WO2019245460A1

WO2019245460A1 - Stratifying cancer patients harbouring oncogenic mutations in the e3 ubiquitin ligase rnf43

Info

Publication number: WO2019245460A1
Application number: PCT/SG2019/050310
Authority: WO
Inventors: David Epstein
Original assignee: National University Of Singapore
Priority date: 2018-06-21
Filing date: 2019-06-21
Publication date: 2019-12-26

Abstract

The present invention relates to stratifying cancer patients harbouring oncogenic mutations in the E3 ubiquitin ligase RNF43. More particularly, the invention relates to stratifying patients based on whether they have a loss of function mutation, a dominant negative mutation or a silent mutation in RNF43, thereby identifying which patients will be likely to respond to Wnt-pathway inhibitors and those that won't respond and can avoid incorrect treatment and possible side effects of Wnt-pathway inhibitor treatment.

Description

STRATIFYING CANCER PATIENTS HARBOURING ONCOGENIC MUTATIONS IN THE

E3 UBIQUITIN LIGASE RNF43

FIELD OF THE INVENTION

The present invention relates to stratifying cancer patients harbouring oncogenic mutations in the E3 ubiquitin ligase RNF43. More particularly, the invention relates to stratifying patients based on whether they have a loss of function mutation, a dominant negative mutation or a silent mutation in RNF43, thereby identifying which patients will be likely to respond to Wnt-pathway inhibitors and those that won’t respond and can avoid incorrect treatment and possible side effects of Wnt-pathway inhibitor treatment.

BACKGROUND OF THE INVENTION

The Wnt signaling pathway is often dysregulated in human cancers. Binding of the Wnt ligands to their cognate cell surface receptors, primarily the Frizzled (Fzd) receptors, and co-receptors LRP5/6 leads to activation of the pathway [reviewed in Nusse & Clevers, Cell, 169(6): 985-999 (2017)]. The level of expression of the Wnt receptors, Frizzled-1 through Frizzled-10, is tightly regulated by post-translational modifications. An E3 ubiquitin ligase, RNF43, regulates cell surface Frizzled abundance via its ubiquitination [Koo et al., Nature. 488: 665-669 (2012)]. Several inactivating mutations in RNF43 are known that sensitize pancreatic cancers to Wnt pathway inhibitors [Jiang et al., PNAS. 1 10: 12649-12654 (2013)]. Hence, loss of RNF43 function is oncogenic and renders tumour cells responsive to Wnt targeted inhibitors, such as the Porcupine enzyme (PORCN) inhibitors [Madan et al., Oncogene. 35: 2197-2207 (2016)].

The Wnt signaling pathway is also central to normal skeletal homeostasis, and the clinical evaluation of a variety of Wnt-pathway directed drugs has revealed that bone degeneration is a major and severe dose-limiting toxicity [Madan et al., Bone Research. 6(1): 17 (2018)]. Importantly, these clinical studies have been conducted in broadly defined cancer patient populations and were not directed to selected patients harbouring Wnt-pathway activating mutations, such as have been recently identified in RNF43. Cancer patients whose tumours harbour RNF43 oncogenic mutations should be candidates for Wnt-targeted therapy with PORCN inhibitors such as those disclosed in WO 2014189466 and WO 20150941 19, the contents of which are incorporated herein by reference in their entirety. More particularly, such patients may benefit from administration of PORCN inhibitors such as 1 ,3-dimethyl-7- ((6-phenylpyridazin-3-yl)glycyl)-3,4,5,7-tetrahydro-1 H-purine-2,6-dione (also known as ETC- 1922159) and/or 4-(2-methyl-6,7-dihydropyrazolo[1 ,5-a]pyrimidin-4(5H)-yl)-4-oxo-N-(6- (pyridin-3-yl)pyridazin-3-yl)butanamide (also known as ETC-2017569). In contrast, cancer patients whose tumours are not driven by Wnt-pathway activation and RNF43 mutation should be spared treatment with PORCN inhibitors. The gene RNF43 is one of over 400 cancer-related genes that are currently screened in cancer DNA sequencing panels, such as that provided to physicians and patients by Foundation Medicine’s, Foundation One™ comprehensive genomic profile test. The stated goals of such genomic tests are to expand treatment options by matching patients with targeted therapies and clinical trials that are relevant to the molecular changes in their tumour, based on the most recent scientific and medical published evidence. However, at present there is little to no information on which of the RNF43 mutations are oncogenic, or whether the majority of the described RNF43 mutations are silent and non-oncogenic [Tsukiyama et al., Mol Cell Biol. 35(1 1): 2007-2023 (2015)]. Delineating which RNF43 mutations cause cancer is essential to providing expanded treatment options for cancer patients who might benefit from treatment with a Wnt-pathway inhibitor.

To enable effective treatment decisions around the use of Wnt pathway inhibitors, it is essential that clinicians have actionable information for the selection of treatments for their patients. More specifically, it is critical for clinicians to be able to identify newly diagnosed cancer patients whose mutations in RNF43 are sensitizing for Wnt activation and would therefore be candidates for treatment with a PORCN-targeted therapy, versus those patients whose RFN43 mutations are non-oncogenic, and therefore would not be a candidate for treatment with a PORCN-targeted therapy. A fundamental problem is that somatic mutations in the RNF43 gene are described across the entire RNF43 protein coding region without evidence of a specific mutational hot spot. Instead, these mutations are essentially equally distributed across the entire length of the gene. A review of DNA sequencing data, from clinical studies comprising 60,000 human tumour samples deposited in the cBioPortal database, reveals that across this large cohort of cancer patients the RNF43 gene exhibits more than 1000 mutations. The oncogenic function of these mutations is not reported and is unknown, so that genomic sequencing of RNF43 currently does not provide actionable treatment information to oncologists. Hence, it is of critical importance to identify which, if any, of these over 1000 known mutations are oncogenic or silent and/or play a role in regulating the function of the Wnt-receptors, Fzd.

