Treatment of cancer with mutations in WNT pathway tumor suppressor genes

Abstract

There is provided a method of treating a cancer having one or more mutations in the tumor suppressor gene AXIN1 and / or APC of the WNT pathway in a subject in need thereof, wherein the method comprises administering to the subject an MEK inhibitor or a pharmaceutically acceptable salt thereof. The MEK inhibitor may be administered as a single agent, or may also be combined with treatment with an immune checkpoint inhibitor, such as a PD-1 and / or PD-L1 inhibitor, or a RAF inhibitor.

Description

Technical Field

[0001] The present disclosure generally relates to the use of mutations in predicting a patient's response to antiproliferative agents, particularly MEK inhibitors. In particular, the presence or absence of mutations in the WNT pathway, particularly in the tumor suppressor genes AXIN1 and / or APC, can be used to predict the response of patients with cancer to treatment with MEK inhibitors (as a single agent or in combination with other RAS / MAPK pathway inhibitors and / or immune checkpoint inhibitors). Therefore, the present disclosure provides a method for treating a specific subclass of cancer patients, wherein a MEK inhibitor is used to identify the patient as having a mutation in the tumor suppressor genes AXIN1 and / or APC of the WNT pathway. This treatment can be a single agent, or it can also be combined with other RAS / MAPK pathway inhibitors and / or immune checkpoint inhibitors (such as PD-1 and / or PD-L1 inhibitors). Background Art

[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 through gain- or loss-of-function mutations appears to be common in many 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. In particular, β-catenin levels are kept low by a multisubunit destruction complex composed of the tumor suppressor genes AXIN1 and adenomatous polyposis coli (APC), as well as the kinases casein kinase 1 (CK1) and glycogen synthase kinase 3β (GSK3β). Inactivating mutations in AXIN1 and APC are frequently observed among 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 carrying AXIN1 and APC loss-of-function mutations are often clinically aggressive and less sensitive to treatment with chemotherapy and / or immunotherapy. For example, from a retrospective analysis, patients with AXIN1 mutant advanced hepatocellular carcinoma (HCC) treated with a combination of atezolizumab and bevacizumab had a poorer response to treatment than non-AXIN1 mutant groups (Zhu, AX et al., Nat Med. [Natural Medicine], 2022, 28: 1599–1611). Therefore, there is a great need for the development of therapeutic agents that can overcome mutation-induced WNT activation, especially for the treatment of AXIN1 and APC mutant cancers, because these mutations are generally considered to have no available drugs (Parsons, MJ et al., Cancer Discov. [Cancer Discovery], 2021, 11 (10): 2413-2429; Bugter, JM et al., Nat Rev Cancer. [Natural Review: 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. Early HCC patients can be successfully treated with surgical resection or liver transplantation. However, the usual late diagnosis of HCC excludes curative treatment, and systemic therapy is the only viable option for patients who cannot undergo surgery. Possible treatments for advanced HCC at present are sorafenib, lenvatinib, a combination of atezolizumab and bevacizumab, regorafenib, ramucirumab, cabozantinib, nivolumab, and pembrolizumab. Sorafenib is an oral multikinase inhibitor approved as a systemic therapy for the treatment of advanced HCC patients. It has been shown that the benefits provided by sorafenib are limited, and new drugs are being developed to overcome resistance to sorafenib and improve patient prognosis, for example, c-MET inhibitor cabozantinib. However, the severity of HCC, the lack of good diagnostic markers and treatment strategies, and clinical heterogeneity make the management of the disease a major challenge. Further difficulties arise because of the long median time to response to first-line checkpoint therapy (approximately 12 weeks), the median overall survival benefit of <6 months, and the limited treatment options for patients classified as Child Pugh B / C.

[0004] The clinical course of HCC patients is highly variable, suggesting that HCC includes several biologically distinct subgroups. The telomerase reverse transcriptase (TERT) promoter has been found to be mutated in more than 50% of HCC tissue samples examined, making it 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 more than 30% of HCC cases. Catenin β1 (CTNNB1) is another of the most frequently mutated genes in HCC, and abnormal activation of β-catenin has been observed in 20%-30% of HCC patients. About 40% of HCC patients carry mutations in the WNT pathway. AXIN1 is the second most frequently mutated gene in this pathway. AXIN1 loss is seen in approximately 11% of HCC patients and is thought to confer intrinsic resistance to immune checkpoint blockade. Despite belonging to the same pathway, genetic alterations in CTNNB1 and AXIN1 were 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. Aberrant activation of the WNT pathway in EOC leads to excessive activity of β-catenin.

[0006] Colorectal cancer (CRC) is one of the most common types of cancer in the world. CRC has recently been further classified into various molecular subtypes, called consensus molecular subtypes (CMS). The CMS classification is not currently used to drive treatment decisions, but is used to distinguish the biological, clinical, and molecular characteristics of CRC, so that clinicians can understand the heterogeneity of CRC. For example, regarding CMS subtypes, CMS2 is called classic and is marked by WNT and MYC signaling activation. It was found that APC mutant CRC tumors are more predominantly concentrated in CMS2 CRCs and often carry co-occurring KRAS and TP53 changes (Bugter, JM et al., Nat Rev Cancer. [Nature Review: Cancer], 2021, 5-21).

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

[0008] Currently, the therapeutic use of MEK inhibitors for cancer is limited to the treatment of BRAF mutant populations including melanoma and non-small cell lung cancer (NSCLC), where treatment is combined with RAF inhibitors. MEK inhibitors are only administered to patients carrying BRAF V600E mutations in combination with RAF inhibitors rather than as single agents, because the use of MEK inhibitors in these patients produces pathway-specific rebound. In addition, the MEK inhibitor selumetinib is also approved for the genetic indication of neurofibromatosis type 1 (NF1). However, the use of MEK inhibitors alone or in combination for genetically defined tumors has not been formally studied, except for cancers carrying RAS / MAPK pathway alterations. In fact, in CRC, MEK inhibition has been shown to activate WNT signaling genetically and pharmacologically, as indicated by increased AXIN2 expression levels after siRNA KD in the APC WT CRC cell line HCT116. However, in colony formation assays, single-agent trametinib was able to reduce the viability of HCT116 cells in a dose-dependent manner (Zhan et al., Nat Comm [Nature Communications], 2019), suggesting that pharmacological induction of WNT activation is not sufficient to drive resistance. A separate group reported that single-agent MEK inhibition itself did not significantly alter AXIN2 expression levels or WNT activity in the more relevant APC mutant CRC cell line COLO320 (Solberg et al., Cancers [Cancer], 2019). Fully elucidating the context specificity between WNT signaling and RAS / MAPK pathway inhibitors may expand the therapeutic use of MEK inhibitors. Summary of the invention

[0009] According to a first aspect, there is provided a method of treating a cancer having 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, a method is provided for using one or more mutations in AXIN1 and / or APC as biomarkers to evaluate the likelihood that a MEK inhibitor or a pharmaceutically acceptable salt thereof will produce an anti-cancer effect in a subject suffering from cancer, the method comprising determining the presence of one or more mutations in AXIN1 and / or APC in the subject; 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 the anti-cancer effect.

