Use of beta-catenin antagonist and MAPK inhibitor

Abstract

Provided are methods of administering a peptide antagonist of β-catenin in combination with an inhibitor of the mitogen-activated protein kinase (MAPK) signaling pathway.

Description

Sapience.026.WOl PATENT USE OF BETA-CATENIN ANTAGONIST AND MAPK INHIBITOR BACKGROUND

[0001] The Wnt / p-catenin pathway, a key oncogenic driver in various cancers, was long deemed undruggable. Historically, attempts at targeting its components resulted in toxicity due to P-catenin’s role in homeostatic functions such as intestinal stem cell renewal and bone osteogenesis.

[0002] The complex formed by the interaction between -catenin and its co-activator, B-cell CLL / lymphoma 9 protein (BCL9), drives oncogene expression in multiple cancers due to aberrent Wnt pathway signaling. ST316 is a P-catenin antagonist peptide that disrupts its interaction with BCL9, targeting oncogenic signaling and immune exclusion while preserving homeostatic functions. ST316 reduces P-catenin transcriptional activity and tumor growth in vitro and in vivo, yet GLP -toxicology studies and Phase 1 clinical trial data confirm no Wnt-inhibiti on-associated toxicity. ST316 has now progressed to a Phase 2 trial in patients with colorectal cancer (CRC), an indication with a high prevalence of Wnt-related mutations.

[0003] The mitogen-activated protein kinase (MAPK) pathway is a complex signaling cascade that transduces signals from cell surface receptors, such as epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (HGFR), platelet growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), and fibroblast growth factor receptor (FGFR) to the nucleus, activating transcription factors such as Elk- 1, c-Myc, c-Jun, and cAMP response element-binding protein (CREB). Mutations in members of the MAPK pathway are among the most common events in cancer, leading to the activation of key signaling molecules, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, which regulate various cellular processes, including proliferation, differentiation, and survival.

[0004] In CRC, the frequency of mutations in both the Wnt / -catenin and MAPK pathways, coupled with the poor prognosis and limited treatment options, highlight the need for novel therapeutic strategies to address this challenging subtype.SUMMARY OF THE INVENTION

[0005] Some of the main aspects of the present invention are summarized below.Additional aspects are described in the Detailed Description of the Invention, Examples, Drawings, and Claims sections of this disclosure. The description in each section of thisSapience.026.WOl PATENTdisclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention.

[0006] Provided is a combination approach for administering a peptide antagonist of P-catenin and a MAPK inhibitor (MAP Ki). Our results particularly support rational combinations of ST316 with clinically relevant MAPK inhibitors as new therapeutic opportunities for the treatment of CRC. BRAF V600E-mutant CRC is a subgroup with especially limited treatment options that can be treated by methods of the invention.

[0007] In one embodiment, a method is provided for treating colorectal cancer (CRC) in a subject, the method comprising combination therapy with: (i) a pharmaceutical composition comprising an effective amount of a peptide antagonist of P-catenin and (ii) a pharmaceutical composition comprising an effective amount of a MAPK inhibitor (MAPKi); wherein the pharmaceutical composition comprising the antagonist of P- catenin and the pharmaceutical composition comprising the MAPKi are administered to the subject together or separately.

[0008] In one embodiment, the CRC tumor comprises wild-type tumor protein 53 (TP53). In one embodiment, the CRC tumor comprises a mutation in a member of the MAPK pathway. In some embodiments, the CRC tumor comprises a mutation is in at least one gene selected from the group consisting of BRAF, KRAS, NRAS, or NF1. In a particular embodiment, the CRC tumor comprises a BRAF V600E mutation.

[0009] In certain embodiments, the peptide antagonist disrupts binding of P-catenin to B-cell CLL / lymphoma 9 protein (BCL9). In one embodiment, the peptide antagonist of P-catenin comprises the D-amino acid sequence FRWLLRQLARLAQLAKLTPVKLTPV (SEQ ID NO: 1). In some embodiments, the peptide antagonist of P-catenin is a cellpenetrating peptide. For example, the peptide antagonist of P-catenin can comprise a cellpenetrating sequence. In a particular embodiment, the peptide antagonist of P-catenin is ST316.

[0010] In some embodiments, the peptide antagonist of P-catenin is administered to the subject at a dose of about 0.25-12 mg / kg.

[0011] In a certain aspect of the invention, the MAPKi is selected from the group consisting of a BRAF inhibitor, a MEK inhibitor, and an EGFR inhibitor. In some embodiments, the MAPKi is selected from the group consisting of binimetinib,Sapience.026.WOl PATENTcobimetinib, dabrafenib, encorafenib, gefitinib, lapatinib, selumetinib, sorafenib, trametinib, and vemurafenib. In one embodiment, the MAP Ki is selected from the group consisting of dabrafenib, encorafenib, and vemurafenib. In a particular embodiment, the MAP Ki is encorafenib.. In a particular embodiment, the MAPKi is selective for BRAF V600E. In certain embodiments, the pharmaceutical composition comprising the MAPKi comprises more than one MAPKi.