There is a need for methods that can improve stratification of patients based on whether they have specific RNF43 mutations that are loss-of-function (LOF), dominant negative or silent.

SUMMARY OF THE INVENTION

The present invention is based on studies in relation to mutations in RNF43 and how the functional effect of a particular mutation may be used in terms of, for example, stratifying a patient to determine whether administration of a Wnt-pathway inhibitor is a suitable treatment for the patient. In a first aspect there is provided an in vitro method of stratifying subjects into classes of predicted sensitivity of tumour cell growth to inhibition by a Wnt-pathway inhibitor, comprising:

(a) assaying a sample comprising or derived from a tumour cell from a subject to determine the sequence of at least a portion of genomic DNA, cDNA or RNA corresponding to RNF43;

(b) determining whether the genomic DNA, cDNA or RNA sequence of RNF43 in the sample harbours one or more mutations;

(c) comparing the one or more RNF43 mutations in the tumour cell sample to the panel of RNF43 mutations listed in Table 1 ; and

(d)(i) recording in a tangible medium that a subject has an RNF43 loss of function mutation, or

(d)(ii) recording in a tangible medium that a subject has an RNF43 dominant negative mutation, or

(d)(iii) recording in a tangible medium that a subject has an RNF43 silent mutation that behaves like wild type RNF43.

In some embodiments the method comprises (a)-(c) and;

(d)(i) recording in a tangible medium that a subject has an RNF43 loss of function mutation and is likely to benefit from therapeutic administration of a Wnt-pathway inhibitor, or

(d)(ii) recording in a tangible medium that a subject has an RNF43 dominant negative mutation and is likely to benefit from therapeutic administration of a Wnt-pathway inhibitor, or

(d)(iii) recording in a tangible medium that a subject has an RNF43 silent mutation that behaves like wild type RNF43 and is unlikely to benefit from therapeutic administration of a Wnt-pathway inhibitor.

Typically the obtained information may be used by a clinician so as to facilitate patient management. For example, the information may be used to direct a treatment, such as whether or not Wnt-pathway inhibitor treatment should be conducted or a different type of treatment should be administered. As not all RNF43 mutations increase cell surface Frizzled expression, it is important to be able to stratify patients based on which particular mutations are loss of function (LOF), which are dominant negative and which are silent.

In some embodiments, the method of stratifying subjects further comprises treating the subjects in class (d)(i) or class (d)(ii) with a Wnt-pathway inhibitor.

In some embodiments, the Wnt-pathway inhibitor is an inhibitor of Porcupine (PORCN). In some embodiments, the Wnt-pathway inhibitor is selected from those disclosed in WO 2014189466 and WO 2015094119.

In some embodiments, the Wnt-pathway inhibitor is 1 ,3-dimethyl-7-((6- phenylpyridazin-3-yl)glycyl)-3,4,5,7-tetrahydro-1 H-purine-2,6-dione (also known as ETC- 1922159) and/or 4-(2-methyl-6,7-dihydropyrazolo[1 ,5-a]pyrimidin-4(5H)-yl)-4-oxo-N-(6- (pyridin-3-yl)pyridazin-3-yl)butanamide (also known as ETC-2017569).

In some embodiments the subject has a tumour selected from the group comprising pancreas, colorectal, gastric, ovarian, esophageal, lung, uterine, melanoma, bladder, breast, renal, prostate, liver, germ cell, cutaneous squamous cell carcinoma and glioblastoma multiforme.

In some embodiments the subject may be stratified according to the tumour type and panel of RNF43 mutations listed in Tables 2 to 15. In particular, stratification may be according to the assigned mutant % inhibition and/or activity and/or predicted effects on FZD.

In a further aspect there is provided a method of detecting, in a tumour cell sample from a subject, an RNF43 loss of function mutation or an RNF43 dominant negative mutation listed in Table 1 , said method comprising:

(a) detecting whether an RNF43 loss of function mutation or an RNF43 dominant negative mutation listed in Table 1 is present in an isolated tumour cell sample by sequencing the genomic DNA, cDNA or RNA corresponding to RNF43 in said tumour cell sample.

In some embodiments the method comprises obtaining the tumour cell sample from the subject prior to step (a).

In some embodiments, the tumour is selected from the group comprising prostate cancer, Ovarian/Fallopian Tube cancer, Penile cancer, Colorectal Adenocarcinoma, Endometrial cancer, Ampullary carcinoma, Colorectal cancer, Breast cancer, Pancreatic cancer, Nerve Sheath tumour, Esophagogastric cancer, Cutaneous melanoma, Gastrointestinal Neuroendocrine tumour, Appendiceal cancer, Mesothelioma, Melanoma, Adenocortical Carcinoma, Skin cancer (non-melanoma), Small Bowel cancer, Bladder cancer, Hepatobiliary cancer, Small cell lung cancer, Upper Tract Urothelial cancer, Prostate cancer, Gastrointestinal stromal cancer, Ovarian cancer, Pheochromocytoma, Non-small cell lung cancer, Soft tissue sarcoma, Embryonal tumour, Cervical cancer, Non-Hodgkin lymphoma, Uterine sarcoma, Renal cell carcinoma, Thymic tumour, Wilms tumour and Glioma.

In some embodiments the tumour cell sample is selected from the group comprising pancreatic, colorectal, gastric, ovarian, esophageal, lung, uterine, melanoma, bladder, breast, renal, prostate, liver, germ cell cutaneous squamous cell carcinoma and glioblastoma multiforme. In some embodiments the RNF43 loss of function mutation or the RNF43 dominant negative mutation is according to the tumour type and panel of RNF43 mutations listed in Tables 2 to 15.

In a further aspect, there is provided use of a Wnt-pathway inhibitor in the manufacture of a medicament for the treatment of a subject with a tumour comprising an RNF43 loss of function mutation or an RNF43 dominant negative mutation listed in Table 1.

In some embodiments, the Wnt-pathway inhibitor is an inhibitor of Porcupine (PORCN).

In some embodiments, the Wnt-pathway inhibitor is selected from those disclosed in WO 2014189466 and WO 2015094119.

