Treating cancers with mutations in WNT pathway tumor suppressors

Abstract

Provided is a method of treating cancer harboring one or more mutations in the WNT pathway tumor suppressors AXIN1 and / or APC in a subject in need thereof, comprising administering to the subject a MEK inhibitor or a pharmaceutically acceptable salt thereof. The MEK inhibitor may be administered as a single agent or in combination with treatment with an immune checkpoint inhibitor, such as an inhibitor of PD-1 and / or PD-L1, or a RAF inhibitor.

Description

[Technical Field]

[0001] The present disclosure relates generally to the use of mutations in predicting patient response to antiproliferative agents, specifically MEK inhibitors. Specifically, the presence or absence of mutations in the WNT pathway, specifically the tumor suppressor AXIN1 and / or APC, can be used to predict response to treatment with MEK inhibitors, either as single agents or in combination with other RAS / MAPK pathway inhibitors and / or immune checkpoint inhibitors, in patients presenting with cancer. Accordingly, the present disclosure provides methods of treating specific subsets of cancer patients identified as having mutations in the WNT pathway tumor suppressor AXIN1 and / or APC using MEK inhibitors. This treatment can be as a single agent or can be combined with other RAS / MAPK pathway inhibitors and / or immune checkpoint inhibitors, such as inhibitors of PD-1 and / or PD-L1. [Background technology]

[0002] WNT signaling is involved in several physiological processes, including embryonic development, stem cell homeostasis, tissue regeneration, and lineage commitment. Aberrant activation of the WNT pathway, either through gain- or loss-of-function mutations, is frequently observed in a wide variety of human cancers. Central to the pathogenesis of WNT-altered tumors is the proteolytic turnover of β-catenin, which functions as a transcriptional coactivator of WNT target genes. Specifically, β-catenin levels are maintained low by a multisubunit destruction complex composed of the tumor suppressors AXIN1 and adenomatous polyposis coli (APC) and the kinases casein kinase 1 (CK1) and glycogen synthase kinase 3β (GSK3β). Inactivating mutations in AXIN1 and APC are frequently observed in many cancers, including hepatocellular carcinoma (HCC), colorectal cancer, liver cancer, bladder cancer, endometrial cancer, melanoma, ovarian cancer, lung cancer, pancreatic cancer, and gastric cancer. Tumors harboring loss-of-function mutations in AXIN1 and APC are often clinically aggressive and less sensitive to chemotherapy and / or immunotherapy. For example, a retrospective analysis of patients with AXIN1-mutated advanced hepatocellular carcinoma (HCC) treated with a combination of atezolizumab and bevacizumab showed a less favorable response rate to this treatment compared with non-AXIN1-mutated patients (Zhu, AX, et al. Nat Med., 2022, 28:1599-1611). Therefore, there is a substantial need to develop therapeutics that can overcome mutation-induced WNT activation, particularly for the treatment of AXIN1- and APC-mutated cancers, as these mutations are often considered undruggable (Parsons, MJ, et al. Cancer Discov., 2021, 11(10):2413-2429; Bugter, JM, et al. Nat Rev Cancer., 2021, 5-21).

[0003] Hepatocellular carcinoma (HCC) is an example of a cancer in which loss-of-function mutations in AXIN1 and / or APC may be present. HCC is the third leading cause of cancer-related death worldwide. Patients with early-stage HCC can be successfully treated with surgical resection or liver transplantation. However, the common delay in diagnosis of HCC precludes curative treatment, and systemic therapy is the only viable option for inoperable patients. Current treatments available for advanced HCC include sorafenib, lenvatinib, the combination of atezolizumab and bevacizumab, regorafenib, ramucirumab, cabozantinib, nivolumab, and pembrolizumab. Sorafenib is an orally available multikinase inhibitor approved as systemic therapy for the treatment of patients with advanced HCC. The benefits offered by sorafenib have been shown to be limited, and new drugs, such as the c-MET inhibitor cabozantinib, are under development to overcome sorafenib resistance and improve patient outcomes. However, the high severity of HCC, the lack of good diagnostic markers and treatment strategies, and clinical heterogeneity make disease management a major challenge. Regarding currently used checkpoint therapies, the median time to response (approximately 12 weeks) and median overall survival benefit are less than 6 months. Furthermore, patients with Child-Pugh B / C classification present further challenges due to limited treatment options.

[0004] The clinical course of HCC patients varies greatly, and HCC comprises several biologically distinct subgroups. Mutations in the telomerase reverse transcriptase (TERT) promoter have been found in over 50% of HCC tissue samples examined, making TERT the most frequently occurring single-nucleotide mutation observed in HCC. Tumor protein 53 (TP53) is the second most frequently mutated gene in HCC, occurring in over 30% of HCC cases. Catenin beta 1 (CTNNB1) is another gene that is highly mutated in HCC, and aberrant activation of β-catenin has been observed in 20–30% of HCC patients. Approximately 40% of HCC patients harbor mutations in the WNT pathway. AXIN1 is the second most frequently mutated gene in this pathway. Loss of AXIN1 is observed in approximately 11% of HCC patients, which is thought to confer inherent resistance to immune checkpoint blockade. Despite belonging to the same pathway, genetic alterations in CTNNB1 and AXIN1 have been found to be mutually exclusive.

[0005] Similarly, mutations in CTNNB1, AXIN, and / or APC have been observed in epithelial ovarian cancer (EOC), particularly in the endometrioid and mucinous subtypes of EOC. EOC is the most lethal female malignancy. In EOC, aberrant activation of the WNT pathway leads to hyperactivity of β-catenin.

[0006] Colorectal cancer (CRC) is one of the most common types of cancer worldwide. Recently, CRC has been further subdivided into various molecular subtypes, known as consensus molecular subtypes (CMS). While the CMS classification is no longer used to drive treatment decisions, it distinguishes the biological, clinical, and molecular characteristics of CRC, thereby facilitating clinicians' awareness of the heterogeneity of CRC. For example, within the CMS subtype, CMS2, known as the canonical subtype, is characterized by activated WNT and MYC signaling. APC-mutated CRC tumors have been found to be more prevalently enriched in CMS2 CRCs, which also tend to harbor concomitant KRAS and TP53 alterations (Bugter, JM, et al. Nat Rev Cancer., 2021, 5-21).

[0007] The RAS / MAPK pathway is an important cellular signaling pathway that plays a major role in proliferation and differentiation processes. Growth factor-induced signals are transmitted by sequential phosphorylation from the serine / threonine kinase Raf to the dual specificity kinase MEK (MAP kinase kinase / ERK kinase) and finally to the kinase ERK (extracellular signal-regulated kinase), thereby influencing gene expression.

[0008] Currently, the use of MEK inhibitors in cancer therapeutic treatment is limited to the treatment of BRAF-mutated populations, including melanoma and non-small cell lung cancer (NSCLC), in combination with RAF inhibitors. MEK inhibitors are administered only in combination with RAF inhibitors to patients harboring the BRAF V600E mutation and are not administered as single agents due to pathway-specific rebound with MEK inhibitors in these patients. In addition, the MEK inhibitor selumetinib has also been approved for the genetically-adapted disease neurofibromatosis type 1 (NF1). However, apart from cancers harboring RAS / MAPK pathway alterations, the use of MEK inhibitors alone or in combination has not been officially investigated in genetically defined tumors. Indeed, MEK inhibition has been shown to activate WNT signaling both genetically and pharmacologically in CRC, as suggested by increased AXIN2 expression levels following siRNA KD in the APC WT CRC cell line HCT116. However, colony formation assays showed that single-agent trametinib was able to dose-dependently reduce the viability of HCT116 cells (Zhan et al., Nat Comm, 2019), suggesting that pharmacological induction of WNT activation is not sufficient to drive resistance. Another group reported that single-agent MEK inhibition alone did not significantly alter AXIN2 expression levels or WNT activity in the more relevant APC-mutated CRC cell line COLO320 (Solberg et al., Cancers, 2019). Full elucidation of the context-specificity between WNT signaling and RAS / MAPK pathway inhibitors may broaden the therapeutic use of MEK inhibitors. Summary of the Invention [Means for solving the problem]

[0009] According to a first aspect, there is provided a method of treating cancer with one or more mutations in AXIN1 and / or APC in a subject in need thereof, the method comprising administering to the subject a MEK inhibitor or a pharmaceutically acceptable salt thereof.