[0012] In one embodiment, the peptide antagonist of P-catenin is ST316. In one embodiment the MAPKi is encorafenib. In a particular embodiment, the peptide antagonist of -catenin is ST316 and the MAPKi is encorafenib.

[0013] In one embodiment, the pharmaceutical composition comprising the peptide antagonist of P-catenin is administered parenterally, for example, intravenously. In one embodiment, the pharmaceutical composition comprising the MAPKi is administered orally.

[0014] In some embodiments, the pharmaceutical composition comprising the peptide antagonist of P-catenin and the pharmaceutical composition comprising the MAPKi are administered to the subject on different days.

[0015] In certain embodiments, the pharmaceutical composition comprising the peptide antagonist of P-catenin is administered once weekly for at least three weeks, or once every two weeks for at least four weeks.

[0016] Also provided is a pharmaceutical composition comprising an effective amount of a peptide antagonist of P-catenin and a pharmaceutical composition comprising an effective amount of a MAPKi for use in a method of combination therapy for treating CRC in a subject.

[0017] In a preferred embodiment of the invention, the subject is a human subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 shows that organoid colorectal cancer (CRC) tumor models with wildtype tumor protein 53 (TP53 WT) displayed higher sensitivity to treatment with ST316 than tumors with a mutation in tumor protein 53 (TP53 Mut).

[0019] FIG. 2A-FIG.2C show that tumors with TP53 WT (FIG.2A) and mutations in the MAPK pathway (FIG.2B, FIG.2C) significantly correlate with sensitivity to ST316.

[0020] FIG. 3 shows the percentage of cell death observed in organoid tumor models following treatment with various concentrations of ST316 and encorafenib.Sapience.026.WOl PATENT

[0021] FIG. 4 shows a Bliss surface model for the CRC cell line, LIM2405, treated with ST316 in combination with MAPKi encorafenib at the indicated concentrations. An apoptosis assay based on Annexin V and Sytox Red was performed at 48 hours posttreatment.

[0022] FIG. 5 shows tumor volume in a LIM2405 CRC tumor model following treatment with vehicle (control), encorafenib (Enc.), ST316, or a combination of encorafenib an ST316 (Combo).DETAILED DESCRIPTION OF THE INVENTION

[0023] In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. 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 to which this invention is related.

[0024] Any headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0025] All references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art.I. Definitions

[0026] The phraseology or terminology in this disclosure is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0027] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.Sapience.026.WOl PATENT

[0028] Furthermore, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0029] Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are included.

[0030] Units, prefixes, and symbols are denoted in their Systeme International d’ Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth. Likewise, a disclosed range is a disclosure of each individual value (i.e., intermediate) encompassed by the range, including integers and fractions. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10 individually, and of 5.2, 7.5, 8.7, and so forth.

[0031] Unless otherwise indicated, the terms “at least” or “about” preceding a series of elements is to be understood to refer to every element in the series. The term “about” depends on the context in which it is used. When preceding a numerical value, it generally includes ± 10% of the recited value. For example, in one context, a concentration of about 1 mg / mL includes 0.9 mg / mLto 1.1 mg / mL. Likewise, a concentration range of about 1% to 10% (w / v) can include 0.9% (w / v) to 11% (w / v).

[0032] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acids of any length, and their salts. The polymer can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. Except where indicated otherwise, e.g., for the abbreviations for the uncommon or unnatural amino acids set forth herein, the three-letter and one-letter abbreviations, as used in the art, are used herein to represent amino acid residues. Except when preceded with a “D” or in lower case, the amino acid is an L-amino acid. Groups or strings of amino acid abbreviations are used to represent peptides. Except where specificallySapience.026.WOl PATENTindicated, peptides are indicated with the N-terminus of the left and the sequence is written from the N-terminus to the C-terminus.

[0033] A “retro inverse” peptide has a reversed amino acid sequence, relative to a reference L-amino acid sequence, and is made up of all D-amino acids (inverting the a- center chirality of the amino acid subunits) to help maintain side-chain topology similar to that of the original L-amino acid peptide.

[0034] An “isolated” molecule is one that is in a form not found in nature, including those which have been purified.

[0035] An “active agent” is an ingredient that is intended to furnish biological activity. The active agent can be in association with one or more other ingredients. An active agent that is a peptide can also be referred to as an “active peptide.”

[0036] An “effective amount” of an active agent is an amount sufficient to carry out a specifically stated purpose.

[0037] The term “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective and which contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile and can comprise a pharmaceutically acceptable carrier, such as physiological saline. Suitable pharmaceutical compositions can comprise one or more of a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), a stabilizing agent (e.g., polyol or amino acid), a preservative (e.g., sodium benzoate), and / or other conventional solubilizing or dispersing agents.

[0038] A “subject” or “individual” or “animal” or “patient” or “mammal,” is any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, sports animals, and laboratory animals including, e.g., humans, non-human primates, canines, felines, porcines, bovines, equines, rodents, including rats and mice, rabbits, etc.