In some embodiments the subjects have a tumour type and RNF43 mutation listed in Tables 2 to 15.

In a further aspect there is provided a method of treatment comprising administering to a subject, with a tumour comprising an RNF43 loss of function mutation or an RNF43 dominant negative mutation listed in Table 1 , an effective amount of a Wnt-pathway inhibitor.

In some embodiments, the method of treatment comprises:

(a) assaying a sample comprising or derived from a tumour cell from a subject to determine the sequence of at least a portion of genomic DNA, cDNA or RNA corresponding to RNF43; (b) determining whether the genomic DNA, cDNA or RNA sequence of RNF43 in the sample harbours one or more mutations;

(d)(i) recommending, prescribing or administering a treatment regimen comprising a Wnt- pathway inhibitor to a subject in whose sample RNF43 is determined to have a loss of function mutation, or

(d)(ii) recommending, prescribing or administering a treatment regimen comprising a Wnt- pathway inhibitor to a subject in whose sample RNF43 is determined to have a dominant negative mutation, or

(d)(iii) recommending, prescribing or administering a treatment regimen not comprising a Wnt-pathway inhibitor to a subject in whose sample RNF43 is determined to have a silent mutation that behaves like wild type RNF43.

In some embodiments the subjects may be stratified for recommended treatment according to a tumour type and RNF43 mutation listed in any one or more of Tables 2 to 15. In a further aspect there is provided a diagnostic kit for detecting whether a subject tumour cell sample has one or more RNF43 loss of function or dominant negative mutations listed in Table 1 , the kit comprising a plurality of oligonucleotides for sequencing of genomic DNA, cDNA or RNA corresponding to RNF43.

In some embodiments, the tumour type and RNF43 mutation is listed in Tables 2 to 15.

It would be understood that oligonucleotide primers and/or probes are structurally and/or chemically modified to, for example, prolong their activity in harsh conditions such as when used in PCR and sequencing methods.

In some embodiments, at least one of the plurality of oligonucleotides is structurally and/or chemically modified from its natural nucleic acid.

In some embodiments, said structural and/or chemical modifications are selected from the group comprising the addition of tags, such as fluorescent tags, radioactive tags, biotin, a 5’ tail, the addition of phosphorothioate (PS) bonds, 2'-0-Methyl modifications and/or phosphoramidite C3 Spacers during synthesis.

In a further aspect there is provided use of a kit according to any aspect of the invention in a method according to any aspect of the invention.

Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference.

BRIEF DESCRIPTION OF FIGURES

Figure 1 shows that RNF43 regulates abundance of multiple Frizzled receptors. (A) Protein abundance of Frizzleds in HEK293 cells transfected with plasmids expressing the indicated HA-tagged Frizzleds. (B)-(F) RNF43 overexpression downregulates cell surface levels of FZD2, 4, 5, 7 and 10 in Pane 08.13 cells. Flow cytometric analysis of Frizzled abundance on Pane 08.13 cells transfected with HA-FZD with or without wild-type RNF43. Data are representative of two independent experiments. HA = haemaggluttinin , AF488 = Alexa Fluor 488 dye.

Figure 2 shows that about 50% of the RNF43 mutations are LOF or dominant negative and they are sensitive to Wnt-pathway inhibition. (A) HEK293 cells expressing Wnt^-catenin reporter were transfected with plasmid expressing WNT3A alone or with the plasmids expressing wild-type (WT) RNF43 or indicated RNF43 mutants. The Wnt^-catenin reporter activity in cells expressing only WNT3A is set to baseline. Data represent percentage inhibition of WNT3A induced reporter activity in the presence of the indicated RNF43 mutants (positioned along the WT RNF43). Loss-of-function (LOF) mutants are defined as the ones with percentage inhibition between -20 to 20. If the percentage inhibition is more than 20%, these mutants are classified as dominant negative (i.e. activating the reporter activity). If the percentage inhibition is less than -20%, the activity of these mutants is comparable to WT. Data are representative of 3 independent experiments. (B) Same as in (A) with only pancreatic cancer specific RNF43 mutations. (C) HEK293 cells expressing Wnt^-catenin reporter were transfected with plasmid expressing WNT3A alone or with the plasmids expressing wild-type (WT) RNF43 or indicated RNF43 mutants. Six hours after transfection, 100 nM ETC-1922159 (ETC-159) was added to the cells and Wnt^-catenin reporter activity was assessed 20 hours after ETC-1922159 (ETC-159) treatment. Data represent relative luciferase activities normalized to mCherry readings. Data are representative of two independent experiments.

Figure 3 shows that RNF43 mutants regulate endogenous FZD levels in HEK293 cells. (A-C) Flow cytometric analysis of endogenous FZD levels in HEK293 cells expressing wild- type RNF43 or indicated mutants. The cells expressing RNF43 were selected based on co expression of EGFP. (A) Mutants S94I, A146G and P1 18T downregulate endogenous FZD levels comparable to wild-type RNF43. (B) I 186T and R286W are activating mutants and increase endogenous cells surface FZD abundance. (C) V287Gfs*7 mutant increases cell surface FZD levels while hotspot mutant G659Vfs*41 partly regulates wild-type RNF43. (D) Mean fluorescent intensity for the indicated mutants based on the flow analysis in A-C. Isotype ctr = Isotype control, EV = Empty Vector, APC = Allophycocyanin.

Figure 4 shows increased levels of Frizzleds on RNF43 mutant patient-derived xenografts (PDX) compared to WT RNF43-expressing tissues. (A) Immunohistochemical (IHC) staining of PAXF1861 PDX with RNF43 mutation (insertion of G at amino acid G372fs) stains positively for Frizzled receptors indicating high abundance on the cell surface. Scale bar, 100 pm. (B) Negative staining of OV0243, PA1457 and PA3127 PDX models having WT RNF43 (I47V and L418M variants) indicates low Frizzled receptor abundance. Scale bar, 100 pm. (C) Treatment with ETC-1922159 (ETC-159) inhibits the growth of RNF43 mutant pancreatic PDX, PAXF1861 , in vivo.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention belongs. Certain terms employed in the specification, examples and appended claims are collected here for convenience.