[0010] According to a second aspect, there is provided a method for assessing the likelihood that a MEK inhibitor, or a pharmaceutically acceptable salt thereof, will produce an anti-cancer effect in a subject suffering from cancer using one or more mutations in AXIN1 and / or APC as biomarkers, the method comprising assaying the subject for the presence of one or more mutations in AXIN1 and / or APC, and if one or more mutations in AXIN1 and / or APC are present in the subject, administering a MEK inhibitor, or a pharmaceutically acceptable salt thereof, to the subject to produce an anti-cancer effect.

[0011] According to a third aspect, there is provided a MEK inhibitor, or a pharmaceutically acceptable salt thereof, for use in treating a cancer with one or more mutations in AXIN1 and / or APC in a subject in need thereof.

[0012] The present disclosure will be more clearly understood from the following description of some of its embodiments, given by way of example only, with reference to the following figures: [Brief explanation of the drawings]

[0013] [Figure 1] Illustrates tumor volume results for treatment with the MEK inhibitor REC-4881 compared to cabozantinib in the HCC AXIN1 mutant LI6612 PDX model. [Figure 2] Illustrates tumor volume results for treatment with the MEK inhibitor REC-4881 and four FDA-approved MEK inhibitors in the HCC AXIN1 mutant LI6612 PDX model. [Figure 3] Figure 1 illustrates tumor volumes for treatment with the MEK inhibitor REC-4881 at 3 mg / kg PO as a single agent or in combination with anti-PD-L1 at 10 mg / kg IP in a B16F10-ova syngeneic melanoma model harboring an APC mutation. [Figure 4A]Figure 1 shows the tumor growth inhibition (TGI) rate of the MEK inhibitor REC-4881 at 3 mg / kg PO in 19 HCC PDX models performed as a mouse clinical trial, with 6 of the models harboring AXIN1 mutations and 13 of the models not harboring AXIN1 mutations. [Figure 4B] The results of progression-free survival (PFS) are shown. [Figure 5A] Figure 1 shows tumor growth inhibition (TGI) by the MEK inhibitor REC-4881 at 3 mg / kg PO in 10 ovarian cancer PDX models performed as mouse clinical trials, five of the models harboring AXIN1 and / or APC mutations and five of the models not harboring AXIN1 and / or APC mutations. [Figure 5B] The results of progression-free survival (PFS) are shown. [Figure 6A] Figure 1 shows tumor growth inhibition (TGI) of the MEK inhibitor REC-4881 at 3 mg / kg PO in a combination study of 19 HCC PDX models and 10 ovarian cancer PDX models performed as a mouse clinical trial. [Figure 6B] The results of progression-free survival (PFS) are shown. [Figure 7] 1 shows tumor volume results for treatment with the MEK inhibitor REC-4881 at 1 mg / kg and 3 mg / kg PO compared to sorafenib in the HCC LI6692 PDX model harboring an AXIN1 mutation. [Figure 8] Pharmacodynamic markers for tumor samples taken from the in vivo PDX study of Example 7: FIG. 8A for pERK / ERK, FIG. 8B for pMEK / MEK, FIG. 8C for SPRY4, FIG. 8D for DUSP6, and FIG. 8E for PPIA. [Figure 9] 1 illustrates pharmacokinetic data from non-tumor-bearing NCG mice administered REC-4881 at 1 mg / kg and 3 mg / kg. [Figure 10] 1 shows viability curves for human colorectal cancer cell lines, APC mutant and wild type, treated with the MEK inhibitor REC-4881 for 72 hours. [Figure 11A]Figure 1 shows that REC-4881 regulates CDKN2A expression in APC mutant and wild-type colorectal cancer cell lines. [Figure 11B] REC-4881 regulates MYC expression. [Figure 12] CD8+ T cell (A) and Treg (B) effects in APC-mutated B16F10-ova mouse melanoma tumors in female C57BL / 6 mice treated with vehicle, anti-PD1 agent, REC-4881 (3 mg / kg), or a combination of the two. [Figure 13] A study of the effect of REC-4881 alone or in combination with anti-PD1 agents on AXIN1-null Hepa1-6 mouse hepatocellular carcinoma (HCC) tumors in female C57BL / 6 mice shows (A) dosing schedule, (B) tumor volumes measured in each arm of the study, (C) an HCC model engineered to knock out AXIN1, and (D) results at days 22 and 24. DETAILED DESCRIPTION OF THE INVENTION

[0014] Provided herein are methods for treating a specific subset of cancers, those cancers harboring one or more mutations in the AXIN1 and / or APC tumor suppressors of the WNT pathway, by administering a MEK inhibitor to a subject. This subset of cancers is clinically aggressive and often less sensitive to chemotherapy and / or immunotherapy treatment. As shown herein, treatment with a MEK inhibitor confers a favorable progression-free survival benefit in models harboring AXIN1 and / or APC mutations compared to models lacking AXIN1 and / or APC mutations. Thus, the present disclosure provides targeted treatments for this specific subset of cancers. Targeting treatments to this specific subset of cancers will facilitate optimal patient-specific treatment, resulting in maximal therapeutic benefit, prolonged survival, minimized treatment costs, and avoidance of potentially undesirable adverse effects from ineffective treatments.

[0015] The method includes administering a MEK inhibitor or a pharmaceutically acceptable salt thereof to a subject having one or more mutations in AXIN1 and / or APC. Thus, the present disclosure provides a MEK inhibitor or a pharmaceutically acceptable salt thereof for use in treating these subjects. It is hypothesized that MEK inhibition can restore the function of AXIN1 and / or APC and suppress the growth of WNT-driven mutant tumors. This is surprising, since it has previously been reported that MEK inhibition in colorectal cancer downregulates AXIN1, thus increasing rather than decreasing WNT signaling.

[0016] Treatment with a MEK inhibitor may be combined with one or more additional treatments, such as treatment with an immune checkpoint inhibitor. In this regard, it is hypothesized that MEK inhibition may sensitize AXIN1 and / or APC mutant tumors to treatment with immune checkpoint inhibitors, such as inhibitors of PD-1 and / or PD-L1. This may provide a treatment for cancers that have previously failed immune checkpoint inhibitor treatment and / or may allow the effective dose of immune checkpoint inhibitors to be reduced.

[0017] MEK inhibitors The term MEK refers to MAP kinase kinase / ERK kinase (MEK), which is part of the Raf / MEK / ERK kinase or RAS / MAPK signaling pathway. MEK phosphorylates and activates MAPK. MEK proteins are encoded by seven different genes, of which MEK1 and MEK2 are the most important. MEK inhibitors, as used herein, are understood to refer to inhibitors of MEK, i.e., any compound that downregulates, reduces, or stops MEK activity and / or function. MEK inhibitors for use in the present disclosure preferably inhibit MEK1 / 2 in a target. MEK inhibitors for use in the present disclosure may be dual inhibitors. In this case, the MEK inhibitor not only inhibits MEK, preferably MEK1 / 2, but also its upstream kinase (i.e., MAPKKK). MEK1 / 2 are MAPKKs in the Ras / Raf pathway, Ras / Raf acts as a MAPKKK, and ERK1 / 2 acts as a MAPK. An example of such a dual inhibitor for use in the present disclosure is PLX-4032. The term "MEK inhibitor," as used herein, is understood to include pharmaceutically acceptable salts thereof. The term "MEK inhibitor," as used herein, can refer to one MEK inhibitor or a combination of two or more MEK inhibitors.

[0018] The MEK inhibitor may be a MEK1 / 2 inhibitor or a pharmaceutically acceptable salt thereof. The MEK inhibitor may be an allosteric inhibitor. The MEK inhibitor may be a selective allosteric inhibitor of MEK1 and MEK2 (MEK1 / 2). The MEK inhibitor may be REC-4881 (TAK-733 or REC-2029 or REC-4881) or a pharmaceutically acceptable salt thereof. TAK-733 is an example of a selective allosteric MEK1 / 2 inhibitor. The MEK inhibitor may be selected from the group consisting of binimetinib (MEK162, ARRY-438162, ARRY-162), cobimetinib (GDC-0973, XL-518, RG7421), selumetinib (AZD6244, ARRY-142,886), trametinib (GSK1120212, JTP-74057), CI-1040, mirdametinib (PD0325901), RO5126766 (CH5126766), RO4987655 (CH4987655), refametinib (RDEA119, BAY869766) and pimasertib (MSC1936369, AS703026) or a pharmaceutically acceptable salt thereof. The MEK inhibitor may be selected from the group consisting of PD98059, PD184352 (2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide), AZD8330, RDEA-119 (BAY-869766), AS703026, and PLX-4032 (Zelboraf® (vemurafenib)) or a pharmaceutically acceptable salt thereof. The MEK inhibitor may be selected from the group consisting of REC-4881, binimetinib, cobimetinib, trametinib, and selumetinib or a pharmaceutically acceptable salt thereof. The MEK inhibitor may be binimetinib. The MEK inhibitor PD98059 inhibits activation of MEK by the kinase Raf. The MEK inhibitor R05126766 is a protein kinase inhibitor specific for Raf and MEK mitogen-activated protein kinases (MAPKs) with potential antineoplastic activity. The Raf / MEK dual kinase inhibitor R05126766 specifically inhibits the kinase activity of Raf and MEK, thereby inhibiting the transcription of target genes that promote malignant transformation of cells.The MEK inhibitor AS703026 is a highly selective and potent allosteric inhibitor of MEK1 / 2.