[0039] Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder. In certain embodiments, a subject is successfully “treated” for a disease or disorder if the patient shows total, partial, or transient alleviation or elimination of at least one symptom or measurable physical parameter associated with the disease or disorder.Sapience.026.WOl PATENT

[0040] A “control patient” is a subject that has not received a treatment of the invention. A “control population” or a “population of control patients” is a group of subjects that have not received a treatment of the invention. A control patient or subject in the control population has the same disease or disorder as the subject being compared to the control patient or control population. For example, a clinical outcome of a cancer patient receiving a pharmaceutical composition or method of the invention is compared with the average (median) outcome of subjects having the same type and / or stage of cancer, who did not receive a phanuaceutical composition or method of the invention. In some embodiments, the control patient or patients in the control population have received a treatment other than a treatment of the invention, for example, a standard-of-care treatment.

[0041] An “antagonist” is a substance that prevents, blocks, inhibits, neutralizes, or reduces a biological activity or effect of another molecule, such as a receptor or ligand.

[0042] The terms “inhibit,” “block,” and “suppress” are used interchangeably and refer to any statistically significant decrease in occurrence or activity, including full blocking of the occurrence or activity. For example, “inhibition” can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in activity or occurrence. An “inhibitor” is a molecule, factor, or substance that produces a statistically significant decrease in the occurrence or activity of a process, pathway, or molecule.

[0043] A “tumor” or “solid tumor” is a mass of neoplastic cells, such as cancer cells.The terms “advanced,” “metastatic,” and “advanced / metastatic" are used interchangeably to describe a cancer in which malignant cells have migrated from the original tumor to another location, for example, another organ, in a patient’s body.

[0044] A “neoplastic cell” or “neoplasm” typically has undergone some form of mutation / transformation, resulting in abnormal growth as compared to normal cells or tissue of the same type. Neoplasms include morphological irregularities, as well as pathologic proliferation. Neoplastic cells can be benign or malignant. Malignant neoplasms, i.e., cancers, are distinguished from benign in that they demonstrate loss of differentiation and orientation of cells, and have the properties of invasion and metastasis.Sapience.026.WOl PATENT II. PeptidesB-Catenin

[0045] The Wnt / p-catenin pathway is a validated target for cancer therapies.Constitutive activation of the Wnt / p-catenin signaling pathway is implicated in carcinogenesis, tumor progression, and poor prognosis of many malignancies (Shang 2017). The complex formed by the interaction between P-catenin and its co-activator, BCL9, drives oncogene expression in multiple cancers due to aberrent Wnt pathway signaling. Disruption of the P-catenin / BCL9 complex has been shown to suppress oncogenic Wnt / p-catenin transcription without impacting P-catenin homeostatic functions (Takada 2012).

[0046] The interaction between P-catenin and BCL9 has previously been considered an “undruggable” target due to the inability of small molecules to inhibit complex formation and the inability of antibodies to gain access to the cytoplasm or nucleus to disrupt the interaction (Takada 2012). Peptide antagonism of P-catenin is distinct from these other approaches because it can specifically target the interaction between P-catenin and BCL9 and is selectively toxic to tumor cells where these proteins are hyperactive. B-Catenin Antagonist Peptides

[0047] In some embodiments, methods of the invention comprise treating a patient having CRC with combination therapy comprising an effective amount of a peptide antagonist of P-catenin and an inhibitor of the mitogen-activated protein kinase (MAPK) signaling pathway. Preferably, the peptide antagonist of P-catenin disrupts interaction of P-catenin with BCL9. Peptide antagonists of P-catenin can be rationally designed, for example, based on the native sequence of BCL9 with modifications to enhance electrostatic interactions between the peptide antagonist and P-catenin. In particular, peptide antagonists of the interaction between P-catenin and BCL9 can be derived from the homology domain 2 (HD2) of BCL9 and designed to interact with the first armadillo repeat (ARM-1) domain of P-catenin, which is a site utilized by BCL9 but not other P- catenin binding partners. WO 2021 / 007158 provides numerous examples of such peptides. Artificial intelligence can be utilized in the design of peptide antagonists of P- catenin.

[0048] The ability of a peptide based on wild-type BCL9 to antagonize the activity of P-catenin, such as to disrupt the interaction of P-catenin with BCL9, can be measured, forSapience.026.WOl PATENTexample, by methods described in WO 2024 / 216237. The cytotoxic activity of a peptide antagonist of P-catenin can be measured in vitro by known assays and / or in vivo using known tumor models; for example, WO 2019 / 136125 and WO 2021 / 007158 describe such assays and models.

[0049] The peptide antagonist of -catenin can be a cell-penetrating peptide. In one embodiment, the peptide comprises a cell-penetrating domain. Numerous cellpenetrating peptide sequences are described and characterized in the literature (see WO 2019 / 136125). In one embodiment, the peptide is a cyclic peptide. Cyclized peptides, for example, using hydrocarbon staples (Bemal 2007; Bird 2017) or other cyclization methods known in the art, can enter cells via passive diffusion, endocytosis / endosomal escape, or other mechanisms (Dougherty 2019). Peptides can also be delivered to cells via mechanisms that exploit cellular receptors, for example, integrin-targeting, RGD-like sequences. Alternatively, peptides can be encapsulated and delivered to cells in vesicles, such as exosomes or liposomes, or in micelles.

