a high molecular risk (HMR), wherein the presence of HMR comprises the presence of a mutation in at least one gene selected from the group consisting of ASXL1, EZH2, SRSF2 and IDH1/2.

17. The use according to any one of claims 1 to 16, further comprising assessing the level of hTERT expression in a biological sample obtained from said patient after administration of said telomerase inhibitor.

18. The use according to claim 17, characterized in that the level of hTERT expression is reduced by 50% or more relative to the baseline level of hTERT expression prior to administration of said telomerase inhibitor.

19. The use according to any one of claims 17-18, further comprising changing the dosage of said telomerase inhibitor, the frequency of dosing, or the course of therapy administered to the subject.

20. Use of a telomerase inhibitor in the treatment of a patient with myelofibrosis, wherein said patient is determined to have a high molecular risk (HMR), wherein the presence of HMR comprises the presence of a mutation in at least one gene selected from the group consisting of the extra sex setae homologue 1 gene (ASXL1), the enhancer of Zeste protein homologue 2 gene (EZH2), the serine-arginine-rich splicing factor 2 gene (SRSF2), and the isocitrate dehydrogenase 1/2 gene (IDH1/2).

21. The use according to item 20, characterized in that the said myelofibrosis is a first

- 33 047547 myelofibrosis.

22. The use according to paragraph 21, characterized in that said myelofibrosis is myelofibrosis that develops after true polycythemia (post-PV MF).

23. The use according to claim 21, characterized in that said myelofibrosis is myelofibrosis that develops after essential thrombocythemia (post-ET MF).

24. Use according to any of paragraphs 20-23, characterized in that said patient has not previously received therapy with JAK inhibitors.

25. The use according to any one of claims 20-23, characterized in that said patient received therapy with JAK inhibitors and was refractory to therapy with JAK inhibitors.

26. The use according to any one of paragraphs 20-23, characterized in that said patient has received therapy with JAK inhibitors and experiences a relapse.

27. The use according to any one of claims 20 to 23, wherein said patient has received JAK inhibitor therapy and has discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

28. The use according to any one of paragraphs 20-27, characterized in that said telomerase inhibitor is imetelstat.

29. The use according to claim 28, characterized in that said imetelstat is imetelstat sodium.

30. The use according to claim 28, wherein said telomerase inhibitor is imetelstat and is administered in 1, 2, 3, 4, 5, 6, 7, 8 or more than 8 dosing cycles, each of which comprises intravenous administration of approximately 7-10 mg/kg imetelstat once every three weeks;

intravenous administration of approximately 7-10 mg/kg imetelstat once a week for three weeks;

intravenous administration of approximately 2.5-10 mg/kg imetelstat once every three weeks or intravenous administration of approximately 0.5-9.4 mg/kg imetelstat once every three weeks.

31. The use according to claim 30, characterized in that each dosing cycle comprises intravenous administration of approximately 7-10 mg/kg imetelstat once every three weeks.

32. The use according to claim 31, characterized in that each dosing cycle comprises intravenous administration of approximately 9.4 mg/kg imetelstat once every three weeks.

33. The use according to any one of claims 20 to 32, further comprising determining the average relative telomere length by analyzing the relative length of telomeric nucleic acids in target cells present in a biological sample from a patient.

34. The use according to any one of claims 20 to 33, further comprising selecting a patient identified as a patient in whose biological sample the average relative telomere length in the target cells is determined to fall within the 50th or lower percentile of a range of relative telomere lengths determined for one or more known standard samples.

35. The use according to any one of claims 20-34, further comprising screening the patient to determine whether said patient has a triple negative status, characterized by the absence of a mutation in each of the genes selected from the group consisting of JAK2, CALR and MPL.

36. The use according to any one of claims 20-35, further comprising assessing the level of hTERT expression in a biological sample obtained from said patient after administration of said telomerase inhibitor.

37. The use according to claim 36, characterized in that the level of hTERT expression is reduced by 50% or more relative to the baseline level of hTERT expression prior to the administration of said telomerase inhibitor.

38. The use according to any one of paragraphs 36, 37, further comprising changing the dosage of said telomerase inhibitor, the frequency of dosing or the course of therapy administered to said subject.

39. Use of a telomerase inhibitor in the treatment of a patient who has myelofibrosis, wherein cells present in a biological sample from said patient have been determined to have a mean relative telomere length that is defined as falling in the 50th percentile or lower of a range of relative telomere lengths determined for one or more known reference standards.

40. The use according to paragraph 39, characterized in that said myelofibrosis is primary myelofibrosis.

41. The use according to claim 40, characterized in that said myelofibrosis is myelofibrosis that develops after true polycythemia (post-PV MF).

42. The use according to claim 40, characterized in that said myelofibrosis is myelofibrosis that develops after essential thrombocythemia (post-ET MF).