[0019] Cancers with one or more mutations in AXIN1 and / or APC (AXIN1 and / or APC mutant cancers) The terms "AXIN1 mutant cancer" and / or "APC mutant cancer" are understood to refer to a cancer in a subject in which the cells of the cancer contain one or more mutations in AXIN1 and / or APC. AXIN1 and APC are tumor suppressors in the WNT pathway. The one or more mutations can result in loss of AXIN1 and / or APC function. AXIN1 and APC mutant cancers form a subset of WNT-driven cancers. The one or more mutations can be mutations in AXIN1. The one or more mutations can be mutations in APC. The one or more mutations can be mutations in AXIN1 and APC. The one or more mutations can be somatic and / or germline mutations. These mutations can be in the AXIN1 and / or APC gene. The one or more mutations can be truncations. The cancer can further contain one or more mutations in the TP53 gene. These mutations can result in loss of TP53 function. The cancer can further contain one or more mutations in BRAF. Alternatively, the cancer may not contain a mutation in BRAF.

[0020] The cancer having the mutation as described above may be selected from the group consisting of hepatocellular carcinoma (HCC), colorectal cancer, liver cancer, bladder cancer, endometrial cancer, melanoma, ovarian cancer, lung cancer, pancreatic cancer, and gastric cancer. The cancer may be HCC, such as AXIN1-mutated HCC and / or APC-mutated HCC. The cancer may be ovarian cancer, such as AXIN1-mutated ovarian cancer and / or APC-mutated ovarian cancer, particularly epithelial ovarian cancer (EOC), especially endometrioid and mucinous subtypes of EOC. The cancer may be melanoma, such as AXIN1-mutated melanoma and / or APC-mutated melanoma. The cancer may exclude cancers with one or more BRAF mutations, such as BRAF-mutated melanoma, NSCLC, and NF1. The cancer may be colorectal cancer (CRC), such as AXIN1-mutated CRC and / or APC-mutated CRC. In particular, the cancer may be consensus molecular subtype 2 (CMS2) CRC. The cancer may be a cancer that does not have a mutation in the RAS / MAPK pathway. The cancer may be a cancer that has not responded to chemotherapy and / or immunotherapy treatment. The cancer may be a cancer that is resistant or refractory to chemotherapy and / or immunotherapy treatment.

[0021] The MEK inhibitor or a pharmaceutically acceptable salt thereof can be provided as first-line or second (or subsequent) line therapy. When the MEK inhibitor is used as second (or subsequent) line therapy, the cancer may be refractory (the previous line of therapy failed) or recurrent (the previous line of therapy initially responded but has shown reduced or no longer shown efficacy). For example, the MEK inhibitor or a pharmaceutically acceptable salt thereof can be provided as second-line therapy in a subject with cancer (e.g., HCC) after sorafenib treatment. In this manner, the subject may have been treated with sorafenib before treatment with the MEK inhibitor or a pharmaceutically acceptable salt thereof. The MEK inhibitor or a pharmaceutically acceptable salt thereof can be provided as second-line therapy in a subject with cancer (e.g., HCC) after lenvatinib treatment. In this manner, the subject may have been treated with lenvatinib before treatment with the MEK inhibitor or a pharmaceutically acceptable salt thereof. The subject may be a case in which first-line therapy failed.

[0022] The method may include evaluating a subject suffering from cancer to identify the genetic or epigenetic makeup of the subject's cancer cells. Specifically, tumors of the cancer subject are systematically investigated, and underlying somatic genetic alterations are identified in terms of sequence, expression, and copy number. Specifically, the method may include analyzing one or more tumors or cancer cells of the cancer subject for the presence of one or more mutations in AXIN1 and / or APC. Routine testing of the mutation status of AXIN1 and / or APC in cancer cells may predict a patient's response to treatment with a MEK inhibitor, thereby facilitating clinicians in determining the best treatment for the patient.

[0023] Combination therapy The treatment (i.e., the MEK inhibitor or a pharmaceutically acceptable salt thereof) may be combined with one or more additional cancer treatments. Thus, the method may further include administering the additional cancer treatment to the subject. The MEK inhibitor or a pharmaceutically acceptable salt thereof and the additional cancer treatment may be administered simultaneously, sequentially, or separately. The MEK inhibitor or a pharmaceutically acceptable salt thereof and the additional cancer treatment may be administered in combination. The MEK inhibitor or a pharmaceutically acceptable salt thereof may be administered simultaneously with, before, or after the additional cancer treatment. The additional treatment may be any other treatment suitable for treating cancer, such as chemotherapy, an immune checkpoint inhibitor, or another RAS / MAPK inhibitor. In certain embodiments, for example, when the cancer is melanoma, the additional treatment is not a RAF inhibitor.

[0024] In particular, the additional cancer treatment may be an immune checkpoint inhibitor, such as a PD-1 and / or PD-L1 inhibitor or a pharmaceutically acceptable salt thereof. It is hypothesized that MEK inhibition may sensitize AXIN1 and / or APC mutant tumors to treatment with immune checkpoint inhibitors, such as PD-1 and / or PD-L1 inhibitors. PD-L1 and PD-1 refer to programmed death-ligand 1 (PD-L1) and its receptor, programmed cell death protein 1 (PD-1), respectively. These are immune checkpoint proteins. PD-1 and PD-L1 inhibitors may serve to inhibit PD-L1 from engaging with its receptor, PD-1. Thus, the method may further include administering to the subject an immune checkpoint inhibitor, such as a PD-1 and / or PD-L1 inhibitor or a pharmaceutically acceptable salt thereof. The MEK inhibitor or a pharmaceutically acceptable salt thereof and the immune checkpoint inhibitor, such as a PD-1 and / or PD-L1 inhibitor, or a pharmaceutically acceptable salt thereof, can be administered simultaneously, sequentially, or separately. The MEK inhibitor or a pharmaceutically acceptable salt thereof and the immune checkpoint inhibitor, such as a PD-1 and / or PD-L1 inhibitor, or a pharmaceutically acceptable salt thereof, can be administered in combination. The MEK inhibitor or a pharmaceutically acceptable salt thereof can be administered simultaneously with, before, or after the immune checkpoint inhibitor, such as a PD-1 and / or PD-L1 inhibitor, or a pharmaceutically acceptable salt thereof. The MEK inhibitor can be a MEK1 / 2 inhibitor, such as REC-4881 (TAK-733) or a MEK1 / 2 inhibitor selected from those listed above. The PD-1 / PD-L1 inhibitor can be an antagonist antibody. The PD-1 / PD-L1 inhibitor can be an antagonist monoclonal antibody.The PD-1 / PD-L1 inhibitor may be selected from the group consisting of pembrolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Libtayo), atezolizumab, durvalumab, avelumab, emvafolimab, BMS-936559, CK-301, CS-1001, SHR-1316 (HTI-1088), CBT-502 (TQB-2450), and BGB-A333, or a pharmaceutically acceptable salt thereof. As used herein, the terms PD-1 inhibitor and PD-L1 inhibitor include pharmaceutically acceptable salts thereof. The immune checkpoint inhibitor may be an inhibitor of PD-1. The immune checkpoint inhibitor may be an inhibitor of PD-L1.

[0025] MEK inhibition may sensitize AXIN1 and / or APC mutant tumors to combination therapy with PD-1 or PD-L1 inhibitors. Thus, a MEK inhibitor and an immune checkpoint inhibitor, such as a PD-1 and / or PD-L1 inhibitor, or a pharmaceutically acceptable salt thereof, may exert a synergistic effect in the treatment of AXIN1 and / or APC mutant cancers that is greater than the additive effect of the MEK inhibitor and the immune checkpoint inhibitor, such as a PD-1 and / or PD-L1 inhibitor, or a pharmaceutically acceptable salt thereof, when administered individually. The therapeutically effective amount of the MEK inhibitor may be less than the amount required to treat AXIN1 and / or APC mutant cancers when the MEK inhibitor is administered without an immune checkpoint inhibitor, such as a PD-1 and / or PD-L1 inhibitor, or a pharmaceutically acceptable salt thereof. Similarly, a therapeutically effective amount of an immune checkpoint inhibitor, such as an inhibitor of PD-1 and / or PD-L1, or a pharmaceutically acceptable salt thereof, may be less than the amount required to treat cancer when the immune checkpoint inhibitor, such as an inhibitor of PD-1 and / or PD-L1, or a pharmaceutically acceptable salt thereof, is administered without the MEK inhibitor.