It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a Wnt ligand" includes a plurality of such Wnt ligands, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.

As used herein, the term“comprising” or“including” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. However, in context with the present disclosure, the term“comprising” or “including” also includes“consisting of”. The variations of the word“comprising”, such as “comprise” and “comprises”, and “including”, such as “include” and “includes”, have correspondingly varied meanings.

The term "subject" is herein defined as vertebrate, particularly mammal, more particularly human. For purposes of research, the subject may particularly be at least one animal model, e.g., a mouse, rat and the like. In particular, for treatment of cancers or tumours such as pancreatic cancer, colorectal cancer, ovarian cancer, oesophagus, ovary squamous cell carcinoma, leukemia, skin cancer and lung adenocarcinoma which have RNF43 LOF or dominant negative mutations, the subject may be a human.

The term "treatment", as used in the context of the invention refers to prophylactic, ameliorating, therapeutic or curative treatment.

The terms “patient” and “patients” include references to mammalian (e.g. human) patients.

As used herein, the term "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.

As used herein, the term "substitution" refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.

The terms "insertion" or "addition," as used herein, refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, to the sequence found in the naturally occurring molecule.

A "mutant" of RNF43, as used herein, refers to an amino acid sequence that is altered by one or more amino acids. The mutant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "non-conservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNASTAR software.

As used herein, Loss-of-function (LOF) mutants are defined as the mutants with little or no inhibition (in the range of -20 to 20% inhibition) of TOPFLASH reporter activity relative to wild type RNF43. Partial LOF mutants are defined as mutants with more than -20% and up to -50% inhibition of reporter activity relative to wild-type RNF43. Dominant negative (DN) mutants are defined as mutants that actually activate TOPFLASH activity by more than 20% relative to a negative control. Mutants with less than -20% inhibition of reporter activity relative to wild-type RNF43 are considered comparable to wild type RNF43.

EXAMPLES

Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, New York (2012).

EXAMPLE 1

Materials and methods

Cell lines, plasmids and Patient-derived xenograft (PDX) FFPE blocks

HEK293 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, L-glutamate, pen/strep and sodium pyruvate. Pane 08.13 cells were cultured in RPMI1640 medium supplemented with 15% fetal bovine serum, L-glutamate, pen/strep, sodium pyruvate and insulin. The HA-FZD constructs were obtained from Jeffrey Rubin’s laboratory. They use the signal peptide of human FZD5, followed by 2XHA tags and then the corresponding mature FZD sequences. FZD1 and FZD2 are rat sequences. FZD5 is a human sequence while the rest are all mouse sequences. PAXF1861 PDX samples were obtained from oncotest. RNF43 sequences were verified by Sanger sequencing. OV0243, PA1457 and PA3127 PDX samples were obtained from CrownBio. RNF43 sequence information was obtained from CrownBio Hubase database.

Generation of RNF43 mutant constructs

Myc-DDK-tagged RNF43 ORF expressing construct was purchased from Origene (Origene, Cat# RC214013) and sequences were verified by Sanger sequencing. The construct was modified to introduce silent mutations by site-directed mutagenesis to make it resistant to CRISPR-Cas9 guide RNA. The nucleotides at amino acid position starting 270 CCT GTG TGT GCC ATC TGT CTG are changed to CCc GTc TGc GCg ATt TGc CTc. This resistant plasmid was used as template to generate other RNF43 mutants. The plasmid was mutated using site-directed mutagenesis following the manufacturer’s protocol (Stratagene). The modified plasmid sequences were verified by Sanger sequencing.

Wnt/3-catenin reporter assay

One day before transfection, cells (100,000 cells/well) were seeded into 24-well plates coated with poly-L-lysine. Cells were transfected using Lipofectamine 2000 (Thermo Fisher) mixed with DNA in a ratio of 3: 1 using the manufacturer’s protocol. For each well 100 ng TOPFIash, 50 ng mCherry, 20 ng PGK-mWNT3A and 20 ng RNF43 plasmid mix was used. After 48 h of transfection, cells were lysed with 100 pi Reporter Lysis Buffer (Promega) supplemented with protease inhibitor cocktail (Roche) for 20 minutes at 4°C with shaking. The cell lysates were transferred to a 96-well black PCR plate and then equal volume of Luciferase assay reagent (Promega) was added for measuring luciferase activities and read on a Tecan infinite 2000 plate reader. mCherry values were used for normalization.

Flow cytometric analysis of HA-tagged FZD cell surface levels in Pane 08.13 cells.

One day before transfection, Pane 08.13 cells were seeded into 6-well plates (coated with poly-L-lysine) at 400,000 cells per well. On the day of transfection, the DNA/liposome mixture was prepared in Opti-MEM medium. 200 ng of HA-FZD (except for HA-FZD10, for which 50 ng of plasmid was used) and 200 ng of RNF43 plasmids were used for transfection with Lipofectamine 2000 transfection reagent. Two days after transfection, single cell resuspension was prepared using TrypLE. The cell number was determined using a hemocytometer and the cells were resuspended in FACS buffer (PBS with 5% FBS, 1 mM EDTA) to make 2x10⁶ cells/ml. 100 pi of cell suspension (2x10⁵ cells) was placed in each Eppendorf tube and 0.5 pi of HA tag monoclonal antibody (16B12) conjugated with Alexa Fluor 488 (A-21284, Thermo Scientific) was added. The tubes were incubated in a cold room for 1 h, protected from light. After incubation, the cells were washed with ice-cold FACS buffer 3 times. Finally, the cells were resuspended in 400 mI of FACS buffer and transferred to flow cytometry tubes with filtered cap. The flow acquisition was done using BD LSRFortessa Cell Analyzer and analyzed with FlowJo 10 software.