43. Use according to any of paragraphs 39-42, characterized in that said patient has not previously received therapy with JAK inhibitors.

44. Use according to any of paragraphs 39-42, characterized in that said patient received therapy

- 34 047547 I am taking JAK inhibitors and was refractory to JAK inhibitor therapy.

45. The use according to any one of paragraphs 39-42, characterized in that said patient has received therapy with JAK inhibitors and experiences a relapse.

46. The use according to any one of paragraphs 39-42, characterized in that said patient received therapy with JAK inhibitors and discontinued therapy with JAK inhibitors due to treatment-related toxicity or intolerance.

47. The use according to any one of paragraphs 39-46, characterized in that said telomerase inhibitor is imetelstat.

48. The use according to claim 47, characterized in that said imetelstat is sodium imetelstat.

49. The use according to claim 47, characterized in that said telomerase inhibitor is imetelstat and is administered during 1, 2, 3, 4, 5, 6, 7, 8 or more than 8 dosing cycles, each of which comprises intravenous administration of approximately 7-10 mg/kg imetelstat once every three weeks;

intravenous administration of approximately 7-10 mg/kg imetelstat once a week for three weeks;

intravenous administration of approximately 2.5-10 mg/kg imetelstat once every three weeks or intravenous administration of approximately 0.5-9.4 mg/kg imetelstat once every three weeks.

50. Use according to claim 49, characterized in that each dosing cycle comprises intravenous administration of approximately 7-10 mg/kg imetelstat once every three weeks.

51. The use according to claim 50, characterized in that each dosing cycle comprises intravenous administration of approximately 9.4 mg/kg imetelstat once every three weeks.

52. The use according to any one of paragraphs 39-51, further comprising determining the average relative telomere length by analyzing the relative length of telomeric nucleic acids in cells present in said biological sample from the patient.

53. The use according to any one of paragraphs 39-52, further comprising assessing the level of hTERT expression in a biological sample obtained from said patient after administration of said telomerase inhibitor.

54. The use according to claim 53, characterized in that the level of hTERT expression is reduced by 50% or more relative to the baseline level of hTERT expression prior to the administration of said telomerase inhibitor.

55. The use according to any one of paragraphs 53-54, further comprising changing the dosage of said telomerase inhibitor, the frequency of dosing, or the course of therapy administered to said subject.

56. A method for selecting a patient who is likely to benefit from treatment with a telomerase inhibitor, comprising testing the patient for triple negative status, which is characterized by the absence of a mutation in each of the JAK2, CALR, and MPL genes; and selecting said patient if he or she has a triple negative status, wherein said selected patient is likely to benefit from treatment with a telomerase inhibitor.

57. The method according to item 56, characterized in that said patient suffers from myelofibrosis.

58. The method according to claim 57, characterized in that said myelofibrosis is primary myelofibrosis.

59. The method according to claim 57, characterized in that said myelofibrosis is myelofibrosis that develops after true polycythemia (post-PV MF).

60. The method according to claim 57, characterized in that said myelofibrosis is myelofibrosis that develops after essential thrombocythemia (post-ET MF).

61. The method according to any one of claims 56-60, characterized in that said patient has not previously received therapy with JAK inhibitors.

62. The method according to any one of paragraphs 56-60, characterized in that said patient has previously received therapy with JAK inhibitors;

previously received JAK inhibitor therapy and it was ineffective or previously received JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

63. The method according to any one of claims 56-60, characterized in that said patient received therapy with JAK inhibitors and was refractory to therapy with JAK inhibitors.

64. The method according to any one of claims 56-60, characterized in that said patient has received therapy with JAK inhibitors and experiences a relapse.

65. The method according to any one of claims 56-60, characterized in that said patient received therapy with JAK inhibitors and discontinued therapy with JAK inhibitors due to treatment-related toxicity or intolerance.

66. The method according to any one of claims 56-65, further comprising administering said inhibitor

- 35 047547 telomerase to the specified patient.

67. The method according to claim 66, characterized in that said telomerase inhibitor is imetelstat.

68. The method according to claim 67, characterized in that said imetelstat is sodium imetelstat.

69. The method according to any one of paragraphs 56-68, further comprising obtaining a sample that contains DNA from the patient.

70. The method according to claim 69, characterized in that said sample contains bone marrow, peripheral blood, or a combination thereof.

71. The method according to claim 70, characterized in that the step of obtaining a sample from a patient includes obtaining a bone marrow sample, a peripheral blood sample, or a combination thereof, and isolating DNA from the bone marrow sample, the peripheral blood sample, or a combination thereof.

72. The method according to claim 70, characterized in that the step of obtaining a sample from a patient includes obtaining a sample of bone marrow from the patient;

isolation of cells from a bone marrow sample and extraction of DNA from the isolated cells.