[0026] In certain embodiments, the additional cancer treatment may be a RAF inhibitor. The term "RAF inhibitor" or "Raf inhibitor" as used herein refers to an inhibitor of RAF kinase, i.e., any compound that downregulates, reduces, or stops RAF activity and / or function. The term "RAF inhibitor" as used herein is understood to include pharmaceutically acceptable salts thereof. The term "RAF inhibitor" as used herein may refer to one RAF inhibitor or a combination of two or more RAF inhibitors.

[0027] The RAF inhibitor can be any RAF inhibitor useful in the treatment of cancer. Examples of RAF inhibitors include AAL-881, ABM-1310, agerafenib, AP-23464, APL-102, ARQ-218, ARQ-680, ARQ-736, ARQ-761, ASN-003, AZ-304, AZ-628, B-Raf and c-RAF dual inhibitor (Redx Pharma), B-raf inhibitor (J&J), b-raf inhibitor (GSK), b-raf inhibitor (Sareum), b-raf kinase inhibitor (Kalypsys), B-Raf kinase inhibitor (Amgen), B-Raf kinase inhibitor (Array BioPharma), B-Raf kinase inhibitor (Array BioPharma-1), B-raf kinase inhibitor (Genentech), B-raf kinase inhibitor (Novartis), B-raf kinase inhibitor (Pfizer-2), B-Raf kinase inhibitor (Wyeth), BAL-3833, BDTX-4933, belbarafenib, BGB-3245, BI-882370, BIIB-024, BRAF inhibitor (Astex), BRAF kinase inhibitor (SelexagenTherapeutics), c-Raf kinase inhibitor (BridgeBio), dabrafenib, Debio-0928, donafenib, DP-2514, DP-2874, DP-4978, EBI-907, EN-3352, encorafenib, FNX-006, GDC-0879, iCo-007, IkT-064, ISIS-5132, KIN-2787, L-779450, LErafAON, LErafAON -ETU, lifirafenib, lifirafenib + mirdametinib, LUT-014, LYN-00204, MCP-110, MG-D-1509, MG-D-1609, N-5355, Nanolipolee-007, naporafenib, NC-1, NCB-0594, NCB-0846, NGN-101, NMS-P285, NMS-P730, ONC-101, pan-RAF kinase inhibitor (Ipsen), pazopanib, pazopanib Zopanib (SCAI), therapeutics, PF-04880594, PF-07284890, PLX-5568, PLX-8394, QLH-11906, Raf antagonist (Pfizer), RAF kinase inhibitor (Astex), Raf kinase inhibitor (Novartis), raf kinase inhibitor (Telik), RAF-265, RAF-709, RAF / VEGFR2 inhibitor (Takeda), Ras / Ra f / MEK / ERK inhibitor+PI3K / AKT / mTOR inhibitor (Celator), REDX-05358, regorafenib, RG-7256, RO-5126766, RO-7276389, RX-208, SAR-397769, SI-001, SJ-C1044, sorafenib, sorafenib beads (Biocompatibles), sorafenib (XSpray), STC-XXXX, substituted purines (Biogen Idec), TAK-632, tetrahydronaphthalene derivative compounds (Millennium), TL-241, toborafenib, UAI-201, UB-941, vemurafenib, VRN-14, WYE-130600, XL-281 and ZK-261991.

[0028] Pharmaceutical Composition To facilitate administration, MEK inhibitors are formulated in various embodiments into physiologically acceptable compositions containing carriers (e.g., vehicles, adjuvants, or diluents). The specific carriers used are limited only by physicochemical limitations, such as solubility and lack of reactivity with the MEK inhibitor, and by the route of administration. Physiologically acceptable carriers are well known in the art. Exemplary pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (see, e.g., U.S. Pat. No. 5,466,468). Injectable formulations are further described, for example, in Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982) and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). Pharmaceutical compositions comprising a MEK inhibitor are, in one aspect, packaged in a container with a package insert providing instructions for use of such pharmaceutical composition. Typically, such instructions include tangible language describing the concentrations of reagents and, in certain embodiments, the relative amounts of excipient components or diluents (e.g., water, saline, or PBS) that may be required to reconstitute the pharmaceutical composition.

[0029] Solid dosage forms for oral administration include capsules, tablets, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert conventional excipient (or carrier), such as sodium citrate or dicalcium phosphate, or (a) a filler or extender, such as starch, lactose, sucrose, mannitol, and silicic acid; (b) a binder, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) a humectant, such as glycerol; (d) a soluble fiber, such as agar, calcium carbonate, potato or tapioca starch, acacia, or the like; The formulation may be mixed with disintegrating agents such as glutaric acid, certain silicic acid complexes, and sodium carbonate; (a) solution retarders such as paraffin; (f) absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glycerol monostearate; (h) adsorbents such as kaolin and bentonite; and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules and tablets, dosage forms may also contain buffering agents. Solid compositions of a similar type may also be used as fillers in soft- and hard-filled gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols, etc.

[0030] Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other known in the art.Solid dosage forms can also contain opacifiers.In addition, solid dosage forms can be embedded compositions, so that these compositions release one or more active compounds in a delayed manner in a specific part of the intestinal tract.Examples of embedding compositions that can be used are polymeric substances and waxes.Active compounds can also be in microencapsulated form, optionally with one or more excipients.

[0031] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame seed oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan or mixtures of these substances.

[0032] Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and perfuming agents. Suspensions can contain, in addition to the active compounds, suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, or mixtures of these substances.

[0033] The compositions used in the methods disclosed herein can be formulated into micelles or liposomes. Such formulations include sterically stabilized micelles or liposomes and sterically stabilized mixed micelles or liposomes. Such formulations can facilitate intracellular delivery, as the lipid bilayer of liposomes and micelles is known to fuse with the plasma membrane of cells, delivering the entrapped contents to intracellular compartments.

[0034] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as injectable solutions, drug-release capsules, and the like. For parenteral administration in aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.

[0035] Single agent The MEK inhibitor can be administered as a single agent for the treatment of cancer as described herein. In this embodiment, no additional treatment for cancer, such as a RAF inhibitor, is administered. However, the treatment can still be administered as a pharmaceutical composition, generally containing a suitable pharmaceutically acceptable excipient, diluent or carrier. The MEK inhibitor can be administered as a single agent for the treatment of melanoma that harbors a mutation in the AXIN1 and / or APC gene. The melanoma can be one that does not have a mutation in the BRAF gene.

[0036] subject Typically, the terms "subject" and "patient" are used interchangeably herein. A subject is typically a mammal, more typically a human.

[0037] A subject suffering from cancer, for example, HCC, may have a TP53 mutation in addition to an AXIN1 and / or APC mutation. A subject suffering from cancer may have an APC mutation in addition to an AXIN1 mutation. A subject suffering from cancer may have a TP53 mutation and an APC mutation in addition to an AXIN1 mutation. A subject may have a BRAF mutation in addition to an AXIN1 and / or APC mutation. Alternatively, a subject may not have a BRAF mutation, for example, if the cancer is melanoma.

[0038] The subject may have failed previous cancer treatment, such as cabozantinib or an immune checkpoint inhibitor. Genetic alterations in AXIN1 have been shown to confer resistance to immune checkpoint inhibitors in small retrospective studies.

[0039] Dosage and Route of Administration The MEK inhibitor or its pharmaceutically acceptable salt may be administered in a therapeutically effective amount, which is an amount sufficient to demonstrate benefit to the subject to which the treatment is administered. Specifically, the MEK inhibitor or its pharmaceutically acceptable salt may be administered at a dose suitable to provide 60-80% average pERK inhibition in the subject. The MEK inhibitor or its pharmaceutically acceptable salt may be administered at a dose suitable to provide approximately 70% average pERK inhibition in the subject. The MEK inhibitor or its pharmaceutically acceptable salt may be administered at a dose suitable to provide greater than 70% average pERK inhibition in the subject. The MEK inhibitor or its pharmaceutically acceptable salt may be administered at a dose suitable to provide approximately 50% trough pERK inhibition in the subject. The MEK inhibitor or its pharmaceutically acceptable salt may be administered at a dose suitable to provide greater than 50% trough pERK inhibition in the subject. Suitable doses may range from 1 to 50 mg of MEK inhibitor per kg of subject body weight (mg / kg). In some embodiments, the dose may be 8-16 mg / kg of the MEK inhibitor.