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

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

[0013] Figure 1 Shows tumor volume results for treatment with the MEK inhibitor REC-4881 compared to cabozantinib in the HCC AXIN1 mutant LI6612 PDX model;

[0014] Figure 2 Presents tumor volume results in the HCC AXIN1 mutant LI6612 PDX model treated with the MEK inhibitor REC-4881 and four FDA-approved MEK inhibitors;

[0015] Figure 3 Shown are tumor volumes in the B16F10-ova syngeneic melanoma model carrying an APC mutation following 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;

[0016] Figure 4A Tumor growth inhibition (TGI) was shown, and Figure 4BProbability of progression-free survival (PFS) results using the MEK inhibitor REC-4881 at 3 mg / kg PO across 19 HCC PDX models conducted as a mouse clinical trial, 6 of which carried AXIN1 mutations and 13 of which did not;

[0017] Figure 5A Tumor growth inhibition (TGI) was shown, and Figure 5B Probability of progression-free survival (PFS) outcomes using the MEK inhibitor REC-4881 at 3 mg / kg PO between 10 ovarian PDX models conducted as a mouse clinical trial, of which 5 models carried AXIN1 and / or APC mutations and 5 models did not carry AXIN1 and / or APC mutations;

[0018] Fig. 6A Tumor growth inhibition (TGI) was shown, and Figure 6B Probability of progression-free survival (PFS) results using 3 mg / kg PO of the MEK inhibitor REC-4881 between a combination study of 19 HCC PDX models and 10 ovarian PDX models conducted as a mouse clinical trial;

[0019] Figure 7 Shown are 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 carrying an AXIN1 mutation;

[0020] FIG8 shows pharmacodynamic markers of tumor samples harvested from the in vivo PDX study of Example 7, Fig. 8A Targeting pERK / ERK; Figure 8B Targeting pMEK / MEK; Figure 8C Targeting SPRY4; Fig.8D Targeting DUSP6; Fig. 8E For PPIA;

[0021] Fig. 9 Pharmacokinetic data for non-tumor bearing NCG mice administered 1 mg / kg and 3 mg / kg of REC-4881 are presented;

[0022] Fig.10 shows the viability curves of human colorectal cancer cell lines (APC mutant and wild type) treated with the MEK inhibitor REC-4881 for 72 hours; and

[0023] Fig.11A shows that REC-4881 regulates CDKN2A expression in APC mutant and wild-type colorectal cancer cell lines, while Fig.11 B shows that REC-4881 regulates the expression of MYC.

[0024] Fig.12 CD8 + Role of T cells (A) and Tregs (B).

[0025] Figure 13 shows the effect of REC-4881 alone or in combination with anti-PD1 agents in AXIN1-less Hepa 1-6 mouse hepatocellular carcinoma (HCC) tumors in female C57BL / 6 mice, (A) dosing schedule; (B) tumor volume measured for each group studied; (C) shows that the HCC model was engineered to knock out AXIN1; (D) results on days 22 and 24. DETAILED DESCRIPTION

[0026] Provided herein is a method for treating a specific cancer subclass by administering a MEK inhibitor to a subject, the specific cancer subclass being a cancer having one or more mutations in the AXIN1 and / or APC tumor suppressor genes of the WNT pathway. This cancer subclass is often clinically aggressive and is less sensitive to treatment using chemotherapy and / or immunotherapy. As shown herein, treatment with a MEK inhibitor produces a favorable progression-free survival benefit for models carrying AXIN1 and / or APC mutations compared to models without AXIN1 and / or APC mutations. Therefore, the present disclosure provides targeted therapy for this specific cancer subclass. Targeted therapy for this specific cancer subclass will help produce the best patient-customized treatment to maximize treatment response, prolong survival, minimize treatment costs, and avoid potential adverse reactions that are not desired for ineffective therapies.

[0027] 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. Therefore, the present disclosure provides a MEK inhibitor or a pharmaceutically acceptable salt thereof for treating these subjects. It is speculated that MEK inhibition can restore the function of AXIN1 and / or APC and attenuate the growth of WNT-driven mutant tumors. This is surprising because it has been previously reported that MEK inhibition in colorectal cancer downregulates AXIN1, thereby increasing rather than decreasing WNT signaling.

[0028] Treatment with a MEK inhibitor can 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 tumors with AXIN1 and / or APC mutations to treatment with immune checkpoint inhibitors, such as inhibitors of PD-1 and / or PD-L1. This may provide treatment for cancers that were previously unresponsive to treatment with immune checkpoint inhibitors and / or may allow for lower effective doses of immune checkpoint inhibitors. MEK inhibitors

[0029] 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 protein is encoded by seven different genes, of which MEK1 and MEK2 are the most important. MEK inhibitors are understood herein to refer to inhibitors of MEK, i.e., any compound that downregulates, reduces or terminates MEK activity and / or function. MEK inhibitors for use in the present disclosure preferably inhibit MEK1 / 2 of the subject. MEK inhibitors for use in the present disclosure may be dual inhibitors-in this case, MEK inhibitors can not only inhibit MEK, preferably MEK1 / 2, but also inhibit its upstream kinase (i.e., MAPKKK). MEK1 / 2 is a MAPKK in the Ras / Raf pathway, whereby Ras / Raf acts as MAPKKK, and ERK1 / 2 acts as MAPK. An example of such a dual inhibitor for use in the present disclosure is PLX-4032. As used herein, the term "MEK inhibitor" is understood to encompass pharmaceutically acceptable salts thereof. As used herein, the term "MEK inhibitor" may refer to one MEK inhibitor, or a combination of two or more MEK inhibitors.

[0030] The MEK inhibitor can be a MEK1 / 2 inhibitor or a pharmaceutically acceptable salt thereof. The MEK inhibitor can be an allosteric inhibitor. The MEK inhibitor can be a selective allosteric inhibitor of MEK1 and MEK2 (MEK1 / 2). The MEK inhibitor can 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 can be selected from the following group: bimetinib (MEK162, ARRY-438162, ARRY-162), cobimetinib (GDC-0973, XL-518, RG7421), selumetinib (AZD6244, ARRY-142,886), trametinib (GSK1120212, JTP-74057), CI-1040, mitradatinib (PD0325901), RO5126766 (CH5126766), RO4987655 (CH4987655), refactinib (RDEA119, BAY 869766) and pimastatin (MSC1936369, AS703026), or a pharmaceutically acceptable salt thereof. The MEK inhibitor can 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 ( (vemurafenib)), or a pharmaceutically acceptable salt thereof. The MEK inhibitor may be selected from the group consisting of REC-4881, bimetinib, cobimetinib, trametinib, and selumetinib, or a pharmaceutically acceptable salt thereof. The MEK inhibitor may be bimetinib. The MEK inhibitor PD98059 inhibits the activation of MEK by the kinase Raf. The MEK inhibitor R05126766 is a protein kinase inhibitor specific for Raf and MEK mitogen-activated protein kinase (MAPK), with potential anti-tumor 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 MEK1 / 2 allosteric inhibitor. Cancers with one or more mutations in AXIN1 and / or APC (AXIN1 and / or APC mutated cancers)

[0031] The term "AXIN1 mutated cancer" and / or "APC mutated cancer" is understood to refer to a cancer in a subject in which cells of the cancer contain one or more mutations in AXIN1 and / or APC. AXIN1 and APC are tumor suppressor genes in the WNT pathway. One or more mutations may result in loss of function of AXIN1 and / or APC. Cancers in which AXIN1 and APC are mutated form a subclass of WNT-driven cancers. The one or more mutations may be mutations in AXIN1. The one or more mutations may be mutations in APC. The one or more mutations may be mutations in AXIN1 and APC. The one or more mutations may be somatic mutations and / or germline mutations. The mutations may be in the AXIN1 and / or APC genes. The one or more mutations may be truncations. The cancer may further comprise one or more mutations present in the TP53 gene. These mutations may result in loss of function of TP53. The cancer may further comprise one or more mutations in BRAF. Alternatively, the cancer may not comprise a mutation in BRAF.