73. The method according to claim 70, characterized in that the step of obtaining a sample from a patient includes obtaining a sample of peripheral blood from said patient;

isolating cells from the specified peripheral blood sample and extracting DNA from the isolated cells.

74. A method for selecting a patient who is likely to benefit from treatment with a telomerase inhibitor, comprising testing the patient to determine whether the patient has an HMR, wherein the presence of an HMR comprises the presence of a mutation in at least one gene selected from the group consisting of ASXL1, EZH2, SRSF2 and IDH1/2; and selecting said patient if the patient has an HMR, wherein said selected patient is likely to benefit from treatment with a telomerase inhibitor.

75. The method according to item 74, characterized in that said patient suffers from myelofibrosis.

76. The method according to claim 75, characterized in that said myelofibrosis is primary myelofibrosis.

77. The method according to claim 75, characterized in that said myelofibrosis is myelofibrosis that develops after true polycythemia (post-PV MF).

78. The method according to claim 75, characterized in that said myelofibrosis is myelofibrosis that develops after essential thrombocythemia (post-ET MF).

79. The method according to any one of claims 74-78, characterized in that said patient has not previously received therapy with JAK inhibitors.

80. The method according to any one of paragraphs 74-78, characterized in that said patient: has previously received therapy with JAK inhibitors;

previously received JAK inhibitor therapy and it was ineffective; or previously received JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

81. The method according to any one of claims 74-78, characterized in that said patient received therapy with JAK inhibitors and was refractory to therapy with JAK inhibitors.

82. The method according to any one of claims 74-78, characterized in that said patient has received therapy with JAK inhibitors and experiences a relapse.

83. The method according to any one of claims 74-78, characterized in that said patient received therapy with JAK inhibitors and discontinued therapy with JAK inhibitors due to treatment-related toxicity or intolerance.

84. The method according to any one of claims 74-83, further comprising administering said telomerase inhibitor to said patient.

85. The method according to claim 84, characterized in that said telomerase inhibitor is imetelstat.

86. The method according to claim 85, characterized in that said imetelstat is sodium imetelstat.

87. The method according to any one of claims 74-86, further comprising obtaining a sample that contains DNA from the patient.

88. The method according to claim 87, characterized in that said sample contains bone marrow, peripheral blood, or a combination thereof.

89. The method according to claim 88, characterized in that the step of obtaining a sample from a patient includes obtaining a bone marrow sample, a peripheral blood sample, or a combination thereof, and isolating DNA from the bone marrow sample, the peripheral blood sample, or a combination thereof.

- 36 047547

90. The method according to claim 88, characterized in that the step of obtaining a sample from a patient includes obtaining a sample of bone marrow from said patient; isolating cells from the bone marrow sample and extracting DNA from the isolated cells.

91. The method according to paragraph 88, characterized in that the step of obtaining a sample from a patient includes obtaining a sample of peripheral blood from said patient;

isolating cells from the specified peripheral blood sample and extracting DNA from the isolated cells.

92. A method for selecting a patient who is likely to benefit from treatment with a telomerase inhibitor, comprising testing the patient for an average relative telomere length by analyzing the relative length of telomeric nucleic acids in target cells present in a biological sample from the patient; and selecting said patient if the average relative telomere length in the target cells present in the biological sample from the patient is determined to fall within the 50th percentile or lower of a range of relative telomere lengths determined for one or more known reference standards, wherein said selected patient is likely to benefit from treatment with the telomerase inhibitor.

93. The method according to item 92, characterized in that said patient suffers from myelofibrosis.

94. The method according to claim 93, characterized in that said myelofibrosis is primary myelofibrosis.

95. The method according to claim 93, characterized in that said myelofibrosis is myelofibrosis that develops after true polycythemia (post-PV MF).

96. The method according to claim 93, characterized in that said myelofibrosis is myelofibrosis that develops after essential thrombocythemia (post-ET MF).

97. The method according to any one of claims 92-96, characterized in that said patient has not previously received therapy with JAK inhibitors.

98. The method according to any one of paragraphs 92-96, characterized in that said patient has previously received therapy with JAK inhibitors;

previously received JAK inhibitor therapy and it was ineffective or previously received JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance.

99. The method according to any one of claims 92-96, characterized in that said patient received therapy with JAK inhibitors and was refractory to therapy with JAK inhibitors.

100. The method according to any one of claims 92-96, characterized in that said patient has received therapy with JAK inhibitors and experiences a relapse.

101. The method according to any one of claims 92-96, characterized in that said patient received therapy with JAK inhibitors and discontinued therapy with JAK inhibitors due to treatment-related toxicity or intolerance.

102. The method according to any one of claims 92-101, further comprising administering said telomerase inhibitor to said patient.