[0040] In one embodiment, the MEK inhibitor is REC-4881 or a pharmaceutically acceptable salt thereof, which is administered at a dose suitable to provide greater than 70% mean pERK inhibition and about 50% trough pERK inhibition in a subject. A suitable dose may be in the range of 8-16 mg of REC-4881 per kg of subject body weight. This dose can achieve an average of greater than 70% inhibition and a trough inhibition level of about 50%.

[0041] The actual dose administered, and the rate and time course of administration, will depend on, and may be determined accordingly, by factors such as the nature and severity of the condition being treated and the age, sex, and weight of the subject being treated, as well as the route of administration. Furthermore, due consideration must be given to the characteristics of the treatment, such as its in vivo plasma lifetime and concentration in the formulation, and the route, site, and rate of delivery. Decisions regarding treatment prescription, e.g., dosage, etc., are ultimately within the responsibility and discretion of general practitioners and other physicians, and will typically take into account the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the treating physician.

[0042] Dosage regimens may include single or multiple doses. The treatments may also be administered simultaneously, sequentially or separately with other therapeutic agents and medications used in the treatment of cancer.

[0043] Therapy may be administered to a subject in need of treatment by any suitable route. In particular, the treatment may be administered systemically. The treatment may be administered orally or parenterally by injection or infusion. Examples of preferred parenteral administration routes include, but are not limited to, intravenous, intracardiac, intraarterial, intraperitoneal, intramuscular, intracavity, subcutaneous, transmucosal, inhalation, and transdermal. Routes of administration may further include enteral, e.g., mucosal (including pulmonary mucosa), and rectal. Therapy may be administered by nanoparticles, microspheres, liposomes, other particulate delivery systems deposited in specific tissues, including blood, or sustained-release formulations.

[0044] Predicting response to MEK inhibitor therapy The present disclosure further provides a method for assessing the likelihood that a MEK inhibitor or a pharmaceutically acceptable salt thereof will produce an anti-cancer effect in a subject suffering from cancer using one or more mutations in AXIN1 and / or APC tumor suppressors of the WNT pathway as biomarkers, the method comprising: assaying the subject for the presence of one or more mutations in AXIN1 and / or APC; and, if one or more mutations in AXIN1 and / or APC are present in the subject, administering a MEK inhibitor or a pharmaceutically acceptable salt thereof to the subject to produce an anti-cancer effect. The one or more mutations may be loss-of-function mutations. The anti-cancer effect may be any effect that is beneficial from treating the subject, including a reduction or inhibition of the progression, severity, and / or duration of cancer or at least one symptom thereof, including a curative, palliative, or preventive effect.

[0045] Accordingly, the present disclosure includes a MEK inhibitor, or a pharmaceutically acceptable salt thereof, for use in treating a cancer having one or more mutations in the AXIN1 and / or APC tumor suppressors of the WNT pathway in a subject in need thereof.

[0046] By systematically investigating the mutational status of cancer cells and / or tumors in cancer subjects, underlying somatic genetic alterations can be identified in terms of sequence, expression, and copy number, and subjects can be treated according to the genetic or epigenetic makeup of their cancer cells.

[0047] definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains.

[0048] As used herein, the term "treatment" and related terms such as "treat" and "treating" refer to the reduction or inhibition of the progression, severity, and / or duration of cancer or at least one symptom thereof. Thus, the term "treatment" refers to any regimen that may benefit a subject. Treatment may include a curative, palliative, or prophylactic effect.

[0049] It will be appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. [Example]

[0050] Example 1 – Efficacy of the MEK inhibitor REC-4881 compared to cabozantinib in the LI6612 AXIN1-mutated HCC PDX model Materials and Methods Tumor inoculation: The LI6612 PDX mouse model, a liver cancer model harboring an AXIN1 mutation, was developed at Crown Biosciences in China. Tumor fragments were collected from stock mice and used for inoculation. Each mouse was subcutaneously inoculated with a primary human tumor xenograft model LI6612 tumor fragment (2–3 mm in diameter) into the right upper flank, and tumors were allowed to develop.

[0051] Randomization: Randomization was performed on patients with a mean tumor size of approximately 170 mm 3 The study began when the rats reached 18 days of age. 50 NCG mice were enrolled in this study. All animals were randomly assigned to one of five study groups. Randomization was performed based on the "matched distribution" method. The randomization date was designated as day 0.

[0052] Tumor Growth Inhibition (TGI): The TGI method is known in the art. After randomization, tumor volumes were measured three times a week using calipers in two dimensions and the volume was expressed in mm using the formula: V = (L x W x W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). 3 The %TGI was calculated for all mice using the formula %TGI = (TV medium - TV treatment) / (TV medium - TV start) × 100 (Wong, H, et al. Clin Cancer Res., 2012, 18(14):3846-3855).

[0053] Treatment Arms: Ten mice per arm were treated for 21 days with either vehicle, cabozantinib, or REC-4881. Cabozantinib was administered PO QD at 10 mg / kg as a suspension, and REC-4881 was administered PO QD at 3 mg / kg as a suspension.

[0054] result The results are shown in Figure 1A (tumor volume) and Table 1 (tumor growth inhibition (TGI)). Figure 1A shows that treatment with REC-4881 at 3 mg / kg PO reduced tumor volume in the HCC AXIN1 mutant LI6612 PDX model, which was resistant to treatment with cabozantinib. Thus, MEK inhibition was superior to treatment with cabozantinib in the HCC AXIN1 mutant model.

[0055] [Table 1]

[0056] Example 2 – Efficacy of MEK inhibitor REC-4881 and four FDA-approved MEK inhibitors in the LI6612 AXIN1 mutant HCC PDX model Materials and Methods Tumor inoculation: The LI6612 PDX mouse model, a liver cancer model harboring an AXIN1 mutation, was developed at Crown Biosciences in China. Tumor fragments were collected from stock mice and used for inoculation. Each mouse was subcutaneously inoculated with a primary human tumor xenograft model LI6612 tumor fragment (2–3 mm in diameter) into the right upper flank, and tumors were allowed to develop.

[0057] Randomization: Randomization was performed on patients with a mean tumor size of approximately 170 mm 3 The study began when the animals reached 60 days of age. Sixty NCG mice were enrolled in this study. All animals were randomly assigned to 60 study groups. Randomization was performed based on the "matched distribution" method. The randomization date was designated as day 0.

[0058] Tumor Growth Inhibition (TGI): The TGI method is known in the art. After randomization, tumor volumes were measured three times a week using calipers in two dimensions and the volume was expressed in mm using the formula: V = (L x W x W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). 3 The %TGI was calculated for all mice using the formula %TGI = (TV medium - TV treatment) / (TV medium - TV start) × 100 (Wong, H, et al. Clin Cancer Res., 2012, 18(14):3846-3855).

[0059] Treatment arms: Ten mice per arm were treated for 15 days with either vehicle, binimetinib, cobimetinib, trametinib, selumetinib, or REC-4881. Note that FDA-approved MEK inhibitor doses may not reflect clinically relevant doses. For example, the dose of binimetinib used in this study was 30 mg / kg BID, whereas a clinically relevant dose of 2.5 mg / kg BID has been reported.

[0060] result The results are shown in Figure 2 (tumor volume) and Table 2 (tumor growth inhibition (TGI)). Figure 2 shows that tumor volume decreased with treatment with the MEK inhibitors REC-4881, binimetinib, cobimetinib, trametinib, and selumetinib.

[0061] [Table 2]

[0062] Example 3 - Efficacy of the MEK inhibitor REC-4881 alone and in combination with anti-PD-1 in a B16F10-ova melanoma syngeneic model (harboring an APC mutation) in female C57BL / 6 mice Materials and Methods Cell culture: B16F10-OVA tumor cells were maintained in vitro in RPMI 1640 supplemented with 10% fetal bovine serum (+1 μg / ml puromycin) in an atmosphere of 5% CO2 in air at 37°C. Cells were harvested during the exponential growth phase and quantified by cell counter before tumor inoculation.