[0050] ST316 is a 25-amino acid peptide antagonist of the interaction between P- catenin and BCL9. ST316 preserves Wnt / -catenin homeostasis, while targeting oncogenic and immune-suppressive -catenin / BCL9 signaling. ST316 is composed entirely of D-amino acids and is highly stable in the presence of proteolytic enzymes. Due to its resistance to protease degradation, ST316 has a long plasma half-life, and should not be processed and presented by antigen presenting cells, thereby avoiding stimulation of an anti-drug antibody (ADA) response.

[0051] ST316 is composed of two domains: (i) a 15 amino acid N-terminal active domain derived from the homology domain 2 (HD2) of BCL9 and designed to interact with the first armadillo repeat (ARM-1) domain of P-catenin, which is a site utilized by BCL9 but not other P-catenin binding partners; and (ii) a 10 amino acid C-terminal domain, which increases solubility and cell penetration. The D-amino acid sequence of ST316 is: FRWLLRQLARLAQLA / f / .7PlW / 7 / JV (SEQ ID NO: 1). The N-terminal active domain is bolded and the C-terminal cell-penetrating domain is italicized. ST316 also comprises an N-terminal octanoyl group.Methods of Preparing B-Catenin Antagonists

[0052] Peptide antagonists of P-catenin can be chemically synthesized, for example, using solid-phase peptide synthesis or solution-phase peptide synthesis, or a combination of both. Synthesis may optionally occur as fragments of the peptide that are subsequentlySapience.026.WOl PATENTcombined either chemically or enzymatically. Alternatively, peptide antagonists of P- catenin can be expressed using recombinant methods.

[0053] Peptides can be purified using methods that include, for example, reversephase high-performance liquid chromatography (RP-HPLC), multicolumn countercurrent solvent gradient purification (MCSGP), and ion-exchange chromatography.III. MAPK Inhibitors

[0054] The methods of the invention involve combination therapy comprising administration of a -catenin antagonist and a MAPKi. Mutations in members of the MAPK pathway are observed in many cancers. In one embodiment, the MAPKi targets a particular mutation in a member of a MAPK signaling pathway.

[0055] BRAF encodes the protein kinase B-Raf. Mutations in BRAF are observed in 10-20% of CRC. The most common BRAF mutation is V600E.

[0056] MEK1 and MEK2 encode MAPK / ERK kinase 1 (MEK1) and MAPK / ERK kinase 2 (MEK2), respectively. MEK is also known as MAPK kinase, MAPKK, and MAP2K.

[0057] The Ras family of oncogenes includes HRAS, KRAS, and NRAS, each of which encodes a small GTPase protein (H-Rase, K-Ras, and N-Ras, respectively).Mutations in RAS genes can lead to uncontrolled cell growth and tumor formation.Common KRAS mutation include G12C, G12D, G12V, and G13D. Common NRAS mutations include G12C, G12D, Q61K, Q61R, and Q61L.

[0058] MAPKi can include, for example, monoclonal antibodies to members of the MAPK pathway and small molecule inhibitors, such as tyrosine kinase inhibitors.

[0059] Because many members of the MAPK pathway are kinases, several MAPKi target more than one member of the pathway. Other MAPKi target upstream receptors, for example, EGFR.

[0060] In some embodiments, MAPKi include, for example, adagrasib, binimetinib, cobimetinib, dabrafenib, daraxonrasib, encorafenib, gefitinib, lapatinib, selumetinib, sorafenib, sotorasib, trametinib, and vemurafenib. In certain embodiments, the MAPKi inhibits BRAF V600E. Examples of MAPKi that are selective for BRAF V600E include, for example, dabrafenib, encorafenib, and vemurafenib.

[0061] In some embodiments, the pharmaceutical composition comprising the MAPKi can comprise more than one MAPKi.Sapience.026.WOl PATENT IV. Compositions and Administration

[0062] In certain aspects, the invention provides a combination approach comprising administration of a composition, e.g., a pharmaceutical composition, comprising an effective amount of a peptide antagonist of P-catenin, such as ST316, and administration of composition, e.g., a pharmaceutical composition, comprising an effective amount of a MAPKi, such as a BRAF inhibitor.

[0063] Because the combination therapy of the invention can be synergistic between the P-catenin antagonist and the MAPKi, the effective amount of each active agent can, in some embodiments, be lower than the effective amount when each active agent is administered individually. For example, the effective amount of each of the peptide antagonist of P-catenin and the MAPKi may be a sub-therapeutic dose.