[0032] Cancers with mutations 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. Cancer may be HCC, for example, HCC with AXIN1 mutations and / or HCC with APC mutations. Cancer may be ovarian cancer, for example, ovarian cancer with AXIN1 mutations and / or ovarian cancer with APC mutations, particularly epithelial ovarian cancer (EOC), particularly endometrioid and mucinous subtypes of EOC. Cancer may be melanoma, for example, melanoma with AXIN1 mutations and / or melanoma with APC mutations. Cancer may exclude cancers with one or more BRAF mutations, for example, melanoma, NSCLC, and NF1 with BRAF mutations. Cancer may be colorectal cancer (CRC), for example, CRC with AXIN1 mutations and / or CRC with APC mutations. In particular, cancer may be consensus molecular subtype 2 (CMS2) CRC. Cancer may be a cancer without mutations in the RAS / MAPK pathway. The cancer may be a cancer that is unresponsive to treatment with chemotherapy and / or immunotherapy. The cancer may be a cancer that is resistant or refractory to treatment with chemotherapy and / or immunotherapy.

[0033] MEK inhibitors or pharmaceutically acceptable salts thereof can be provided as first-line or second (or subsequent) line treatments. When a MEK inhibitor is a second (or subsequent) line treatment, the cancer may be refractory (no response to the previous therapy line) or recurrent (initial response to the previous therapy line, but showing reduced efficacy or no efficacy). For example, a MEK inhibitor or a pharmaceutically acceptable salt thereof can be provided as a second-line treatment for a subject with cancer (e.g., HCC) after sorafenib treatment. As such, the subject may have been treated with sorafenib before being treated with a MEK inhibitor or a pharmaceutically acceptable salt thereof. A MEK inhibitor or a pharmaceutically acceptable salt thereof can be provided as a second-line treatment for a subject with cancer (e.g., HCC) after lenvatinib treatment. As such, the subject may have been treated with lenvatinib before being treated with a MEK inhibitor or a pharmaceutically acceptable salt thereof. The subject may have been unresponsive to first-line treatment.

[0034] The method may include evaluating a subject with cancer to identify the genetic or epigenetic makeup of the subject's cancer cells. In particular, the tumor of a cancer subject may be systematically investigated to identify potential somatic genetic changes in sequence, expression, and copy number. In particular, the method may include analyzing the presence of one or more mutations in AXIN1 and / or APC of one or more tumors or cancer cells of a cancer subject. The mutation status of AXIN1 and / or APC in cancer cells may be routinely tested to predict the patient's response to treatment with a MEK inhibitor, and thereby help clinicians make decisions on the best treatment for the patient. Combination therapy

[0035] Treatment (i.e., MEK inhibitor or a pharmaceutically acceptable salt thereof) can be combined with one or more additional cancer treatments. As such, the method may further include the step of administering additional cancer treatments to the subject. MEK inhibitor or a pharmaceutically acceptable salt thereof can be administered simultaneously, sequentially or separately with additional cancer treatments. MEK inhibitor or a pharmaceutically acceptable salt thereof and additional cancer treatments can be administered in combination. MEK inhibitor or a pharmaceutically acceptable salt thereof can be administered before, after or concurrently with additional cancer treatments. Additional treatment can be any other treatment suitable for treating cancer, for example, chemotherapy, immune checkpoint inhibitors or other RAS / MAPK inhibitors. In certain embodiments, additional treatment is not a RAF inhibitor, for example, when the cancer is a melanoma.

[0036] In particular, additional cancer treatments may be immune checkpoint inhibitors (such as inhibitors of PD-1 and / or PD-L1 or pharmaceutically acceptable salts thereof). It is speculated that MEK inhibition may sensitize tumors with AXIN1 and / or APC mutations to treatment with immune checkpoint inhibitors (such as inhibitors of PD-1 and / or PD-L1). 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 can act to inhibit the association of PD-L1 with its receptor PD-1. Therefore, the method may further include administering immune checkpoint inhibitors such as inhibitors of PD-1 and / or PD-L1 or pharmaceutically acceptable salts thereof to the subject. MEK inhibitors or pharmaceutically acceptable salts thereof may be administered simultaneously, sequentially or separately with immune checkpoint inhibitors such as inhibitors of PD-1 and / or PD-L1 or pharmaceutically acceptable salts thereof. A MEK inhibitor or a pharmaceutically acceptable salt thereof can be administered in combination with an immune checkpoint inhibitor such as an inhibitor of PD-1 and / or PD-L1 or a pharmaceutically acceptable salt thereof. A MEK inhibitor or a pharmaceutically acceptable salt thereof can be administered before, after or concurrently with an immune checkpoint inhibitor such as an inhibitor of PD-1 and / or PD-L1 or a pharmaceutically acceptable salt thereof. The MEK inhibitor can be a MEK1 / 2 inhibitor, for example, 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. PD-1 / PD-L1 inhibitors can be selected from the group consisting of pembrolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Libtayo), atezolizumab, durvalumab, avelumab, envoli monoclonal antibody, BMS-936559, CK-301, CS-1001, SHR-1316 (HTI-1088), CBT-502 (TQB-2450), and BGB-A333, or pharmaceutically acceptable salts thereof. As used herein, the terms PD-1 inhibitor and PD-L1 inhibitor include pharmaceutically acceptable salts thereof. Immune checkpoint inhibitors can be inhibitors of PD-1. Immune checkpoint inhibitors can be inhibitors of PD-L1.

[0037] MEK inhibition can sensitize tumors with AXIN1 and / or APC mutations to combined treatment with inhibitors of PD-1 or PD-L1. As such, MEK inhibitors and immune checkpoint inhibitors such as PD-1 and / or PD-L1 inhibitors or pharmaceutically acceptable salts thereof can have a synergistic effect in the treatment of cancers with AXIN1 and / or APC mutations that is greater than the additive effect of each of the MEK inhibitor and immune checkpoint inhibitors such as PD-1 and / or PD-L1 inhibitors or pharmaceutically acceptable salts thereof when administered alone. If the MEK inhibitor is administered without an immune checkpoint inhibitor such as an inhibitor of PD-1 and / or PD-L1 or a pharmaceutically acceptable salt thereof, the therapeutically effective amount of the MEK inhibitor may be less than the amount required to treat cancers with AXIN1 and / or APC mutations. Similarly, if an immune checkpoint inhibitor, such as an inhibitor of PD-1 and / or PD-L1, or a pharmaceutically acceptable salt thereof, is administered without a MEK inhibitor, the therapeutically effective amount of the 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.

[0038] In certain embodiments, the additional cancer treatment may be a RAF inhibitor. As used herein, the term "RAF inhibitor" or "Raf inhibitor" refers to an inhibitor of RAF kinase, i.e., any compound that downregulates, reduces or terminates RAF activity and / or function. As used herein, the term "RAF inhibitor" is understood to encompass pharmaceutically acceptable salts thereof. As used herein, the term "RAF inhibitor" may refer to a RAF inhibitor, or a combination of two or more RAF inhibitors.