One day before transfection, tHEK293 cells were seeded into 12-well plates (coated with poly-L-lysine). On the day of transfection, the DNA/liposome mixture was prepared in Opti-MEM medium. 200 ng of HA-FZD plasmids were used for transfection with TurboFect transfection reagent. Two days after transfection, cells were harvested and lysed with 4% SDS. The lysates were sonicated for 5 minutes to break genomic DNA. The protein concentration was measured using BCA protein assay and 35 pg of total protein lysates were used for SDS-PAGE and western blotting. The membrane was probed with anti-HA tag antibody (Cell Signaling).

Flow cytometric analysis of endogenous FZD cell surface levels in HEK293 cells after mutant

RNF43 expression.

One day before transfection, HEK293 cells were seeded into 6-well plates (coated with poly-L-lysine). On the day of transfection, the DNA/liposome mixture was prepared in Opti- MEM medium. 100 ng of pEGFP and 100 ng of RNF43 plasmids were used for transfection with TurboFect transfection reagent. Two days after transfection, a single cell resuspension was prepared using TrypLE. The cell number was determined using a hemocytometer and cells were resuspended in FACS buffer (PBS with 5% FBS, 1 mM EDTA) to make 5x10⁶ cells/ml. 100 mI of cell suspension (5x10⁵ cells) was taken and added to V-bottom 96-well plate. Pan-FZD antibody OMP18R5 was diluted 10 times with PBS. A master mix was prepared with 1 mI of diluted antibody and 99 mI of FACS buffer for each well. Human lgG2 lamba protein (0.5 mg/ml) was used as isotype control. The plate was incubated in a cold room for 1 h. After incubation, the cells were washed with ice cold FACS buffer 2 times. Secondary antibody anti human Fc fragment APC-conjugated (Jackson lab) was added using 1 in 300 dilutions in FACS buffer (100 mI per well). The plate was incubated in a cold room for 30 minutes, protected from light. After incubation, the cells were washed with ice cold FACS buffer 3 times. Finally, cells were resuspended in 400 mI of FACS buffer and transferred to flow cytometry tubes with filtered cap. The flow acquisition was done using BD LSRFortessa Cell Analyzer and around 200,000 cells were acquired and analyzed with FlowJo 10 software. The analysis was gated on GFP positive population.

Immunohistochemical staining

FFPE blocks were sectioned at 5 pm and incubated in a 60°C oven for 1 h before standard de-waxing procedure. The slides were boiled in Tris-EDTA (pH 9.0) buffer in microwave for 20 minutes for antigen-retrieval. After cooling and washing in TBS, the samples were incubated with 3% H₂0₂ for 10 minutes to block the endogenous peroxidase activity. Pan-FZD antibody was diluted in 3% BSA in TSBT (0.1 % Tween-20) and the samples were incubated with the primary antibody overnight at 4°C. The next day, the samples were washed three times in TBS before incubating with anti-human IgG secondary antibody conjugated with HRP for 1 h at room temperature. After that, the samples were washed 3 times in TBS and DAB chromogen was added to develop the colour. The samples were counterstained by Gill No.2 hematoxylin solution and underwent dehydration procedure before being mounted using DPX mounting medium.

Xenograft mouse model with Wnt inhibitor treatment

Patient-derived solid tissue fragments were subcutaneously implanted in NCr nude mice. All groups were matched for tumour size with equal variance before treatment. ETC- 1922159 formulated in 50% PEG400 (vol/vol) in water was administered by oral gavage at a dosing volume of 10 mI/g body weight. T umours were harvested and weighed at the end of the treatment.

EXAMPLE 2

RNF43 regulates abundance of multiple Frizzled receptors on the cell surface:

There are 10 Frizzled receptors in the human genome and there is no report demonstrating whether or not RNF43 regulates all these Frizzled proteins. When the expression of HA-tagged Frizzled proteins in HEK293 cells was measured by immunoblot, it was observed that FZD 2, 4, 5, 7 and 10 were expressed at higher levels in HEK293 cells compared to other FZDs (Figure 1A). To test if RNF43 can regulate the cell surface abundance of these Frizzled proteins, FZD 2, 4, 5, 7 and 10 were expressed in Pane 08.13, a pancreatic cancer cell line that has low endogenous levels of Frizzled proteins. The HA-tagged Frizzled proteins were co-expressed with or without wild-type RNF43 proteins. As analyzed by flow cytometry (Figure 1 B-F), co-expression of wild-type RNF43 reduced the surface levels of all the five Frizzled proteins. This demonstrates that RNF43 acts as a universal regulator of the abundance of cell surface Frizzled receptors.

EXAMPLE 3

Systematic study of RNF43 mutations reveals 50% of the mutations in RNF43 are loss- of-function or dominant negative

RNF43 mutations found in various cancers are spread across the entire length of the gene without hot spots, except the frequently occurring frameshift mutation G659Vfs*41. To test the activity of these mutants in regulating Wnt signaling, an analysis of approximately one hundred RNF43 mutants was performed. RNF43 mutants were generated by site-directed mutagenesis to compare their ability to regulate WNT3A induced Wnt/p-catenin reporter activity. As shown in Figure 2A, expression of wild-type RNF43 led to 80-90% reduction of the Wnt/p-catenin reporter activity (‘WT’) with the reporter activity in the presence of Wnt ligands alone set to zero. Surprisingly, hot spot mutation G659Vfs*41 suppressed Wnt/p-catenin reporter activity to the same extent as wild-type RNF43, and was hence not Loss-Of-Function (LOF) as seen in Figure 2B. A large fraction of the RNF43 mutations analyzed were either LOF, i.e.: no inhibition of reporter activity (set to -20% to 20%), or dominant negative, i.e.: activating the reporter activity (> 20%). RNF43 has an extracellular protease-associated (PA) domain that has been shown to interact with the Wnt agonist R-spondin, and an intracellular RING domain that exhibits an E3 ubiquitin ligase function [Chen et a!., Genes & Development, 27(12): 1345-1350 (2013); Zebisch et al., Nature Communications, 4: 2787 (2013)]. Approximately seventy-five percent (75%) of the mutations in the region preceding the PA domain (amino acid 87-186) were identified to be LOF or dominant negative. Almost all the mutations in the RING domain (272-313) were LOF or dominant negative. Truncation mutants after amino acid position 330 (R330* and R371*) seem to maintain the activity. As the frequency of mutations in the RING domain or before are more prevalent, taken together approximately 50% of the mutations in RNF43 are LOF (Table 1).