[0064] The peptide antagonist of P-catenin can be dosed based on the patient’s weight. The peptide antagonist of P-catenin, such as ST316, can be administered to a patient at a dose of about 0.25 mg / kg to about 12 mg / kg. In certain embodiments, ST316 is administered at a dose of about 0.25 mg / kg, about 0.5 mg / kg, about 0.75 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 4.5 mg / kg, about 5 mg / kg, about 5.5 mg / kg, about 6 mg / kg, about 6.5 mg / kg, about 7 mg / kg, about 7.5 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, or about 12 mg / kg. These amounts can also serve as endpoints for a range of doses to be administered, for example, about 0.75 mg / kg to about 7 mg / kg, about 2 mg / kg to about 4 mg / kg, etc.

[0065] Alternatively, the peptide antagonist of P-catenin can be administered at a fixed dose. The peptide antagonist of P-catenin, such as ST316, can be administered to a patient at a dose of about 500 mg to about 1500 mg. In certain embodiments, ST316 is administered at a dose of about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, or about 1500 mg. These amounts can also serve as endpoints for a range of doses to be administered, for example, about 600 ng to about 1100 mg, about 750 mg to about 900 mg, etc.

[0066] The composition comprising a peptide antagonist of P-catenin is preferably administered parenterally. Parenteral routes of administration include intravenous (IV), intramuscular, intraperitoneal, intrathecal, and subcutaneous. In certain embodiments theSapience.026.WOl PATENTcomposition comprising the peptide antagonist of P-catenin is administered to the subject by intravenous infusion.

[0067] Most commonly, the composition comprising a peptide antagonist of P-catenin and the composition comprising a MAPKi are different compositions. If the peptide antagonist and the MAPKi are comprised in separate compositions, the compositions can be administered to the subject at the same time or at different times, including on different days.

[0068] Each of the composition comprising a peptide antagonist and the composition comprising a MAPKi is typically administered to the subject more than once. In one embodiment, administration of the peptide antagonist of P-catenin can occur once weekly for a duration of at least one week (i.e., one administration), at least two weeks (i.e., two administrations), at least three weeks (i.e., three administrations), at least six weeks (i.e., six administrations), at least nine weeks, at least twelve weeks, at least three months, at least six months, at least nine months, or at least twelve months. In another embodiment, administration can occur once every two weeks for a duration of at least four weeks (i.e., two administrations), at least eight weeks (i.e., four administrations), at least twelve weeks, at least three months, at least six months, at least nine months, or at least twelve months. In some embodiments, a patient can be administered the peptide antagonist of P- catenin once weekly for a duration of at least one week, two weeks, three weeks, six weeks, nine weeks, twelve weeks, three months, six months, nine months, or twelve months, followed by administration once every two weeks for at least four weeks, eight weeks, twelve weeks, three months, six months, nine months, or twelve months.

[0069] In one embodiment, administration of the MAPKi can occur at least once weekly for a duration of at least three weeks (e.g., three administrations), six weeks (e.g., six administrations), nine weeks, twelve weeks, three months, six months, nine months, or twelve months. In another embodiment, administration can daily for a duration of at least about: four weeks, six weeks, eight weeks, twelve weeks, three months, six months, nine months, or twelve months.

[0070] For purposes of the present disclosure, “combination therapy” means that the treatment period comprising administration of the peptide antagonist of P-catenin overlaps with the treatment period comprising administration of the MAPKi.Sapience.026.WOl PATENTV. Methods of Use

[0071] Subjects in need of the methods of the invention are patients diagnosed with CRC. For example, the subject can have a locally advanced solid CRC tumor or a metastatic inoperable CRC tumor or an advanced unresectable CRC tumor.

[0072] In one embodiment, the tumor harbors one or more abnormalities in the Wnt / p-catenin signaling pathway. By way of non-limiting example, the one or more abnormalities in the Wnt / p-catenin signaling pathway can comprise one or more mutations in one or more of adenomatous polyposis coli (APC). APC membrane recruitment protein 1 (AMER1), Axin2, P-catenin, RING finger protein 43 (RNF43), and / or T cell factor 7 (TCF7).

[0073] In one embodiment, the tumor harbors one or more abnormalities in the MAPK signaling pathway. By way of non-limiting example, the one or more abnormalities in the MAPK signaling pathway can comprise one or more mutations in one or more of BRAF, ERK, MEK, and / or RAS genes.

[0074] In particular embodiments of the invention, combination therapy with a peptide antagonist of P-catenin, such as ST316, and a MAPKi can inhibit tumor growth, reduce tumor volume, or a combination thereof.