[0063] Tumor inoculation: Each mouse received 2 x 10 B16F10-OVA tumor cells (2 x 10) in 0.1 ml of PBS in the right lower flank region. 5 ) was inoculated subcutaneously and tumors were allowed to develop.

[0064] Randomization: Randomization was performed on patients with a mean tumor size of 81 mm 3 The study began when the tumor volume reached 100 μg / kg. A total of 40 mice were randomly enrolled in the study and assigned to four groups of 10 mice per group. Randomization was performed using the "Matched distribution" method (Study Director™ software, version 3.1.399.19). Dose administration occurred immediately after randomization, on day 10, and 10 days after tumor inoculation.

[0065] Tumor Growth Inhibition (TGI): Methods for measuring TGI are known in the art. After randomization, tumor volumes were measured three times a week using calipers in two dimensions and the volume was expressed in mm using the formula: V = (L x W x W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). 3 The %TGI was calculated for all mice using the formula %TGI = (TV medium - TV treatment) / (TV medium - TV start) × 100 (Wong, H, et al. Clin Cancer Res., 2012, 18(14):3846-3855).

[0066] Treatment Arms: Ten mice per arm were treated with either anti-PD-1, REC-4881, or the combination of REC-4881 and anti-PD-1 for 13 days. REC-4881 was administered at 3 mg / kg PO QD for a total of 13 doses, while anti-PD-1 was administered at 10 mg / kg IP BIW for a total of 4 doses.

[0067] result The results are shown in Figure 3 (tumor volume) and Table 3 (tumor growth inhibition (TGI)). Figure 3 shows that tumor volume decreased with treatment with the MEK inhibitor REC-4881, anti-PD-1 alone, or the combination of both treatments.

[0068] [Table 3]

[0069] Example 4 - Efficacy of the MEK inhibitor REC-4881 in 19 HuPrime hepatocellular carcinoma xenograft models in NCG mice for AXIN1 mutations compared to non-AXIN1 mutations Materials and Methods Design: The study was conducted at Crown Biosciences in a PDX Mouse Clinical Trial (MCT) format as a 3x3x3 design.

[0070] Objective: The objective of this study was to evaluate the in vivo efficacy of REC-4881 in 6 AXIN1 mutant and 13 non-AXIN1 mutant HCC PDX models to determine whether there is an association between AXIN1 mutation status and treatment response with REC-4881.

[0071] Tumor inoculation: Tumor fragments were collected from stock mice and used for inoculation of mice. Primary human tumor xenograft model tumor fragments (2–3 mm in diameter) were subcutaneously inoculated into the right upper flank of each mouse, and tumors were allowed to develop.

[0072] Randomization: Randomization was performed on patients with an average tumor size of approximately 100–200 mm3 The study began when the rats reached 100 mg / kg / day. Six mice were enrolled in each model. All animals were randomly assigned to two study groups for each model. Randomization was performed based on the "matched distribution" method. The randomization date was designated as day 0.

[0073] Treatment arms: vehicle (n=3) or REC-4881 (n=3) administered at 3 mg / kg PO QD for up to 21 days.

[0074] Tumor Growth Inhibition (TGI): The TGI method is known in the art. After randomization, tumor volumes were measured three times a week using calipers in two dimensions and the volume was expressed in mm using the formula: V = (L x W x W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). 3 The %TGI was expressed in units. %TGI was calculated for all mice using the formula %TGI = (TV vehicle - TV treatment) / (TV vehicle - TV start) x 100 (Wong, H, et al. Clin Cancer Res., 2012, 18(14):3846-3855). TGI was calculated for all mice in all models on the last day of treatment. In both the vehicle and REC-4881 arms, if any mice died due to reaching the humane endpoint or before the completion of treatment (before 21 days), %TGI was calculated on the day all mice were alive to ensure normalization.

[0075] Data Analysis: Methods for analyzing mouse clinical trials (MCTs) and estimating progression-free survival (PFS) from large-scale xenograft studies are known in the art. MCTs were described as clustered longitudinal studies by analyzing the association between response and treatment using a linear mixed model (LMM), which explicitly models heterogeneity in growth and drug response across and within mouse models. PFS was defined as the tumor volume doubling time at any time point over time and was determined by linear interpolation of tumor growth data (Guo, S., et al. BMC Cancer., 2019, 19:718; Gao, H., et al. Nat Med., 2015, 21:1318-1325). Kaplan-Meier curves were calculated and generated using the Python programming language, lifelines, and significance was tested using the log-rank test with an alpha threshold of 0.05.

[0076] result The results are shown in Figure 4A (tumor growth inhibition rate (TGI)), Figure 4B (progression-free survival probability) and Table 4 (TGI). Figure 4B shows that treatment of HCC with REC-4881 resulted in a favorable PFS benefit in models harboring AXIN1 mutations compared with mice lacking AXIN1 mutations (AXIN1 wild-type).

[0077] [Table 4]

[0078] Example 5 – Efficacy of the MEK inhibitor REC-4881 in 10 HuPrime ovarian cancer xenograft models in NOD / SCID mice Materials and Methods Design: The study was conducted at Crown Biosciences in a PDX Mouse Clinical Trial (MCT) format as a 3x3x3 design.

[0079] Objective: The objective of this study was to evaluate the in vivo efficacy of REC-4881 in five AXIN1 and / or APC mutant and five non-AXIN1 and / or non-APC mutant models to determine whether there is an association between AXIN1 and / or APC mutation status and treatment response with REC-4881.

[0080] Tumor inoculation: Tumor fragments were collected from stock mice and used for inoculation of mice. Primary human tumor xenograft model tumor fragments (2–3 mm in diameter) were subcutaneously inoculated into the right upper flank of each mouse, and tumors were allowed to develop.

[0081] Randomization: Randomization was performed on patients with an average tumor size of approximately 100–200 mm 3 The study began when the rats reached 100 mg / kg / day. Six mice were enrolled in each model. All animals were randomly assigned to two study groups for each model. Randomization was performed based on the "matched distribution" method. The randomization date was designated as day 0.

[0082] Treatment arms: vehicle (n=3) or REC-4881 (n=3) administered at 3 mg / kg PO QD for up to 21 days.

[0083] Tumor Growth Inhibition (TGI): The TGI method is known in the art. After randomization, tumor volumes were measured three times a week using calipers in two dimensions and the volume was expressed in mm using the formula: V = (L x W x W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). 3The %TGI was expressed in units. %TGI was calculated for all mice using the formula %TGI = (TV vehicle - TV treatment) / (TV vehicle - TV start) x 100 (Wong, H, et al. Clin Cancer Res., 2012, 18(14):3846-3855). TGI was calculated for all mice in all models on the last day of treatment. In both the vehicle and REC-4881 arms, if any mice died due to reaching the humane endpoint or before the completion of treatment (before 21 days), %TGI was calculated on the day all mice were alive to ensure normalization.

[0084] Data Analysis: Methods for analyzing mouse clinical trials (MCTs) and estimating progression-free survival (PFS) from large-scale xenograft studies are known in the art. MCTs were described as clustered longitudinal studies by analyzing the association between response and treatment using a linear mixed model (LMM), which explicitly models heterogeneity in growth and drug response across and within mouse models. PFS was defined as the tumor volume doubling time at any time point over time and was determined by linear interpolation of tumor growth data (Guo, S., et al. BMC Cancer., 2019, 19:718; Gao, H., et al. Nat Med., 2015, 21:1318-1325). Kaplan-Meier curves were calculated and generated using the Python programming language, lifelines, and significance was tested using the log-rank test with an alpha threshold of 0.05.

[0085] result The results are shown in Figure 5A (tumor growth inhibition (TGI)), Figure 5B (progression-free survival probability) and Table 5 (TGI). Figure 5B shows that treatment of ovarian cancer with REC-4881 confers a favorable PFS benefit in models harboring AXIN1 and / or APC mutations compared to mice lacking AXIN1 and / or APC mutations (AXIN1 and / or APC wild-type).

[0086] [Table 5]

[0087] Example 6 - Combination Efficacy Analysis of MEK Inhibitor REC-4881 in 29 HuPrime PDX Mouse Models (11 AXIN1 and / or APC Mutants, 18 Non-AXIN1 and / or Non-APC Mutants) Materials and Methods Methods: Data sets from both HCC and ovarian cancer PCTs were combined into one %TGI waterfall plot, and the correlation between mutation status and response and PFS was analyzed as previously discussed (Examples 4 and 5, Figures 4 and 5).

[0088] result Figure 6B demonstrates that treatment with REC-4881 provides a more significant PFS benefit in models harboring AXIN1 and / or APC mutations when data from HCC (Figure 4C) and ovarian cancer (Figure 5C) mouse clinical trial studies are combined.