[0039] The RAF inhibitor can be any RAF inhibitor that can be used for cancer treatment. The RAF inhibitor can be selected from the following group: 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 (Johnson & Johnson (J&J)), b-raf inhibitor (GlaxoSmithKline (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), BioPharma-1), B-raf kinase inhibitors (Genentech), B-Raf kinase inhibitors (Novartis), B-raf kinase inhibitors (Pfizer-2), B-Raf kinase inhibitors (Wyeth), BAL-3833, BDTX-4933, belvarafenib, BGB-3245, BI-882370, BIIB-024, BRAF inhibitors (Astex), BRAF kinase inhibitors (Selexagen Therapeutics), c-Raf kinase inhibitors (BridgeBio), dabrafenib, Debio-0928, donafenib, DP-2514, DP-2874, DP-4978 , EBI-907, EN-3352, conafenib, FNX-006, GDC-0879, iCo-007, IkT-064, ISIS-5132, KIN-2787, L-779450, LErafAON, LErafAON-ETU, lifilafenib, lifilafenib + mitatinib, LUT-014, LYN-00204, MCP-110, MG-D-1509, MG-D-160 9. 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 (SCAI), herapeutics, 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 / Raf / MEK / ERK inhibitor + PI3 K / AKT / mTOR inhibitors (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, tetralin-derived compounds (Millennium), TL-241, tovorafenib, UAI-201, UB-941, vemurafenib, VRN-14, WYE-130600, XL-281, and ZK-261991. Pharmaceutical composition

[0040] For ease of administration, in many aspects, MEK inhibitors are formulated into physiologically acceptable compositions comprising a carrier (e.g., a vehicle, an adjuvant, or a diluent). The specific carrier employed is limited only by physical-chemical considerations (such as solubility and lack of reactivity with MEK inhibitors) and by the route of administration. Physiologically acceptable carriers are well known in the art. Illustrative pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (e.g., see U.S. Patent No. 5,466,468). Injectable formulations are further described in, for example, Pharmaceutics and Pharmacy Practice, JB Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pp. 238-250 (1982) and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pp. 622-630 (1986). In one aspect, a pharmaceutical composition comprising a MEK inhibitor is placed in a container together with packaging material providing instructions for the use of such a pharmaceutical composition. Typically, such instructions include a tangible expression describing the relative amounts of excipient components or diluents (e.g., water, saline or PBS) that may be required to reconstitute the pharmaceutical composition in certain embodiments.

[0041] 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) fillers or extenders, for example, starches, lactose, sucrose, mannitol, and silicic acid; (b) binders, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (a) solution retarding agents, for example, paraffin; (f) absorption promoters, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin and bentonite; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules and tablets, the dosage form may also contain buffering agents.Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar and high molecular weight polyethylene glycols.

[0042] Tablets, lozenges, capsules, pills, and solid dosage forms of granules can be prepared with coatings and shells (such as enteric coatings and other coatings known in the art). Solid dosage forms may also contain opacifiers. In addition, solid dosage forms may be embedded compositions so that they release one or more active compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedded compositions that can be used are polymeric substances and waxes. The active compound may also be in a microencapsulated form optionally with one or more excipients.

[0043] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame seed oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, or mixtures of these substances, etc.

[0044] In addition to such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatics. In addition to the active compound, the suspension may also contain 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.

[0045] The compositions used in the methods disclosed herein can be formulated in 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 it is known that the lipid bilayer of liposomes and micelles fuse with the plasma membrane of cells and deliver the embedded contents to intracellular compartments.

[0046] After formulation, the solution will be administered in a manner compatible with the dosage formulation and in, for example, a therapeutically effective amount. The formulation is easily administered in a variety of dosage forms (e.g., injectable solutions, drug release capsules, etc.). For parenteral administration in aqueous solutions, for example, the aqueous solution should be appropriately buffered if necessary and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Single drug

[0047] MEK inhibitors can be administered as a single agent for treating cancer, as described herein. In this embodiment, no additional cancer treatment, such as RAF inhibitors, is administered. However, the treatment can still be administered as a pharmaceutical composition, which will generally contain a suitable pharmaceutically acceptable excipient, diluent or carrier. MEK inhibitors can be administered as a single agent for treating melanomas carrying mutations in the AXIN1 and / or APC genes. Melanoma may not contain mutations in the BRAF gene. Subjects

[0048] Typically, the terms "subject" and "patient" are used interchangeably herein. A "subject" is typically a mammal, more typically a human.

[0049] In addition to AXIN1 and / or APC mutations, a subject with cancer (e.g., HCC) may also have a TP53 mutation. In addition to AXIN1 mutations, a subject with cancer may also have an APC mutation. In addition to AXIN1 mutations, a subject with cancer may also have a TP53 mutation and an APC mutation. In addition to AXIN1 and / or APC mutations, a subject may also have a BRAF mutation. Alternatively, a subject may not have a BRAF mutation, for example, in the case where the cancer is melanoma.

[0050] The subject may not have responded to prior cancer treatment (e.g., prior treatment with cabozantinib or with an immune checkpoint inhibitor).In small retrospective studies, genetic alterations in AXIN1 have been shown to confer resistance to immune checkpoint inhibitors. Dosage and route of administration

[0051] The MEK inhibitor or its pharmaceutically acceptable salt may be administered in a therapeutically effective amount, which is an amount sufficient to show a benefit to the subject to which the treatment is administered. In particular, the MEK inhibitor or its pharmaceutically acceptable salt may be administered at a suitable dose to provide an average pERK inhibition of 60%-80% in the subject. The MEK inhibitor or its pharmaceutically acceptable salt may be administered at a suitable dose to provide an average pERK inhibition of about 70% in the subject. The MEK inhibitor or its pharmaceutically acceptable salt may be administered at a suitable dose to provide an average pERK inhibition of greater than 70% in the subject. The MEK inhibitor or its pharmaceutically acceptable salt may be administered at a suitable dose to provide an inhibition of about 50% of the valley pERK in the subject. The MEK inhibitor or its pharmaceutically acceptable salt may be administered at a suitable dose to provide an inhibition of greater than 50% of the valley pERK in the subject. A suitable dose may be in the range of 1-50 mg MEK inhibitor / kg subject body weight (mg / kg). In some embodiments, the dose may be 8-16 mg / kg MEK inhibitor.

[0052] In one embodiment, the MEK inhibitor is REC-4881 or a pharmaceutically acceptable salt thereof, and is administered at a suitable dose to provide an average pERK inhibition of greater than 70% and a trough pERK inhibition of about 50% in a subject. A suitable dose may be in the range of 8-16 mg REC-4881 / kg subject body weight. This dose allows for an average inhibition level of >70% and a trough inhibition level of about 50%.

[0053] The dose actually administered, as well as the rate and time course of administration, will depend on the nature and severity of the condition being treated, as well as factors such as the age, sex and weight of the subject being treated, and the route of administration, and may be determined with appropriate reference to the above. The nature of the treatment, for example, its in vivo plasma lifetime and concentration in the formulation, as well as the route of delivery, site and rate should further be appropriately considered. Treatment prescriptions, such as decisions about dosage, are ultimately within the responsibility of general practitioners and other physicians and are at their discretion, and 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 practicing physicians.

[0054] The dosage regimen may include a single administration or multiple administration doses.The treatment may further be administered simultaneously, sequentially or separately with other therapies and drugs used to treat cancer.