[0075] Efficacy of treatment can be evaluated by one or more known measures. For example, patients subjected to methods of the invention can experience outcomes including extended survival, improved progression-free survival, improved duration of response, longer remission, reduced risk of relapse, and / or improved tumor response to treatment with the combination therapy of the invention, compared with the same outcome(s) in patients not subjected to methods of the invention, i.e., control patients. An outcome in a patient treated by a method of the invention can be compared, for example, to the median outcome in a population of control patients. The population of control patients can be administered, for example, a regimen selected from the group consisting of a placebo, surgery, radiation, chemotherapy, immunotherapy, hormone-based therapy, or targeted therapy. In another embodiment, a patient subjected to the combination therapy of the invention can be compared to a population of control patients administered treatment with only one of a peptide antagonist of P-catenin or a MAPKi. Comparisons can be analyzed statistically using, for example, the Wilcoxon signed rank test or the Kaplan-Meier method.Sapience.026.WOl PATENT

[0076] Tumor response to treatment can be assessed, for example, by measuring tumor burden and / or tumor regression. Response to treatment compares one or more measures of efficacy after a treatment regimen, as compared to baseline, e.g., prior to treatment with combination therapy. A baseline assessment is preferably performed within 24, 48, or 72 hours, or within 1, 2, 3, or 4 weeks prior to the first treatment. In a one preferred embodiment, a baseline assessment is performed within one week prior to the first ST316 treatment.

[0077] ‘Tumor burden” is the total mass or total size of cancerous tissue in a patient’s body. Tumor response can be evaluated by measures including objective response rate, including partial response and / or complete response, stable disease, disease control rate, duration of disease control, and duration of response. These parameters can be determined, for example, by revised Response Evaluation Criteria in Solid Tumors (RECIST 1.1) (Eisenhauer 2009).

[0078] Objective response rate assesses reduction of tumor size, for example, tumor diameter, which can be determined by clinical examination and / or imaging. Where a patient has multiple tumors, tumor size can optionally be expressed as the average diameter of all tumors or by the sum of diameters of all tumors. Superficial tumors can be measured clinically, for instance, using calipers or by photography and ruler measurement. Imaging methods include computed tomography (CT), typically with contrast; X-ray; magnetic resonance imaging (MRI); and positron emission tomography (PET), such as (18)F-fluorodeoxyglucose PET. In one preferred embodiment, CT is utilized to assess tumor response. Accordingly, in one aspect, the invention provides a method of reducing tumor burden, i.e., tumor mass and / or tumor size, in a patient, the method comprising administering to the patient combination therapy comprising a peptide antagonist of -catenin, such as ST316, and a MAPKi. Reduction in tumor burden is measured relative to baseline.

[0079] In certain embodiments, particularly those in which assessment is by RECIST 1.1, disease control rate defines the level of tumor response as the best of the following: complete response (CR), which is the disappearance of tumor(s); partial response (PR), which is a decrease of at least 30%, in the size of tumor(s); stable disease (SD), in which the change in tumor size is decreased by less than 30% or increased by less than 20%; or disease progression, which is an increase of at least 20%, in tumor size and / or new lesions. Patients treated by methods of the invention can experience CR, PR, or SD.Sapience.026.WOl PATENT

[0080] Duration of disease control is the length of time from achievement of a response (CR or PR) or SD until disease progression. Duration of response is the length of time from the achievement of a response until disease progression, i.e.. the period in which a tumor does not grow or spread, or death. Duration of response in patients receiving combination therapy of the invention can be, for example, at least 4, 6, 8, 10, or 12 weeks, at least 4, 6, 8, 10, 12, 16, 18, or 24 months, or at least 3, 4, or 5 years. Patients treated by methods of the invention can experience increased duration of disease control or increased duration of response. Accordingly, in one aspect, the invention provides a method of increasing the duration of response in a patient, the method comprising administering to the patient combination therapy comprising a peptide antagonist of [>- catenin and a MAP Ki. Increase in duration of disease control or duration of response is measured relative to the median duration of disease control or duration of response, respectively, in a control population.

[0081] Survival can be assessed as overall survival, i.e., the length of time a patient lives, as progression-free survival, i.e., the length of time a patient is treated without progression or worsening of the disease, or as event-free survival, i.e., the length of time that a patient remains free of complications or negative events such as relapse or disease progression. Survival is from the date that treatment commences. Overall survival, median overall survival, progression-free survival, median progression-free survival, event-free survival, and median event-free survival can be calculated, for example, by Kaplan-Meier analysis, based on the response to treatment.

[0082] Accordingly, in one aspect, the invention provides a method of increasing overall survival in a patient, the method comprising administering to the patient combination therapy comprising a peptide antagonist of P-catenin and a MAPKi.Increase in overall survival can be measured relative to the median overall survival in a control population. Alternatively, an increase compared to a control population in the percentage of patients who survive for a given time period (e.g., 6 months or 12 months) indicates an increase in overall survival.

[0083] In another aspect, the invention provides a method of increasing progression- free survival in a patient, the method comprising administering to the patient combination therapy comprising a peptide antagonist of P-catenin and a MAPKi. Increase in progression-free survival can be measured relative to the median progression-free survival in a control population. Alternatively, an increase compared to a controlSapience.026.WOl PATENTpopulation in the percentage of patients who are relapse-free for a given time period (e.g., 6 months or 12 months) indicates an increase in progression-free survival.