[0089] Example 7 – Efficacy and Pharmacodynamic Evaluation of the MEK Inhibitor REC-4881 in the LI6692 HCC PDX Model in NCG Mice Carrying an AXIN1 Mutation Materials and Methods Tumor inoculation: The LI6692 PDX model, from Crown Biosciences, China, is a liver cancer model harboring an AXIN1 mutation. Tumor fragments were harvested from stock mice and used for inoculation. Each mouse was subcutaneously inoculated with a primary human tumor xenograft model LI6692 tumor fragment (2–3 mm in diameter) into the right upper flank, and tumors were allowed to develop.

[0090] Randomization: Randomization was performed on patients with a mean tumor size of approximately 169 mm for efficacy studies. 3The study began when the serotonin concentration reached 100 mg / kg / day. Thirty-six mice were enrolled in the efficacy study and 23 in the pharmacodynamics (PD) study. All animals were randomly assigned to one of four study groups. Randomization was performed based on the "matched distribution" method. The randomization date was designated as day 0.

[0091] Efficacy Treatment Arms: Nine mice per arm were treated with either vehicle, 1 mg / kg REC-4881, 3 mg / kg REC-4881, or 30 mg / kg sorafenib for 21 days. Both sorafenib and REC-4881 were administered as suspensions.

[0092] Pharmacodynamic Treatment Arms: Five mice per arm were treated with 1 mg / kg REC-4881, 3 mg / kg REC-4881, or 30 mg / kg sorafenib for two days. Both sorafenib and REC-4881 were administered as suspensions. Eight mice were enrolled in the vehicle arm.

[0093] Tumor Growth Inhibition (TGI): The TGI method is known in the art. After randomization, tumor volumes were measured three times a week using calipers in two dimensions and the volume was expressed in mm using the formula: V = (L x W x W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). 3 The %TGI was calculated for all mice using the formula %TGI = (TV medium - TV treatment) / (TV medium - TV start) × 100 (Wong, H, et al. Clin Cancer Res., 2012, 18(14):3846-3855).

[0094] Pharmacodynamic Study Objectives: The objective of this PD portion was to evaluate protein expression ERK, pERK, MEK, and pMEK as assessed by Western blot and gene expression of DUSP6, SPRY4, and PPIA using SYBR green-based PCR in tumor samples of the LI6692 PDX model following compound treatment.

[0095] Western Blot Procedure: Load 1.50 μg / lane of each PDX tumor protein lysate or 20 μg / lane of each PBMC protein lysate and splenocyte lysate onto a precast gel (26-well, 4-15%, Bio-Rad Criterion TGX). 2. Apply a constant voltage (60 V) to the gel and stop the electrophoresis when the loading dye approaches the bottom edge. 3. The PVDF membrane is pre-activated with methanol for 2 minutes, and then the membrane and filter paper are pre-wetted with cold transfer buffer. 4. Assemble the filter paper-gel-membrane-filter paper "sandwich" and transfer the proteins at 280 mA for approximately 2 hours. 5. Once the transfer is complete, immerse the membrane in 5% milk in TBST and block the membrane for 1 hour at room temperature on a shaker. 6. Dilute the primary antibody in 5% milk in TBST to the recommended dilution as per the antibody datasheet, then incubate the membrane with the primary antibody overnight at 4°C with gentle shaking. 7. Wash the membrane with TBST 3 x 5 min. 8. Incubate the membrane with the appropriate secondary antibody diluted in 5% milk in TBST for 1 hour at room temperature on a nutator. 9. Wash membrane 3 x 5 min with TBST. 10. Detect the target protein using the Odyssey system or the Tanon 5200 chemiluminescence imaging system.

[0096] [Table 6]

[0097] qPCR procedure: Total RNA purification 1. Chop the tumor sample into small pieces (approximately 30 mg). 2. Add 350 μL Buffer RLT, then shock the sample tube mounted in the Tissue lyser II instrument at 30 shocks / second for 5 minutes before proceeding to the next step. 3. Add 1 volume of 70% ethanol to the lysate and mix thoroughly by pipetting. Do not centrifuge. 4. Transfer up to 700 μL of the sample, including any precipitate, to an RNeasy Mini spin column in a 2 mL collection tube (supplied). Close the lid and centrifuge at ≥13,200 rpm for 15 seconds. Discard the flow-through. 5. Add 350 μL Buffer RW1 to the RNeasy column. Close the lid and centrifuge at 13,200 rpm for 15 seconds. Discard the flow-through. 6. Add 10 μL DNase I stock solution to 70 μL Buffer RDD. Mix by gently inverting the tube and centrifuge briefly. 7. Add DNase I incubation mix (80 μL) directly to the RNeasy column membrane and place the tube on the benchtop (20–30°C) for 15 minutes. 8. Add 350 μL Buffer RW1 to the RNeasy column. Close the lid and centrifuge at 13,200 rpm for 15 seconds. Discard the flow-through. 9. Add 500 μL Buffer RPE to the spin column. Close the lid gently and centrifuge at 13,200 rpm for 2 minutes to wash the spin column membrane. Discard the flow-through. 10. Add 500 μL Buffer RPE to the spin column. Close the lid gently and centrifuge at 13,200 rpm for 2 minutes to wash the spin column membrane. 11. Place the RNeasy spin column into a new 1.5 mL collection tube (supplied). Add 50 μL RNase-free water directly to the center of the spin column membrane. Close the lid gently and centrifuge at full speed for 1 minute to elute the RNA.

[0098] RNA QC 1. Total RNA integrity was quantified using NanoDrop. Only high-quality RNA samples were used for future cDNA synthesis and qPCR assays.

[0099] result The results are shown in Figure 7 (tumor volume) and Table 8 (tumor growth inhibition (TGI)). These results demonstrate that treatment with the MEK inhibitor REC-4881 was non-inferior to treatment with sorafenib in mice with AXIN1-mutant HCC. Figure 8 illustrates pharmacodynamic markers from tumor samples collected in the in vivo PDX study (Figure 7), demonstrating a decrease in markers of pERK (Figure 8A), SPRY4 (Figure 8C), and DUSP6 (Figure 8D) after 2 days of treatment with REC-4881 administered PO at 1 mg / kg and 3 mg / kg, with no change in PPIA as a control (Figure 8E). This figure demonstrates the on-target and pathway engagement and pathway rebound reported for MEK inhibitors, with REC-4881 at 1 mg / kg and 3 mg / kg exhibiting an increase in pMEK after 2 days of treatment (Figure 8B).

[0100] [Table 8]

[0101] Example 9 – Pharmacokinetics of the MEK inhibitor REC-4881 after oral administration to female NCG mice Materials and Methods Design: Single doses of REC-4881 at 1 mg / kg and 3 mg / kg PO were administered to female NCG mice. Three mice per treatment arm. Plasma samples were collected at 0.5, 1, 2, 4, 8, 12, and 24 hours after dose administration.

[0102] Bioanalytical Assays: PK analysis methods are known in the art. Data are presented as mean ± SD per time point per treatment for each mouse.

[0103] result Figure 9 demonstrates that REC-4881 can achieve clinically meaningful and effective exposures.

[0104] Example 10 – MEK inhibition for the treatment of APC-mutated colorectal cancer Materials and Methods Human colorectal cancer cell lines HCT116, Colo-205, and HT29 were treated with 0.002, 0.02, 0.2, and 2 μM REC-4881 for 24 hours. NHCE cells were treated with 0.37, 1.11, 3.33, and 10 μM REC-4881 for 24 hours. Cells were washed to remove REC-4881 and medium, and cell viability was assayed using the CellTiter-Glo® 2.0 Cell Viability Assay (Promega; Madison, WI).

[0105] result

[0106] [Table 9]

[0107] As shown in Figure 10, the MEK inhibitor REC-4881 preferentially reduced the viability of APC mutant colorectal cancer cells (HT-29, SW48, and COLO-205) compared to normal human colon epithelium (NHCE) or APC wild-type colorectal cancer cells (HCT-116), demonstrating greater selectivity than selumetinib (see Table 9). These data demonstrate that the selectivity range of REC-4881 is significantly greater than 1000-fold, potentially enabling its use in targeting colorectal cancer cells harboring APC mutations. In addition, REC-4881 exhibited significantly greater potency and selectivity than selumetinib in APC mutant cells (see Table 9).