[0055] Treatment can be administered to a subject in need of treatment via any suitable route. In particular, treatment can be administered systemically. Treatment can be administered orally or parenterally by injection or infusion. Examples of preferred routes for parenteral administration include, but are not limited to, intravenous, intracardiac, intraarterial, intraperitoneal, intramuscular, intracavitary, subcutaneous, transmucosal, inhalation, and transdermal administration. The route of administration may further include enteral administration, such as mucosal (including lungs) and rectal administration. Treatment may be administered via nanoparticles, microspheres, liposomes, other microparticle delivery systems, or sustained-release formulations placed in certain tissues (including blood). Predicting response to therapy with MEK inhibitors

[0056] The present disclosure further provides a method for evaluating the possibility that a MEK inhibitor or a pharmaceutically acceptable salt thereof will produce an anti-cancer effect in a subject with cancer using one or more mutations in the AXIN1 and / or APC tumor suppressor genes of the WNT pathway as a biomarker, the method comprising determining the presence of one or more mutations in AXIN1 and / or APC in the subject; 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 the 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 for treating the subject. This includes reducing or inhibiting the progression, severity and / or duration of cancer or at least one symptom thereof, and includes curative, palliative or preventive effects.

[0057] Thus, 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 suppressor genes of the WNT pathway in a subject in need thereof.

[0058] The mutational status of cancer cells and / or tumors in a cancer subject can be systematically surveyed to identify underlying somatic genetic changes in sequence, expression, and copy number, and the subject can be treated based on the genetic or epigenetic makeup of the cancer cells. definition

[0059] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of this invention.

[0060] As used herein, the term "treatment" and related terms such as "treat" and "treating" mean reducing or inhibiting the progression, severity, and / or duration of a cancer or at least one symptom thereof. Thus, the term "treatment" refers to any regimen that can benefit a subject. Treatment can include curative, palliative, or preventive effects.

[0061] It should be understood that, for clarity, certain features of the present invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for simplicity, different features of the present invention described in the context of a single embodiment may also be provided individually or in any suitable sub-combination. Examples Example 1 - Efficacy of MEK Inhibitor REC-4881 Compared with Cabozantinib in the LI6612 AXIN1 Mutant HCC PDX Model Materials and Methods

[0062] Tumor Inoculation: The LI6612 PDX mouse model is a liver cancer model that carries an AXIN1 mutation and is managed by Crown Biosciences in China. Tumor fragments from stock mice were harvested and used for inoculation into mice. Primary human tumor xenograft model LI6612 tumor fragments (2-3 mm in diameter) were subcutaneously inoculated into the right upper flank of each mouse for tumor development.

[0063] Randomization: Randomization starts at a mean tumor size of approximately 170 mm 3 The study started at 14:00 p.m. 50 NCG mice were enrolled in the study. All animals were randomly assigned to 5 study groups. Randomization was performed based on the "matched distribution" method. The day of randomization was denoted as day 0.

[0064] Tumor Growth Inhibition (TGI): Methods for TGI are known in the art. Tumor volume was measured in two dimensions using calipers three times per week after randomization and expressed in mm using the following formula: 3 Volume is expressed as: V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L). %TGI for all mice was calculated using the formula %TGI = (TV vehicle - TV treatment) / (TV vehicle - TV initial) * 100 (Wong, H et al., Clin Cancer Res., 2012, 18(14): 3846-3855).

[0065] Treatment Groups: 10 mice per group were treated with vehicle, cabozantinib, or REC-4881 for 21 days. Cabozantinib was administered as a suspension at 10 mg / kg PO QD, and REC-4881 was administered as a suspension at 3 mg / kg PO QD. result

[0066] The results are shown in Figure 1 A (tumor volume) and Table 1 (tumor growth inhibition (TGI)). Figure 1 A shows that tumor volume was reduced with REC-4881 at 3 mg / kg PO in the HCC AXIN1 mutant LI6612 PDX model and was resistant to treatment with cabozantinib. Thus, MEK inhibition was superior to treatment with cabozantinib in the HCC AXIN1 mutant model. Table 1 - Tumor Growth Inhibition PO-oral, QD-once daily; n-number of subjects; TGI-tumor growth inhibition Example 2 - Efficacy of MEK Inhibitor REC-4881 and 4 FDA-Approved MEK Inhibitors in the LI6612 AXIN1 Mutant HCC PDX Model Materials and Methods

[0067] Tumor Inoculation: The LI6612 PDX mouse model is a liver cancer model that carries an AXIN1 mutation and is managed by Crown Biopharmaceuticals in China. Tumor fragments from stock mice were harvested and used for inoculation into mice. Primary human tumor xenograft model LI6612 tumor fragments (2-3 mm in diameter) were subcutaneously inoculated into the right upper flank of each mouse for tumor development.

[0068] Randomization: Randomization starts at a mean tumor size of approximately 170 mm 3 The study started at 1:00 p.m. The study enrolled 60 NCG mice. All animals were randomly assigned to 60 study groups. Random assignment was based on the "matched distribution" method. The day of random assignment was denoted as day 0.

[0069] Tumor Growth Inhibition (TGI): Methods for TGI are known in the art. Tumor volume was measured in two dimensions using calipers three times per week after randomization and expressed in mm using the following formula: 3Volume is expressed as: V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L). %TGI for all mice was calculated using the formula %TGI = (TV vehicle - TV treatment) / (TV vehicle - TV initial) * 100 (Wong, H et al., Clin Cancer Res., 2012, 18(14): 3846-3855).

[0070] Treatment Groups: Ten mice per group were treated with vehicle, bimetinib, cobimetinib, trametinib, selumetinib, or REC-4881 for 15 days. Note that doses of FDA-approved MEK inhibitors may not reflect clinically relevant doses. For example, the dose of bimetinib used in this study was 30 mg / kg BID, while the clinically relevant dose is reported as 2.5 mg / kg BID. result

[0071] The results are shown in Figure 2 (tumor volume) and Table 2 (tumor growth inhibition (TGI)). Figure 2 Shown is a reduction in tumor volume with treatment with the MEK inhibitors REC-4881, binimetinib, cobimetinib, trametinib, and selumetinib. Table 2 PO - oral, BID - twice daily; QD - once daily; n - number of subjects; TGI - tumor growth inhibition Example 3 - Efficacy of the MEK inhibitor REC-4881 as a single agent and in combination with anti-PD-1 in a B16F10-ova melanoma syngeneic model (with APC mutation) in female C57BL / 6 mice Materials and Methods

[0072] Cell culture: B16F10-OVA tumor cells were maintained in vitro in RPMI1640 (+1 ug / ml puromycin) supplemented with 10% fetal bovine serum in an atmosphere of 5% CO2 in air at 37°C. Cells in the exponential growth phase were harvested and quantified by cell counter before tumor inoculation.

[0073] Tumor inoculation: Each mouse was subcutaneously inoculated with B16F10-OVA tumor cells (2×10 5 ), for tumor development.

[0074] Randomization: Randomization started from a mean tumor size of 81 mm 3A total of 40 mice were randomly enrolled in the study and assigned to 4 groups of 10 mice each. Random assignment was performed based on the "matched distribution" method (Study Director TM software, version 3.1.399.19). Dosing was performed immediately on day 10 after random assignment (10 days after tumor inoculation).

[0075] Tumor Growth Inhibition (TGI): Methods for measuring TGI are known in the art. Tumor volume was measured in two dimensions using calipers three times per week after randomization and expressed in mm using the following formula: 3 Volume is expressed as: V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L). %TGI for all mice was calculated using the formula %TGI = (TV vehicle - TV treatment) / (TV vehicle - TV initial) * 100 (Wong, H et al., Clin Cancer Res., 2012, 18(14): 3846-3855).