[0084] In a further aspect, the invention provides a method of increasing event-free survival in a patient, the method comprising administering to the patient a peptide antagonist of 0-catenin and a MAP Ki. Increase in event-free survival is measured relative to the median event-free survival in a control population. Alternatively, an increase compared to a control population in the percentage of patients who are event-free for a given time period (e.g., 6 months or 12 months) indicates an increase in event-free survival.

[0085] A patient is successfully treated according to the methods of the invention if the patient experiences or displays at least one of the following outcomes after administration of combination therapy:- undetectability of the tumor (or at least one tumor, if multiple tumors are present at baseline);- at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% reduction in tumor size compared to baseline;- no significant increase (e.g., less than 20% or less than 30%) in tumor size compared to baseline;- significantly increased duration of response, optionally compared with median duration of response of a population of control patients;- significantly increased progression-free survival, optionally compared with median progression-free survival of a population of control patients;- significantly increased overall survival, optionally compared with median overall survival of a population of control patients.EXAMPLES

[0086] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure.Example 1. Study in Organoids Demonstrates Rationale for P-Catenin Antagonist MAPKi Combination

[0087] We evaluated the anti-tumor activity of ST316 in 60 tumor organoids with diverse genetic backgrounds, including CRC tumor-derived organoids, to identifySapience.026.WOl PATENTbiomarkers associated with treatment responses. Organoids were treated with escalating doses of ST316 and the IC50S were determined. RNA sequencing and whole exome sequencing (WES) data were available for all models (untreated condition) and used to identify transcriptional signatures, as well as gene mutations, enriched in high-sensitivity and low-sensitivity models.

[0088] Models with wild-type TP53 (TP53 WT) were most responsive to ST316 (FIG. 1, FIG. 2A) Further analysis of the TP53 WT group demonstrated that organoids with the highest sensitivity to ST316 (lowest ICsos) were enriched for mutations in the MAPK pathway genes, KRAS, BRAF, NRAS, and NF1 (FIG. 2B, FIG. 2C). BRAF and TP53 mutations tend to be mutually exclusive, so most BRAF-mutant patients typically are TP53 WT. This genetic background was the most susceptible to ST316 in the organoids study. Our results indicate co-dependency between the MAPK and Wnt / p- catenin pathways.Example 2. p-Catenin Antagonist MAPKi Combination Demonstrates Synergistic AntiTumor Effect

[0089] BRAF mutations occur in approximately 10% of CRC patients. This group has a poor outcome, and new treatment options are needed (Ahronian 2015; Pashirzad 2021). Given the importance of MAPK signaling in cancer, we evaluated dual inhibition of P-catenin and BRAF by combining ST316 with the BRAF inhibitor encorafenib in BRAF-mutant organoids. This combination showed additive or synergistic anti-tumor effects in three tumor models (FIG. 3).

[0090] We confirmed these findings by performing checkerboard assays of ST316 in combination with encorafenib. Results confirmed that ST316 synergizes with MAPKi inhibition in CRC (FIG.4).

[0091] We next tested the combination of ST316 and encorafenib in a subcutaneous tumor model using CRC cells with a V600E mutation in the BRAF gene. Briefly, LIM2405 cells suspended 1 : 1 in Matrigel were implanted via subcutaneous injection into the axilla of Nu / J mice (Jackson Laboratories). ST316 was administered subcutaneously at a dose of 5 mg / kg, Ix / week. Encorafenib was administered daily via oral gavage at 20 mg / kg. Dosing was initiated on day 6 post-tumor inoculation, with average starting tumor volume of about 200 mm3. Tumor volume was monitored three times weekly. Results are shown in FIG.5. Subtherapeutic doses of ST316 and encorafenib minimizeSapience.026.WOl PATENTefficacy of the monotherapies; however, our results show synergy between ST316 and encorafenib in the combination group.

[0092] P-catenin has been linked to resistance to BRAF inhibitors. Our results show that combination therapies targeting both P-catenin and BRAF can overcome such resistance and provide a promising new therapeutic approach for this difficult-to-treat cancer.Example 3. 0-Catenin Antagonist Is Effective in Patients with Mutation in MAPK Pathway