103. The method according to claim 102, characterized in that said telomerase inhibitor is imetelstat.

104. The method according to claim 103, characterized in that said imetelstat is sodium imetelstat.

105. The method according to any one of paragraphs 92-104, further comprising obtaining a sample that contains DNA from the patient.

106. The method according to claim 105, characterized in that said sample contains bone marrow, peripheral blood, or a combination thereof.

107. The method according to claim 106, characterized in that the step of obtaining a sample from a patient includes obtaining a bone marrow sample, a peripheral blood sample, or a combination thereof, and isolating DNA from the bone marrow sample, the peripheral blood sample, or a combination thereof.

108. The method according to claim 106, characterized in that the step of obtaining a sample from a patient includes obtaining a sample of bone marrow from said patient;

isolating cells from a bone marrow sample; and extracting DNA from the isolated cells.

109. The method according to item 106, characterized in that the step of obtaining a sample from a patient includes obtaining a sample of peripheral blood from said patient;

isolating cells from the specified peripheral blood sample and extracting DNA from the isolated cells.

110. A method for monitoring therapeutic efficacy in a subject with myelofibrosis (MF), comprising measuring the expression level of hTERT in a biological sample obtained from said patient following administration of a telomerase inhibitor; and

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Claims (5)

comparing the level of hTERT expression in said biological sample with the baseline level of hTERT expression prior to administration of said telomerase inhibitor, wherein a decrease in the level of hTERT expression by 50% or more in said biological sample identifies a subject who is likely to benefit from treatment with said telomerase inhibitor. 111. The method according to claim 110, characterized in that the measured or estimated level of hTERT expression is represented by the level of hTERT RNA expression. 112. A method for identifying a patient with myelofibrosis (MF) for treatment with a telomerase inhibitor, comprising measuring the level of hTERT expression in a biological sample obtained from said patient after administration of a telomerase inhibitor; and comparing the level of hTERT expression in said biological sample with a baseline level of hTERT expression before administration of said telomerase inhibitor, wherein a decrease in the level of hTERT expression in said biological sample identifies a patient who is likely to benefit from treatment with said telomerase inhibitor. 113. The method according to claim 112, characterized in that said reduction in the level of hTERT expression is 50% or more. Although specific embodiments have been described in sufficient detail by way of illustration and example for clarity of understanding, it will be apparent in light of the disclosed principles that certain changes and modifications can be made therein without departing from the spirit or scope of the appended claims. Accordingly, the foregoing is merely illustrative of the principles of the present invention. Various arrangements may be devised which, although not explicitly described or shown herein, embody the principles of the present invention and which are within the scope and spirit of the present invention. Furthermore, all examples and symbols used herein are intended essentially to assist the reader in understanding the principles of the present invention and the ideas contributed by the present inventors to develop the art, and should not be construed as limiting the invention to the specifically recited examples and conditions. Furthermore, all statements in this document which describe the principles, aspects and embodiments of the present invention, as well as specific examples thereof, include both structural and functional equivalents thereof. Furthermore, such equivalents are intended to include both currently known equivalents and equivalents that will be developed in the future, i.e., any developed elements that perform the same function, regardless of structure. Accordingly, it is not intended that the scope of the present invention be limited to the exemplary embodiments shown and described herein. In fact, the scope and essence of the present invention are defined by the appended claims. CLAUSE OF THE INVENTION 1. A method for selecting a patient with myelofibrosis who is likely to benefit from treatment with a telomerase inhibitor, including: (a) testing a patient for triple negative status, which is characterized by the absence of a mutation in each of the genes: Janus kinase 2 (JAK2), calreticulin (CALR), and thrombopoietin receptor (MPL); and (b) selecting a patient based on the presence of triple negative status, wherein the selected patient is likely to benefit from treatment with a telomerase inhibitor, wherein the telomerase inhibitor is imetelstat or a pharmaceutically acceptable salt thereof, and the telomerase inhibitor is administered to the patient at a dose that ranges from 4.5 to 11.7 mg/kg. 2. The method according to claim 1, characterized in that myelofibrosis is selected from the group consisting of primary myelofibrosis, myelofibrosis that develops after true polycythemia (post-IP MF), myelofibrosis that develops after essential thrombocythemia (post-ET MF). 3. The method according to any one of claims 1, 2, characterized in that the patient has not previously received therapy with JAK inhibitors. 4. The method according to any one of claims 1, 2, characterized in that the patient received therapy with JAK inhibitors and was refractory to therapy with JAK inhibitors; received JAK inhibitor therapy and relapsed or received JAK inhibitor therapy and discontinued JAK inhibitor therapy due to treatment-related toxicity or intolerance. 5. The method according to any one of claims 1-4, characterized in that the telomerase inhibitor is imetelstat sodium. -