[0076] Treatment groups: 10 mice per group were treated with anti-PD-1, REC-4881, or a combination of REC-4881 + 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. result

[0077] The results are shown in Figure 3 (tumor volume) and Table 3 (tumor growth inhibition (TGI)). Figure 3 Tumor volume reduction is shown with treatment with the MEK inhibitor REC-4881, anti-PD-1, or a combination of both treatments. Table 3 PO - oral, IP - injection; BID - twice daily; QD - once daily; n - number of subjects; TGI - tumor growth inhibition Example 4 - Efficacy of MEK Inhibitor REC-4881 in 19 HuPrime Hepatocellular Carcinoma Xenograft Models in AXIN1 Mutated vs. Non-AXIN1 Mutated NCG Mice Materials and Methods

[0078] Design: This study was conducted at Crown Biopharmaceuticals in a 3×3×3 design as a PDX mouse clinical trial (MCT).

[0079] Objective: The objective of this study was to evaluate the in vivo efficacy of REC-4881 in 6 AXIN1 mutant HCC PDX models and 13 non-AXIN1 mutant HCC PDX models to determine if an association between AXIN1 mutation status and treatment response with REC-4881 is observed.

[0080] Tumor Inoculation: Tumor fragments from stock mice were harvested and used for inoculation into mice. Primary human tumor xenograft model tumor fragments (2-3 mm in diameter) were inoculated subcutaneously in the right upper flank of each mouse for tumor development.

[0081] Randomization: Randomization starts from a mean tumor size of approximately 100-200 mm 3 The study started at 1:00 p.m. 6 mice were enrolled in each model. All animals were randomly assigned to 2 study groups per model. Random assignment was performed based on the "matched distribution" method. The day of random assignment was denoted as day 0.

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

[0083] Tumor Growth Inhibition (TGI): Methods for TGI are known in the art. Tumor volume was measured in two dimensions using calipers three times per week after randomization and expressed in mm using the following formula: 3 The volume is expressed as: V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L). The %TGI of all mice was calculated using the formula %TGI = (TV vehicle-TV treatment) / (TV vehicle-TV initial) * 100. (Wong, H et al., Clin Cancer Res. [Clinical Cancer Research], 2012, 18 (14): 3846-3855). The TGI of all mice between all models was calculated on the last day of treatment. If any mouse between the vehicle and REC-4881 groups died before 21 days due to reaching the humane endpoint or before the completion of treatment, the %TGI was calculated on the day when all mice were alive to ensure standardization.

[0084] Data analysis: Methods for analyzing mouse clinical trials (MCT) and estimating progression-free survival (PFS) from large xenograft studies are known in the art. MCT is described as a clustered longitudinal study using a linear mixed model (LMM) to analyze the association between response and treatment, which clearly models the heterogeneity of growth and drug response between mouse models and between mice within a mouse model. PFS is defined as the tumor volume doubling time at any time point and is obtained by linear interpolation of tumor growth data. (Guo, S. et al., BMC Cancer. [BMC Cancer], 2019, 19: 718; Gao, H. et al., Nat Med. [Natural Medicine], 2015, 21: 1318-1325). The calculated Kaplan-Meier curve is generated with Python programming language and lifelines, and the log-rank test with an alpha threshold of 0.05 is used to test significance. result

[0085] The results are shown in Figure 4A (Tumor Growth Inhibition (TGI)), Figure 4B (probability of progression-free survival) and Table 4 (TGI). Figure 4B It was shown that treatment of HCC with REC-4881 resulted in a favorable PFS benefit for models carrying AXIN1 mutations compared to mice without AXIN1 mutations (AXIN1 wild-type). Table 4 TVVehicle – tumor volume in the vehicle group; TVTreatment – ​​tumor volume in the treatment group; TVInitial – tumor volume at the start of the study; TGI – tumor growth inhibition Example 5 - Efficacy of MEK Inhibitor REC-4881 in 10 HuPrime Ovarian Cancer Xenograft Models in NOD / SCID Mice Materials and Methods

[0086] Design: This study was conducted at Crown Biopharmaceuticals in a 3×3×3 design as a PDX mouse clinical trial (MCT).

[0087] Objective: The objective of this study was to evaluate the in vivo efficacy of REC-4881 in 5 AXIN1 and / or APC mutant models and 5 non-AXIN1 and / or non-APC mutant models to determine if an association between AXIN1 and / or APC mutation status and treatment response with REC-4881 is observed.

[0088] Tumor Inoculation: Tumor fragments from stock mice were harvested and used for inoculation into mice. Primary human tumor xenograft model tumor fragments (2-3 mm in diameter) were inoculated subcutaneously in the right upper flank of each mouse for tumor development.

[0089] Randomization: Randomization starts from a mean tumor size of approximately 100-200 mm 3 The study started at 1:00 p.m. 6 mice were enrolled in each model. All animals were randomly assigned to 2 study groups per model. Random assignment was performed based on the "matched distribution" method. The day of random assignment was denoted as day 0.

[0090] Treatment groups: Vehicle (n=3) or REC-4881 (n=3) was administered at 3 mg / kg PO QD for up to 21 days.

[0091] Tumor Growth Inhibition (TGI): Methods for TGI are known in the art. Tumor volume was measured in two dimensions using calipers three times per week after randomization and expressed in mm using the following formula: 3 The volume is expressed as: V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L). The %TGI of all mice was calculated using the formula %TGI = (TV vehicle-TV treatment) / (TV vehicle-TV initial) * 100. (Wong, H et al., Clin Cancer Res. [Clinical Cancer Research], 2012, 18 (14): 3846-3855). The TGI of all mice between all models was calculated on the last day of treatment. If any mouse between the vehicle and REC-4881 groups died before 21 days due to reaching the humane endpoint or before the completion of treatment, the %TGI was calculated on the day when all mice were alive to ensure standardization.

[0092] Data analysis: It is known in the art to analyze mouse clinical trials (MCT) and to estimate progression-free survival (PFS) from large xenograft studies. MCT is described as a clustered longitudinal study using a linear mixed model (LMM) to analyze the association between response and treatment, which clearly models the heterogeneity of growth and drug response between mouse models and between mice within a mouse model. PFS is defined as the tumor volume doubling time at any time point, and is obtained by linear interpolation of tumor growth data ((Guo, S. et al., BMC Cancer. [BMC Cancer], 2019, 19: 718. Gao, H. et al., Nat Med. [Natural Medicine], 2015, 21: 1318-1325). The calculated Kaplan-Meier curve is generated with Python programming language and lifelines, and the log-rank test with an alpha threshold of 0.05 is used to test significance. result

[0093] The results are shown in Figure 5A (Tumor Growth Inhibition (TGI)), Figure 5B (Probability of progression-free survival) and Table 5 (TGI). Figure 5B It was shown that treatment of ovarian cancer with REC-4881 resulted in a favorable PFS benefit for models carrying AXIN1 and / or APC mutations compared to mice without AXIN1 and / or APC mutations (AXIN1 and / or APC wild type). Table 5 TVVehicle – tumor volume in the vehicle group; TVTreatment – ​​tumor volume in the treatment group; TVInitial – tumor volume at the start of the study; TGI – tumor growth inhibition Example 6-29 HuPrime PDX Mouse Models (11 AXIN1 and / or APC Mutants, 18 Non-AXIN1 and / or Non-APC Mutants) Combination Efficacy Analysis of MEK Inhibitor REC-4881 Materials and Methods