The following table summarizes all the RNF43 mutants that the study has profiled, with information including the mutation types in which the mutant RNF43 has been identified, their percentage inhibition in Wnt/p-catenin reporter assays (representative results of three independent experiments), which subgroups (WT, LOF or dominant negative) they belong to, as well as the predicted effects on Fzd levels. Table 1. Summary of RNF43 mutants’ activity.

The following tables provide known RNF43 mutants grouped into the tumour types in which the mutant RNF43 has been identified, our data on percentage inhibition in Wnt/p-catenin reporter assays (representative results of three independent experiments), which subgroups they belong to (WT, LOF or dominant negative), as well as the predicted effects on Fzd levels. Table 2. Summary of RNF43 mutants’ activity in pancreas tumour.

Table 3. Summary of RNF43 mutants’ activity in colorectal tumour.

Table 4. Summary of RNF43 mutants’ activity in gastric tumour.

Table 5. Summary of RNF43 mutants’ activity in ovarian tumour.

Table 6. Summary of RNF43 mutants’ activity in esophageal tumour.

Table 7. Summary of RNF43 mutants’ activity in lung tumour.

Table 8. Summary of RNF43 mutants’ activity in uterine tumour.

Table 9. Summary of RNF43 mutants’ activity in melanoma.

Table 11. Summary of RNF43 mutants’ activity in breast tumour.

Table 12. Summary of RNF43 mutants’ activity in renal tumour.

Table 13. Summary of RNF43 mutants’ activity in prostate tumour.

Table 14. Summary of RNF43 mutants’ activity in liver tumour.

Table 15. Summary of RNF43 mutants’ activity in other tumours.

EXAMPLE 4

RNF43 Pancreatic cancer specific mutations are sensitive to Wnt inhibitor

Specific RNF43 mutations found in pancreatic ductal carcinoma (PDAC) patients were analyzed and more than half of the mutations were either LOF or dominant negative in the Wnt^-catenin reporter assay (Figure 2B). In order to test whether these RNF43 mutants were sensitive to Wnt inhibition, selected mutants were exposed to Wnt inhibitor ETC-1922159 using the Wnt^-catenin reporter assay. These included missense mutants (i.e. S94I, V162M, G166V, I186T, R286W and D300G), and frameshift mutants (V287Gfs*7 and G659Vfs*41). In all cases, regardless of whether these mutants were functional or not, Wnt inhibition abolished the reporter activities completely (Figure 2C). This implied that a Wnt ligand is required for signaling in the presence of RNF43 mutations, and Wnt inhibition could be a plausible treatment in cancers with RNF43 LOF or dominant negative mutations.

EXAMPLE 5

The activity of RNF43 mutants correlates with their ability to regulate Frizzled abundance

The activity of RNF43 mutants in regulating Wnt^-catenin reporter was then correlated with their ability to regulate endogenous cell surface Frizzled abundance. HEK293 cells have relatively high levels of Frizzleds on the cell surface (Figure 3A, solid black line). The Frizzled levels were measured by flow cytometry using an antibody (pan-FZD antibody) that recognizes Frizzleds 1 , 2, 5, 7 and 8. Consistent with the ability of WT RNF43 to reduce Wnt^-catenin reporter activity, expression of wild-type RNF43 significantly reduced Frizzled abundance on the cell surface (Figure 3A, dashed line). Similarly, P118T, A146G and S94I mutants behaved like wild-type in their ability to reduce Frizzled abundance on the cell surface (Figure 3A), which was also consistent with their reporter activity. On the other hand, PA domain mutant I186T and RING domain mutant R286W that were activating in reporter assay, upregulated surface Frizzled receptors (Figure 3B). Frameshift mutation V287Gfs*7, in the RING domain, significantly increased surface Frizzled receptors, consistent with the reporter assay results. Hot spot mutation G659Vfs*41 still decreased surface Frizzled levels (Figure 3D). In conclusion, the results from both assays were consistent, and they demonstrated that a large percentage of RNF43 mutations found in human cancers are potentially activating the Wnt signaling pathway by elevating multiple cell surface Frizzled receptors.

EXAMPLE 6

Detection of Frizzled abundance using immunohistochemical staining (IHC)

The possibility of using a pan-FZD antibody for immunohistochemical staining (IHC) as a diagnostic marker to identify tumours with RNF43 mutations was explored using pancreatic cancers Patient-Derived Xenograft (PDX) tumour formalin-fixed paraffin- embedded (FFPE) samples with different RNF43 status. PAXF1861 PDX tumour has an insertion of G at amino acid position 372 of RNF43. PA1457 and PA3127 and OV0243 (an ovarian cancer PDX) have wild-type RNF43. The IHC results show that PAXF1861 has strong positive staining (Figure 4(A)), while the wild-type RNF43 PDXs have weaker or negative staining (Figure 4(B)). These results indicate that it is plausible to use Frizzled staining as a secondary test to identify cancer patients carrying RNF43 mutants. The data could potentially be extended to detect patients having colorectal cancers with R-spondin translocations, which also leads to the upregulation of Frizzled on the cell surface.

EXAMPLE 7

Treatment with ETC-1922159 inhibits the growth of RNF43 mutant patient-derived xenografts (PDX) in vivo

Patient-derived solid tissue fragments were subcutaneously implanted in NCr nude mice. All groups were matched for tumour size with equal variance before treatment. The Porcupine inhibitor ETC-1922159, formulated in 50% PEG400 (vol/vol) in water as the vehicle, was administered by oral gavage at a dosing volume of 10 mI/g body weight. The mice were treated for 14 days with 75 mg/kg ETC-1922159 administered every day. Tumours were harvested and weighed at the end of the treatment on day 14. As shown in Figure 4(C), treatment with ETC-1922159 inhibited the growth of RNF43 mutant pancreatic PDX, PAXF1861 in vivo.