[0093] In a Phase II study conducted in CRC patients in a second- to third-line setting, ST316 was administered in combination with standard of care Folfiri / Folfox, Bevacizumab, or Lonsurf. ST316 was dosed at 4-8 mg / kg, once weekly or every two weeks. Consistent with the findings of the organoids study, CRC patients with NRAS mutations responded to treatment with ST316. Results are shown in Table 1.Table 1Data analyzed Responders (PR) Non-Responders (PD, SD) NRAS mutation 3 0NRAS WT 5 22Fisher Exact Test P-value = 0.0138; PR = partial response; PD = progressive disease; SD = stable diseaseREFERENCESAhronian L, et al. Clinical Acquired Resistance to RAF Inhibitor Combinations in BRAF-Mutant Colorectal Cancer through MAPK Pathway Alterations. Cancer Discov. 2015; 5:358-367.Bernal F, et al. Reactivation of the p53 Tumor Suppressor Pathway by a Stapled p53 Peptide. J. Am. Chem. Soc. 2007: 129:2456-2457.Bird GH, et al. Biophysical Determinants for Cellular Uptake of Hydrocarbon-Stapled Peptide Helices. Nat. Chem. Biol. 2017; 12:845-852.Dougherty PG, et al. Understanding Cell Penetration of Cyclic Peptides. Chem. Rev. 2019; 119(17): 10241-10287.Eisenhauer EA, et al. New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur J Cancer 2009; 45(2):228-247.Lee S, et al. Targeting MAPK Signaling in Cancer: Mechanisms of Drug Resistance and Sensitivity. Int. J. Mol. Sci. 2020; 21: 1102; doi:10.3390 / ijms21031102.Sapience.026.WOl PATENTPashirzad M, et al. The Therapeutic Potential of MAPK / ERK Inhibitors in the Treatment of Colorectal Cancer. 2021; 21:932-943.Shang S, et al. The regulation of -catenin activity and function in cancer; therapeutic opportunities. Oncotarget 2017; 8(20):33972-33989.Takada K, et al. Targeted disruption of the BCL9 / -catenin complex inhibits oncogenic Wnt signaling. Sci Transl Med. 2012 Aug 22; 4(148).***The present invention is further described by the following claims.

Claims

Sapience.026.WOl PATENT CLAIMS1. A method of treating a colorectal cancer (CRC) tumor in a subject, the method comprising combination therapy with: (i) a pharmaceutical composition comprising an effective amount of a peptide antagonist of P-catenin and (ii) a pharmaceutical composition comprising an effective amount of a mitogen-activated protein kinase (MAPK) pathway inhibitor; wherein the pharmaceutical composition comprising the antagonist of P-catenin and the pharmaceutical composition comprising the MAPK pathway inhibitor (MAP Ki) are administered to the subject together or separately.

2. The method of claim 1, wherein the CRC tumor comprises wild-type tumor protein 53 (TP53).

3. The method of claim 1 or claim 2, wherein the CRC tumor comprises a mutation in a member of the MAPK pathway.

4. The method of any preceding claims, wherein the CRC tumor comprises a mutation is in at least one gene selected from the group consisting of BRAF, NRAS, or NFL 5. The method of any preceding claim, wherein the CRC tumor comprises a BRAF V600E mutation.

6. The method of any preceding claim, wherein the peptide antagonist of P-catenin disrupts binding of P-catenin to B-cell CLL / lymphoma 9 protein (BCL9).

7. The method of any preceding claim, wherein the peptide antagonist of P-catenin comprises the D-amino acid sequence FRWLLRQLARLAQLAKLTPVKLTPV (SEQ ID NO: 1).

8. The method of any preceding claim, wherein the peptide antagonist of P-catenin is a cell-penetrating peptide.

9. The method of any preceding claim, wherein the peptide antagonist of P-cateninis ST316.

10. The method of any preceding claim, wherein the peptide antagonist of P-catenin is administered to the subject at a dose of about 0.25-12 mg / kg.

11. The method of any preceding claim, wherein the MAPKi is selected from the group consisting of a BRAF inhibitor, an MEK inhibitor, and an EGGR inhibitor.Sapience.026.WOl PATENT12. The method of any preceding claim, wherein the MAPKi is selected from the group consisting of binimetinib, cobimetinib, dabrafenib, encorafenib, gefitinib, lapatinib, selumetinib, sorafenib, trametinib, and vemurafenib.

13. The method of any preceding claim, wherein the MAPKi is selected from the group consisting of dabrafenib, encorafenib, and vemurafenib.

14. The method of any preceding claim, wherein the MAPKi is encorafenib.

15. The method of any preceding claim, wherein the MAPKi is selective for BRAF V600E.

16. The method of any preceding claim, wherein the pharmaceutical composition comprising the MAPKi comprises more than one MAPKi.

17. The method of any preceding claim, wherein the pharmaceutical composition comprising the antagonist of P-catenin is administered intravenously.

18. The method of any preceding claim, wherein the pharmaceutical composition comprising the MAPKi is administered orally.

19. The method of any preceding claim, wherein the pharmaceutical composition comprising the antagonist of P-catenin and the pharmaceutical composition comprising the MAPKi are administered to the subject on different days.

20. The method of any one of claims 1 to 19, wherein the pharmaceutical composition comprising the antagonist of P-catenin is administered at least once weekly for at least three weeks.

21. The method of any one of claims 1 to 19, wherein the pharmaceutical composition comprising the antagonist of P-catenin is administered once every two weeks for at least four weeks.

22. A pharmaceutical composition comprising an effective amount of a peptide antagonist of P-catenin and (ii) a pharmaceutical composition comprising an effective amount of a mitogen-activated protein kinase (MAPK) pathway inhibitor for use in a method of combination therapy for treating colorectal cancer (CRC) in a subject.

23. The pharmaceutical composition of claim 22, wherein the antagonist of P-cateninis ST316.Sapience.026.WOl PATENT24. The pharmaceutical composition of claim 22, wherein the MAPKi is encorafenib.

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