[0094] Methods: Data sets from HCC and ovarian cancer PCT were combined into one %TGI waterfall plot, where association of mutation status with response and PFS were analyzed similarly to previous discussions (Examples 4 and 5, Figures 4 and 5). result

[0095] Figure 6BIt was demonstrated that when data from HCC ( FIG. 4C ) and ovarian ( FIG. 5C ) mouse clinical trial studies were combined, treatment with REC-4881 resulted in a more significant PFS benefit in models carrying AXIN1 and / or APC mutations. Example 7 - Efficacy and Pharmacodynamic Evaluation of MEK Inhibitor REC-4881 in the LI6692 HCC PDX Model of NCG Mice with AXIN1 Mutation Materials and Methods

[0096] Tumor Inoculation: The LI6692 PDX model is a liver cancer model carrying an AXIN1 mutation and is from Crown Biotech in China. Tumor fragments from stock mice were harvested and used for inoculation into mice. Primary human tumor xenograft model LI6692 tumor fragments (2-3 mm in diameter) were subcutaneously inoculated in the right upper flank of each mouse for tumor development.

[0097] Randomization: Randomization started from a mean tumor size of approximately 169 mm 3 0 for efficacy studies. 36 mice were enrolled for efficacy studies and 23 mice were enrolled for pharmacodynamic (PD) studies. All animals were randomly assigned to 4 study groups. Random assignment was performed based on the "matching distribution" method. The day of random assignment was denoted as day 0.

[0098] Efficacy treatment groups: 9 mice per group were treated with vehicle, 1 mg / kg of REC-4881, 3 mg / kg of REC-4881, or 30 mg / kg of sorafenib for 21 days. Both sorafenib and REC-4881 were administered as suspensions.

[0099] Pharmacodynamics Treatment Groups: 5 mice per group were treated with 1 mg / kg of REC-4881, 3 mg / kg of REC-4881, or 30 mg / kg of sorafenib for 2 days. Both sorafenib and REC-4881 were administered as suspensions. 8 mice were enrolled in the vehicle group.

[0100] Tumor Growth Inhibition (TGI): Methods for TGI are known in the art. Tumor volume was measured in two dimensions using calipers three times per week after randomization and expressed in mm using the following formula: 3Volume is expressed as: V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L). %TGI for all mice was calculated using the formula %TGI = (TV vehicle - TV treatment) / (TV vehicle - TV initial) * 100 (Wong, H et al., Clin Cancer Res., 2012, 18(14): 3846-3855).

[0101] Pharmacodynamic Study Objectives: The objectives of the PD part are to evaluate the protein expression ERK, pERK, MEK and pMEK as assessed by Western blot, and the gene expression of DUSP6, SPRY4 and PPIA using SYBR green-based qPCR in tumor samples of the LI6692 PDX model after compound treatment.

[0102] Western blotting procedure: 1. Load 50 μg / lane of each PDX tumor protein lysate or 20 μg / lane of each PBMC protein lysate and spleen cell lysate onto precast gels (26-well, 4%-15%, Bio-Rad Criterion TGX). 2. Run the gel at a constant voltage (60 V) and stop when the loading dye approaches the lower edge. 3. Pre-activate the PVDF membrane in methanol for 2 min, then pre-wet the membrane and filter paper in cold transfer buffer. 4. Assemble the filter paper-gel-membrane-filter paper "sandwich" and perform protein transfer at 280 mA for about 2 h. 5. After the transfer was completed, the membrane was immersed in TBST with 5% milk and blocked on a shaker at RT for 1 h. 6. Dilute the primary antibody in TBST with 5% milk at the recommended dilution according to the antibody datasheet, and incubate the membrane with the primary antibody overnight at 4°C with gentle shaking. 7. Wash the membrane 3 x 5 min in TBST. 8. Incubate the membrane with appropriate secondary antibodies diluted in TBST with 5% milk for 1 h at RT on a nutator. 9. Wash the membrane 3 x 5 min in TBST. 10. Detect target protein using Odyssey system or Tanon 5200 chemiluminescent image analysis system. Table 6. Primary antibodies against 4 targets.

[0103] qPCR procedure: Purification of total RNA 1. Cut the tumor sample into small pieces (about 30 mg). 2. Then, add 350 μL of Buffer RLT to the sample tube containing the tissue lyser II device, electroporate at 30 times / s for 5 minutes, and proceed 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 sample (including any precipitate) to an RNeasy Mini spin column placed in a 2 mL recovery tube (included). Close the lid and centrifuge at ≥13200 rpm for 15 s. Discard the flow-through. 5. Add 350 μL of Buffer RW1 to the RNeasy column. Close the cap and centrifuge at 13200 rpm for 15 seconds. Discard the flow-through. 6. Add 10 μL of DNase I stock solution to 70 μL of Buffer RDD. Mix by gently inverting the tube and centrifuge briefly. 7. Add DNase I incubation mixture (80 μL) directly to the RNeasy column membrane and place the tube on the bench (20°C-30°C) for 15 min. 8. Add 350 μL of Buffer RW1 to the RNeasy column. Close the cap and centrifuge at 13200 rpm for 15 seconds. Discard the flow-through. 9. Add 500 μL of Buffer RPE to the spin column. Gently close the cap and centrifuge at 13200 rpm for 2 min to wash the spin column membrane. Discard the flow-through. 10. Add 500 μL of Buffer RPE to the spin column. Gently close the lid and centrifuge at 13200 rpm for 2 min to wash the spin column membrane. 11. Place the RNeasy spin column in a new 1.5 mL recovery tube (provided). Add 50 μL of RNase-free water directly to the center of the spin column membrane. Gently close the lid and centrifuge at full speed for 1 min to elute the RNA. RNAQC 1. Quantify the integrity of total RNA using NanoDrop. Only high-quality RNA samples should be used for future cDNA synthesis and qPCR assays. result

[0104] The results are shown in Figure 7(tumor volume) and Table 8 (tumor growth inhibition (TGI)). These results indicate that treatment with the MEK inhibitor REC-4881 is non-inferior to treatment with sorafenib in mice with AXIN1 mutated HCC. Figure 8 shows the results from in vivo PDX studies ( Figure 7 ) showed that pERK ( Fig. 8A )、SPRY4( Figure 8C ) and DUSP6( Fig.8D ) decreased, while PPIA, which served as a control, did not change ( Fig. 8E ). The figure demonstrates on-target and pathway engagement as well as pathway rebound reported with MEK inhibitors, with 1mg / kg and 3mg / kg of REC-4881 demonstrating increased pMEK two days after treatment ( Figure 8B ). Table 8 PO-oral, QD-once daily; n-number of subjects; TGI-tumor growth inhibition Example 9 - Pharmacokinetics of MEK inhibitor REC-4881 after oral administration to female NCG mice Materials and Methods

[0105] Design: Female NCG mice were administered a single dose of REC-4881 at 1 mg / kg and 3 mg / kg PO. 3 mice per treatment group. Plasma sampling was performed at 0.5, 1, 2, 4, 8, 12, and 24 hours after dosing.

