WO2019245460A1 - Stratification de patients cancéreux hébergeant des mutations oncogènes dans l'ubiquitine ligase e3 rnf43 - Google Patents

Stratification de patients cancéreux hébergeant des mutations oncogènes dans l'ubiquitine ligase e3 rnf43 Download PDF

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WO2019245460A1
WO2019245460A1 PCT/SG2019/050310 SG2019050310W WO2019245460A1 WO 2019245460 A1 WO2019245460 A1 WO 2019245460A1 SG 2019050310 W SG2019050310 W SG 2019050310W WO 2019245460 A1 WO2019245460 A1 WO 2019245460A1
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cancer
rnf43
tumour
wnt
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David Epstein
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National University Of Singapore
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • 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.
  • 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)].
  • Frizzled-1 through Frizzled-10 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)].
  • 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.
  • 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).
  • 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
  • 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 OneTM 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.
  • 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.
  • 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.
  • an in vitro method of stratifying subjects into classes of predicted sensitivity of tumour cell growth to inhibition by a Wnt-pathway inhibitor comprising:
  • the method comprises (a)-(c) and;
  • the obtained information may be used by a clinician so as to facilitate patient management.
  • 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.
  • a treatment such as whether or not Wnt-pathway inhibitor treatment should be conducted or a different type of treatment should be administered.
  • LEF loss of function
  • the method of stratifying subjects further comprises treating the subjects in class (d)(i) or class (d)(ii) with a Wnt-pathway inhibitor.
  • 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.
  • 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).
  • 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.
  • 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.
  • the subject may be stratified according to the tumour type and panel of RNF43 mutations listed in Tables 2 to 15.
  • stratification may be according to the assigned mutant % inhibition and/or activity and/or predicted effects on FZD.
  • a tumour cell sample from a subject comprising:
  • the method comprises obtaining the tumour cell sample from the subject prior to step (a).
  • 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
  • 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.
  • 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.
  • 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.
  • the Wnt-pathway inhibitor is an inhibitor of Porcupine (PORCN).
  • the Wnt-pathway inhibitor is selected from those disclosed in WO 2014189466 and WO 2015094119.
  • 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).
  • 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
  • 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.
  • 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.
  • the subjects have a tumour type and RNF43 mutation listed in Tables 2 to 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.
  • the method of treatment comprises:
  • the Wnt-pathway inhibitor is an inhibitor of Porcupine (PORCN).
  • the Wnt-pathway inhibitor is selected from those disclosed in WO 2014189466 and WO 2015094119.
  • 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).
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • tumour type and RNF43 mutation is listed in Tables 2 to 15.
  • 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.
  • At least one of the plurality of oligonucleotides is structurally and/or chemically modified from its natural nucleic acid.
  • 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.
  • tags such as fluorescent tags, radioactive tags, biotin, a 5’ tail
  • PS phosphorothioate
  • kit according to any aspect of the invention in a method according to any aspect of the invention.
  • 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.
  • LEF Loss-of-function
  • ETC-1922159 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.
  • FIG. 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.
  • 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.
  • 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.
  • 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.
  • subject is herein defined as vertebrate, particularly mammal, more particularly human.
  • the subject may particularly be at least one animal model, e.g., a mouse, rat and the like.
  • the subject may be a human.
  • treatment refers to prophylactic, ameliorating, therapeutic or curative treatment.
  • patient and “patients” include references to mammalian (e.g. human) patients.
  • 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.
  • substitution refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
  • 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 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • Pane 08.13 cells were seeded into 6-well plates (coated with poly-L-lysine) at 400,000 cells per well.
  • 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.
  • 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 2x10 6 cells/ml. 100 pi of cell suspension (2x10 5 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.
  • tHEK293 cells were seeded into 12-well plates (coated with poly-L-lysine).
  • 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).
  • HEK293 cells were seeded into 6-well plates (coated with poly-L-lysine).
  • 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 5x10 6 cells/ml.
  • FACS buffer PBS with 5% FBS, 1 mM EDTA
  • 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.
  • 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% H 2 0 2 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
  • 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.
  • RNF43 regulates abundance of multiple Frizzled receptors on the cell surface:
  • Frizzled receptors there are 10 Frizzled receptors in the human genome and there is no report demonstrating whether or not RNF43 regulates all these Frizzled proteins.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • LEF Loss-Of-Function
  • 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.
  • PA protease-associated
  • Truncation mutants after amino acid position 330 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).
  • pan-FZD antibody for immunohistochemical staining (IHC) as a diagnostic marker to identify tumours with RNF43 mutations was explored using pancreatic cancers
  • IHC immunohistochemical staining
  • 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)).
  • 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.
  • 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.
  • treatment with ETC-1922159 inhibited the growth of RNF43 mutant pancreatic PDX, PAXF1861 in vivo.
  • 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.
  • Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. Nature. 488: 665-669.

Abstract

La présente invention concerne la stratification de patients cancéreux hébergeant des mutations oncogènes dans l'ubiquitine ligase E3 RNF43. Plus précisément, l'invention concerne la stratification de patients selon qu'ils présentent une mutation par perte de fonction, une mutation dominante négative ou une mutation silencieuse dans RNF43, en identifiant ainsi les patients qui sont susceptibles de répondre à des inhibiteurs de la voie Wnt et ceux qui ne répondraient pas, et il est possible d'éviter un traitement incorrect et d'éventuels effets latéraux d'un traitement par un inhibiteur de la voie Wnt.
PCT/SG2019/050310 2018-06-21 2019-06-21 Stratification de patients cancéreux hébergeant des mutations oncogènes dans l'ubiquitine ligase e3 rnf43 WO2019245460A1 (fr)

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