Summary

The activity of a large set of human cancer related RNF43 mutants, identified in public domain cancer genome databases, were analysed using a Wnt^-catenin reporter assay. The analysis revealed that ~ 50% of RNF43 mutations found in cancers do not compromise the function of RNF43. The remaining mutants either led to loss of RNF43 activity or acted as dominant negative mutations and further potentiated Wnt signaling pathway activation. Moreover, the activity of these mutants correlated with their ability to regulate the abundance of cell surface levels of Frizzled receptors as detected by flow cytometry. Immunohistochemical staining, using a pan-frizzled antibody, of patient derived xenografts with RNF43 loss of function mutation confirmed the increase in abundance of Frizzled levels. Further, inhibition of Wnt signaling using a PORCN inhibitor, ETC-1922159 prevented the activation of the Wnt/b- catenin reporter activity by these mutants and inhibited the growth of patient derived xenografts harbouring these mutations.

Any listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that such document is part of the state of the art or is common general knowledge.

References

1) Chen, P.-H., Chen, X., Lin, Z., Fang, D., & He, X. (2013). The structural basis of R- spondin recognition by LGR5 and RNF43. Genes & Development, 27(12): 1345-1350.

2) Jiang, X., Hao, H.-X., Growney, J. D., Woolfenden, S., Bottiglio, C., Ng, N., et al. (2013). Inactivating mutations of RNF43 confer Wnt dependency in pancreatic ductal

adenocarcinoma. PNAS. 1 10: 12649-12654.

3) Koo, B.-K., Spit, M., Jordens, I., Low, T. Y., Stange, D. E., van de Wetering, M., et al. (2012). Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. Nature. 488: 665-669.

4) Madan, B., Ke, Z., Harmston, N., Ho, S. Y., Frois, A. O., Alam, J., et al. (2016). Wnt addiction of genetically defined cancers reversed by PORCN inhibition. Oncogene. 35: 2197- 2207.

5) Madan, B., McDonald, M. J., Foxa, G. E., Diegel, C. R., Williams, B. O., & Virshup, D. M. (2018). Bone loss from Wnt inhibition mitigated by concurrent alendronate therapy. Bone Research. 6(1): 17.

6) Nusse, R., & Clevers, H. (2017). Wnt^-Catenin Signaling, Disease, and Emerging

Therapeutic Modalities. Cell, 169(6): 985-999.

7) Tsukiyama T, Fukui A, Terai S, Fujioka Y, Shinada K, Takahashi H, et al. (2015). Molecular Role of RNF43 in Canonical and Noncanonical Wnt Signaling. Mol Cell Biol. 35(11): 2007- 2023.

8) Zebisch, M., Xu, Y., Krastev, C., Macdonald, B. T., Chen, M., Gilbert, R. J. C., et al.

(2013). Structural and molecular basis of ZNRF3/RNF43 transmembrane ubiquitin ligase inhibition by the Wnt agonist R-spondin. Nature Communications, 4: 2787.

Claims

1. An in vitro method of stratifying subjects into classes of predicted sensitivity of tumour cell growth to inhibition by a Wnt-pathway inhibitor, comprising:

2. The method of claim 1 , comprising (a)-(c) and;

3. The method of claim 1 or 2, further comprising treating subjects in class (d)(i) or class (d)(ii) with a Wnt-pathway inhibitor.

4. The method of claim 3, wherein the Wnt-pathway inhibitor is an inhibitor of Porcupine (PORCN).

5. The method of claim 4, wherein the Wnt-pathway inhibitor is 1 ,3-dimethyl-7-((6- phenylpyridazin-3-yl)glycyl)-3,4,5,7-tetrahydro-1 H-purine-2,6-dione and/or 4-(2-methyl-6,7- dihydropyrazolo[1 ,5-a]pyrimidin-4(5H)-yl)-4-oxo-N-(6-(pyridin-3-yl)pyridazin-3-yl)butanamide.

6. The method of any one of claims 1 to 5, wherein the tumour is selected from the group comprising prostate cancer, Ovarian/Fallopian Tube cancer, Penile cancer, Colorectal Adenocarcinoma, Endometrial cancer, Ampullary carcinoma, Colorectal cancer, Breast cancer, Pancreatic cancer, Nerve Sheath tumour, Esophagogastric cancer, Cutaneous melanoma, Gastrointestinal Neuroendocrine tumour, Appendiceal cancer, Mesothelioma, Melanoma, Adenocortical Carcinoma, Skin cancer (non-melanoma), Small Bowel cancer, Bladder cancer, Hepatobiliary cancer, Small cell lung cancer, Upper Tract Urothelial cancer, Prostate cancer, Gastrointestinal stromal cancer, Ovarian cancer, Pheochromocytoma, Non small cell lung cancer, Soft tissue sarcoma, Embryonal tumour, Cervical cancer, Non-Hodgkin lymphoma, Uterine sarcoma, Renal cell carcinoma, Thymic tumour, Wilms tumour and Glioma.

7. A method of detecting, in a tumour cell sample from a subject, an RNF43 loss of function mutation or an RNF43 dominant negative mutation listed in Table 1 , said method comprising:

8. The method of claim 7, wherein the tumour is selected from the group comprising prostate cancer, Ovarian/Fallopian Tube cancer, Penile cancer, Colorectal Adenocarcinoma, Endometrial cancer, Ampullary carcinoma, Colorectal cancer, Breast cancer, Pancreatic cancer, Nerve Sheath tumour, Esophagogastric cancer, Cutaneous melanoma, Gastrointestinal Neuroendocrine tumour, Appendiceal cancer, Mesothelioma, Melanoma, Adenocortical Carcinoma, Skin cancer (non-melanoma), Small Bowel cancer, Bladder cancer, Hepatobiliary cancer, Small cell lung cancer, Upper Tract Urothelial cancer, Prostate cancer, Gastrointestinal stromal cancer, Ovarian cancer, Pheochromocytoma, Non-small cell lung cancer, Soft tissue sarcoma, Embryonal tumour, Cervical cancer, Non-Hodgkin lymphoma, Uterine sarcoma, Renal cell carcinoma, Thymic tumour, Wlms tumour and Glioma.