[0106] Bioanalytical Assays: Methods for PK analysis are known in the art. Data are presented as mean + / - SD for each treatment per mouse per time point. result

[0107] Fig. 9 This suggests that REC-4881 can achieve clinically relevant effective exposures. Example 10 - MEK inhibition for the treatment of colorectal cancer with APC mutations Materials and Methods

[0108] 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 h. NHCE cells were treated with 0.37, 1.11, 3.33, and 10 μM REC-4881 for 24 h. Cells were washed free of REC-4881 and medium and treated with Cell viability was measured by 2.0 cell viability assay (Promega; Madison, WI). result Table 9. ECs with reduced viability in human colorectal cancer cell lines 50 strain NHCE HCT-116 SW-48 HT-29 COLO-205 REC-4881(nM) >2000 >2000 0.86 2.07 0.54 Selumetinib(nM) >2000 >2000 15.9 123.7 27.1

[0109] like Fig.10 As shown, compared with normal human colon epithelium (NHCE) or APC wild-type colorectal cancer cells (HCT-116), MEK inhibitor REC-4881 showed a preferential reduction in the activity of APC mutant colorectal cancer cells (HT-29, SW48 and COLO-205), wherein the selectivity was higher than that of selumetinib (see Table 9). These data show that REC-4881 has a selectivity range far exceeding 1000 times and can be used to target colorectal cancer cells carrying APC mutations. In addition, relative to selumetinib, REC-4881 showed greater potency and selectivity in APC mutant lines (see Table 9). Example 11 - REC-4881 regulates genes downstream of β-catenin and other disease-related pathways in CRC Materials and Methods

[0110] Human colorectal cancer cell lines NHCE, HCT116, Colo-205, and HT29 were treated with 0.002, 0.02, 0.2, and 2 μM REC-4881 for 24 h. NHCE cells were treated with 0.37, 1.11, 3.33, and 10 μM REC-4881 for 24 h. Cells were washed without REC-4881 and medium, and RNA was extracted. RNA was isolated and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to analyze MYC transcripts ( Fig.11 A) and CDKN2A transcripts ( Fig.11 B) Amplification and measurement were performed using 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 as internal references for the amount of starting RNA. result

[0111] like Fig.11 As shown in A, REC-4881 dose-dependently increased CDKN2A in all colon epithelial cells tested. CDKN2A gene expression was most pronounced in COLO-205 and HT-29APC mutant cell lines. Fig.11 As shown in B, treatment of normal human colon epithelium (NHCE), APC wild-type colorectal cancer cells (HCT-116) and APC mutant colorectal cancer cells (HT-29 and COLO-205) with REC-4881 for 24 hours resulted in a dose-dependent decrease in MYC expression. HT-29 cells treated with REC-4881 showed the greatest decrease in MYC expression compared to other CRC cell lines tested. It is speculated that the specific background of APC and AXIN1 mutant cancers results in an increase in cell cycle and MYC genes, whereby MEK inhibition can reduce the activity of these downstream targets in a selective manner. Example 12 - REC-4881 alone or with anti-PD-1 in an APC mutant B16F10-ova syngeneic melanoma mouse model

[0112] Using the APC mutant B16F10-ova synthetic melanoma mouse model, REC-4881 alone or in combination with anti-PD1 agents was administered to female C57BL / 6 mice. Tumor samples were harvested after 13 days of treatment and CD8 + T cells and T regulatory cells. Methods: Immune cell analysis by flow cytometry. Tumor tissues were collected 4 hours after the last dose. N = 3 pools of three mice per group.

[0113] Mice administered REC-4881 alone or with anti-PD1 agents showed CD8 + Increased T cells and decreased T regulatory cells (respectively Fig.12 A and 12B). These data indicate that REC-4881 is able to modulate clinically relevant immune cell markers as a single agent or in combination with anti-PD1. Example 13 - REC-4881 alone or with anti-PD-1 in the Hepa 1-6 synergistic hepatocellular carcinoma (HCC) model ● Cell culture: Hepa 1-6AXIN1-KO CL No. 1 tumor cells were maintained in vitro with Dulbecco's Modified Eagle Medium (DMEM), 10% non-heat-inactivated fetal bovine serum (FBS), and 1% penicillin / streptomycin / L-glutamine (PSG). Tumor inoculation: Each mouse was subcutaneously inoculated with AXIN1-free tumor cells (5×10 6 ), for tumor development. ● Random Assignment: All mice are assigned to study groups based on caliper estimates of tumor burden. Mice are assigned to ensure that the mean tumor burden of all groups is within 10% of the total mean tumor burden of the study population. Tumor Growth Inhibition (TGI): The method of TGI is known in the art. Tumor volume was measured in two dimensions using calipers three times a week after randomization, and the volume was expressed in mm3 using the following formula: V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L). The %TGI of all mice was calculated using the formula %TGI = (TV vehicle-TV treatment) / (TV vehicle-TV initial) * 100 (Wong, H et al., Clin Cancer Res. [Clinical Cancer Research], 2012, 18 (14): 3846-3855). Treatment groups: 10 mice per group were treated with anti-PD-1, REC-4881, bevacizumab, or a combination of REC-4881 + anti-PD-1 or bevacizumab + anti-PD-1 for 19 days. 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 blotting: The method of Western blotting is known in the art. Primary antibodies: Axin (C76H11) rabbit mAb No. 2087 and GAPDH (D16H11) Rabbit mAb No. 5174. Dilute the following secondary antibodies 1:1000 in blocking buffer: Anti-rabbit IgG, HRP-linked antibody No. 7074. Dilute ECL 1:5000 in blocking buffer, image exposed for 5 min Predicted MW: Axin-1 110 kDa, GAPDH 37 kDa

[0114] 1 mg / kg and 3 mg / kg of REC-4881 (as a single agent or in combination with 10 mg / kg IP of anti-PD-1) were orally administered to the Hepa 1-6 syngeneic liver model ( Fig.13A and Fig. 13B ), which was engineered to KO AXIN1( Fig. 13C ). This figure demonstrates that in this model, REC-4881 has potent single-agent activity and can produce a more rapid response when combined with anti-PD-1 ( Fig.13D )。

Claims

1. A method of treating a cancer having one or more mutations in AXIN1 and / or APC in a subject in need thereof, the method comprising: - administering to the subject a therapeutically effective amount of a MEK inhibitor or a pharmaceutically acceptable salt thereof to treat the cancer.

2. The method of claim 1, wherein the MEK inhibitor is selected from the group consisting of REC-4881, bimetinib, 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 of any one of claims 1-3, 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. The method of claim 4, wherein the cancer is melanoma.

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

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

10. The method of any one of claims 1-9, wherein the method further comprises the step of evaluating a subject having cancer for the presence or absence of one or more mutations in AXIN1 and / or APC.

11. The method of any one of claims 1-10, wherein the method further comprises the step of administering to the subject one or more additional cancer treatments.

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

13. The method of claim 12, wherein the immune checkpoint inhibitor is an inhibitor of PD-1 and / or PD-L1 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. The method of claim 11, wherein the one or more additional therapies comprises treatment with a RAF inhibitor.

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

17. The method of any one of claims 1-16, wherein the cancer is a relapsed cancer or a refractory cancer.

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

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

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

21. A method of using one or more mutations in AXIN1 and / or APC as biomarkers in a subject suffering from cancer to evaluate the likelihood that a MEK inhibitor, or a pharmaceutically acceptable salt thereof, will produce an anti-cancer effect in the subject, the method comprising: - determining the presence of one or more mutations in AXIN1 and / or APC in the subject; as well as - 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 the anti-cancer effect.

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