[0108] Example 11 – REC-4881 regulates genes downstream of β-catenin and other disease-related pathways in CRC Materials and Methods Human colorectal cancer cell lines NHCE, HCT116, Colo-205, and HT29 were treated with REC-4881 at 0.002, 0.02, 0.2, and 2 μM for 24 hours. NHCE cells were treated with REC-4881 at 0.37, 1.11, 3.33, and 10 μM for 24 hours. Cells were washed to remove REC-4881 and medium, and RNA was extracted. RNA was isolated, and MYC (Figure 11A) and CDKN2A (Figure 11B) transcripts were amplified and measured using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Amplification of glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase 1, glucuronidase β, tyrosine 3-monooxygenase / tryptophan 5-monooxygenase-activating protein ζ, and TATA box-binding protein transcripts was used as an internal standard for the amount of starting RNA.

[0109] result As shown in Figure 11A, REC-4881 dose-dependently increased CDKN2A in all colonic epithelial cells tested. CDKN2A gene expression was most pronounced in COLO-205 and HT-29 APC mutant cell lines. As shown in Figure 11B, 24-hour treatment of normal human colonic epithelium (NHCE), APC wild-type colorectal cancer cells (HCT-116), and APC mutant colorectal cancer cells (HT-29 and COLO-205) with REC-4881 resulted in a dose-dependent decrease in MYC expression. REC-4881-treated HT-29 cells showed the greatest decrease in MYC expression compared with the other CRC cell lines tested. It is hypothesized that the specific context of APC and AXIN1 mutant cancers leads to an increase in cell cycle genes and MYC genes, and therefore MEK inhibition may selectively reduce the activity of these downstream targets.

[0110] Example 12 –REC-4881 alone or in combination with anti-PD-1 in an APC-mutated B16F10-ova syngeneic melanoma mouse model Using an APC-mutated B16F10-ova syngeneic melanoma mouse model, REC-4881 was administered alone or in combination with an anti-PD1 agent to female C57BL / 6 mice. Tumor samples were collected after 13 days of treatment and CD8 + T cells and T regulatory cells were assessed. Methods: Immune cell profiling by flow cytometry. Tumor tissues were collected 4 hours after the last dose. N=3 pools of 3 mice per group.

[0111] Mice treated with REC-4881 alone or with anti-PD1 agents showed increased CD8 + We observed an increase in T cells and a decrease in T regulatory cells (Figures 12A and 12B, respectively). These data demonstrate that REC-4881 has the ability to modulate clinically relevant immune cell markers either as a single agent or in combination with anti-PD1.

[0112] Example 13 –REC-4881 alone or in combination with anti-PD-1 in the Hepa1-6 syngeneic hepatocellular carcinoma (HCC) model Cell culture: Hepa1-6 AXIN1-KO CL#1 tumor cells were maintained in vitro in Dulbecco's modified Eagle's medium (DMEM), 10% non-heat-inactivated fetal bovine serum (FBS), and 1% penicillin / streptomycin / L-glutamine (PSG). Tumor inoculation: Each mouse was inoculated with AXIN1-null tumor cells (5 × 10) in 0.2 ml of PBS into the right upper flank. 6 ) was inoculated subcutaneously and tumors were allowed to develop. Randomization: All mice were assigned to study groups based on caliper estimates of tumor burden. Mice were distributed to ensure that the mean tumor burden in all groups was within 10% of the mean tumor burden in the overall study population. Tumor growth inhibition (TGI): The TGI method is known in the art. After randomization, tumor volumes were measured three times weekly using calipers in two dimensions, and the volume was expressed in mm using the formula: V = (L × W × W) / 2, where V is tumor volume, L is tumor length (the longest tumor dimension), and W is tumor width (the longest tumor dimension perpendicular to L). %TGI was calculated for all mice using the formula %TGI = (TV vehicle - TV treatment) / (TV vehicle - TV start) × 100 (Wong, H, et al. Clin Cancer Res., 2012, 18(14):3846-3855). Treatment arms: 10 mice per arm were treated for 19 days with either anti-PD-1, REC-4881, bevacizumab, or a combination of REC-4881 + anti-PD-1 or bevacizumab + anti-PD-1. REC-4881 was administered at 1 mg / kg or 3 mg / kg PO QD for a total of 19 doses, anti-PD-1 was administered at 10 mg / kg IP BIW for a total of 6 doses, and bevacizumab was administered at 10 mg / kg IP BIW for a total of 6 doses. Western Blot: Western blotting is known in the art. Primary antibodies: Axin (C76H11) rabbit mAb #2087 and GAPDH (D16H11) XP® rabbit mAb #5174, diluted 1:1000 in blocking buffer. Secondary antibodies: anti-rabbit IgG, HRP-conjugated antibody #7074, diluted 1:5000 in blocking buffer. ECL image 5 minute exposure. Expected MW: Axin-1 110 kDa, GAPDH 37 kDa.

[0113] REC-4881 was administered orally at 1 mg / kg and 3 mg / kg as a single agent or in combination with anti-PD-1 at 10 mg / kg IP in a Hepa1-6 syngeneic liver model (Figures 13A and 13B), which was engineered to knockout AXIN1 (Figure 13C). This figure demonstrates that REC-4881 has potent single-agent activity and can produce a more rapid response when combined with anti-PD-1 in this model (Figure 13D).

Claims

1. 1. A method of treating a cancer having one or more mutations in axin1 and / or APC in a subject in need thereof, comprising: administering to said subject a therapeutically effective amount of a MEK inhibitor, or a pharmaceutically acceptable salt thereof, to treat said cancer. A method comprising:

2. 2. The method of claim 1, wherein the MEK inhibitor is selected from the group consisting of REC-4881, binimetinib, cobimetinib, selumetinib, and trametinib, or a pharmaceutically acceptable salt thereof.

3. The method of claim 2, wherein the MEK inhibitor is REC-4881 or a pharmaceutically acceptable salt thereof.

4. The method according to any one of claims 1 to 3, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, colorectal cancer, liver cancer, bladder cancer, endometrial cancer, melanoma, ovarian cancer, lung cancer, pancreatic cancer and gastric cancer.

5. The method of claim 4, wherein the cancer is hepatocellular carcinoma.

6. The method of claim 4 , wherein the cancer is ovarian cancer.

7. 5. The method of claim 4, wherein the cancer is melanoma.

8. 5. The method of claim 4, wherein the cancer is colorectal cancer.

9. 9. The method of claim 8, wherein the colorectal cancer is classified as a CMS2 colorectal cancer.

10. The method of any one of claims 1 to 9, further comprising assessing the subject suffering from cancer for the presence of one or more mutations in axin1 and / or APC.

11. The method of any one of claims 1 to 10, further comprising administering to the subject one or more additional treatments for the cancer.

12. 12. The method of claim 11, wherein the one or more additional therapies comprises treatment with an immune checkpoint inhibitor.

13. The method of claim 12, wherein the immune checkpoint inhibitor is a PD-1 and / or PD-L1 inhibitor or a pharmaceutically acceptable salt thereof.

14. The method of claim 13, wherein the MEK inhibitor is REC-4881 or a pharmaceutically acceptable salt thereof.

15. 12. The method of claim 11, wherein the one or more additional therapies comprises treatment with a RAF inhibitor.

16. 16. The method of any one of claims 1 to 15, wherein the MEK inhibitor or a pharmaceutically acceptable salt thereof is provided as a second-line treatment, the subject having previously received first-line treatment with an immune checkpoint inhibitor for the cancer, and the subject has failed the treatment with the immune checkpoint inhibitor.

17. The method of any one of claims 1 to 16, wherein the cancer is recurrent or refractory.

18. The method of any one of claims 1 to 17, wherein the MEK inhibitor or a pharmaceutically acceptable salt thereof is administered as a single agent.

18. 19. The method of any one of claims 1 to 18, wherein the MEK inhibitor, or a pharmaceutically acceptable salt thereof, is administered to the subject at a dose suitable to provide a mean pERK inhibition of greater than 70% and a trough pERK inhibition of about 50% in the subject.

19. 20. The method of claim 18, wherein the MEK inhibitor is administered in the range of 8-16 mg of MEK inhibitor per kg of the subject's body weight.

20. 1. A method for assessing the likelihood that a MEK inhibitor, or a pharmaceutically acceptable salt thereof, will produce an anti-cancer effect in a subject suffering from cancer, using one or more mutations in axin1 and / or APC as biomarkers, comprising: - assaying said subject for the presence of one or more mutations in axin1 and / or axin1 receptor; - if one or more mutations in axin1 and / or APC are present in said subject, administering to said subject a MEK inhibitor or a pharmaceutically acceptable salt thereof to produce said anti-cancer effect. A method comprising:

Images