9. The method of any one of the preceding claims, wherein the subject may be stratified according to the tumour type and panel of RNF43 mutations listed in any one of Tables 2 to 15.

10. Use of a Wnt-pathway inhibitor in the manufacture of a medicament for the treatment of a subject with a tumour comprising an RNF43 loss of function mutation or an RNF43 dominant negative mutation listed in Table 1.

11. The use according to claim 10, wherein the Wnt-pathway inhibitor is an inhibitor of Porcupine (PORCN).

12. The use according to claim 11 , wherein the Wnt-pathway inhibitor is 1 ,3-dimethyl-7-((6- phenylpyridazin-3-yl)glycyl)-3,4,5,7-tetrahydro-1 H-purine-2,6-dione and/or 4-(2-methyl-6,7- dihydropyrazolo[1 ,5-a]pyrimidin-4(5H)-yl)-4-oxo-N-(6-(pyridin-3-yl)pyridazin-3-yl)butanamide.

13. The use according to any one of claims 10 to 12, wherein said subject has a tumour selected from the group comprising prostate cancer, Ovarian/Fallopian Tube cancer, Penile cancer, Colorectal Adenocarcinoma, Endometrial cancer, Ampullary carcinoma, Colorectal cancer, Breast cancer, Pancreatic cancer, Nerve Sheath tumour, Esophagogastric cancer, Cutaneous melanoma, Gastrointestinal Neuroendocrine tumour, Appendiceal cancer, Mesothelioma, Melanoma, Adenocortical Carcinoma, Skin cancer (non-melanoma), Small Bowel cancer, Bladder cancer, Hepatobiliary cancer, Small cell lung cancer, Upper Tract Urothelial cancer, Prostate cancer, Gastrointestinal stromal cancer, Ovarian cancer, Pheochromocytoma, Non-small cell lung cancer, Soft tissue sarcoma, Embryonal tumour, Cervical cancer, Non-Hodgkin lymphoma, Uterine sarcoma, Renal cell carcinoma, Thymic tumour, Wilms tumour and Glioma.

14. The use according to any one of claims 10 to 13, wherein the tumour and RNF43 mutation is listed in any one of Tables 2 to 15.

15. A method of treatment comprising administering, to a subject with a tumour comprising an RNF43 loss of function mutation or an RNF43 dominant negative mutation listed in Table 1 , an effective amount of a Wnt-pathway inhibitor.

16. The method of claim 15 comprising:

(d)(i) recommending, prescribing or administering a treatment regimen comprising a Wnt- pathway inhibitor to a subject in whose sample RNF43 is determined to have a loss of function mutation, or (d)(ii) recommending, prescribing or administering a treatment regimen comprising a Wnt- pathway inhibitor to a subject in whose sample RNF43 is determined to have a dominant negative mutation, or

(d)(iii) recommending, prescribing or administering a treatment regimen not comprising a Wnt inhibitor to a subject in whose sample RNF43 is determined to have a silent mutation that behaves like wild type RNF43.

17. The method of claim 15 or 16, wherein the Wnt-pathway inhibitor is an inhibitor of Porcupine (PORCN).

18. The method of any one of claims 15 to 17, wherein the Wnt-pathway inhibitor is 1 ,3- dimethyl-7-((6-phenylpyridazin-3-yl)glycyl)-3,4,5,7-tetrahydro-1 H-purine-2,6-dione and/or 4- (2-methyl-6,7-dihydropyrazolo[1 ,5-a]pyrimidin-4(5H)-yl)-4-oxo-N-(6-(pyridin-3-yl)pyridazin-3- yljbutanamide.

19. The method of any one of claims 15 to 18, wherein the tumour is selected from the group comprising prostate cancer, Ovarian/Fallopian Tube cancer, Penile cancer, Colorectal Adenocarcinoma, Endometrial cancer, Ampullary carcinoma, Colorectal cancer, Breast cancer, Pancreatic cancer, Nerve Sheath tumour, Esophagogastric cancer, Cutaneous melanoma, Gastrointestinal Neuroendocrine tumour, Appendiceal cancer, Mesothelioma, Melanoma, Adenocortical Carcinoma, Skin cancer (non-melanoma), Small Bowel cancer, Bladder cancer, Hepatobiliary cancer, Small cell lung cancer, Upper Tract Urothelial cancer, Prostate cancer, Gastrointestinal stromal cancer, Ovarian cancer, Pheochromocytoma, Non small cell lung cancer, Soft tissue sarcoma, Embryonal tumour, Cervical cancer, Non-Hodgkin lymphoma, Uterine sarcoma, Renal cell carcinoma, Thymic tumour, Wilms tumour and Glioma.

20. The method of any one of claims 15 to 19, wherein the tumour and RNF43 mutation is according to any one of Tables 2 to 15.

21. A diagnostic kit for detecting whether a subject tumour cell sample has one or more RNF43 loss of function or dominant negative mutations listed in Table 1 , the kit comprising a plurality of oligonucleotides for sequencing of genomic DNA, cDNA or RNA corresponding to RNF43.

22. The diagnostic kit of claim 21 , wherein at least one of the plurality of oligonucleotides is structurally and/or chemically modified from its natural nucleic acid.

23. The diagnostic kit of claim 22, wherein said structural and/or chemical modifications are selected from the group comprising the addition of tags, such as fluorescent tags, radioactive tags, biotin, a 5’ tail, the addition of phosphorothioate (PS) bonds, 2'-0-Methyl modifications and/or phosphoramidite C3 Spacers during synthesis.

24. Use of a kit of any one of claims 21 to 23 in a method according to any one of claims 1 to 9.