Methods for predicting response to a ras(ON) inhibitor and combination therapies

Abstract

The present disclosure is directed to methods of identifying subjects as likely or unlikely to have a durable response to a RAS(ON) inhibitor based on certain characteristics of the subjects. In addition, the present disclosure provides therapies for treating cancer, including combination therapies useful for treating subjects identified as not likely to have a durable response to a RAS(ON) inhibitor monotherapy.

Description

[0001] PATENT

[0002] ATTORNEY DOCKET NO.: 51432-056WO2

[0003] METHODS FOR PREDICTING RESPONSE TO A RAS(ON) INHIBITOR AND COMBINATION THERAPIES

[0004] Field

[0005] The present disclosure provides methods for treating subjects with cancers having specific characteristics using a RAS(ON) inhibitor compound and combination therapies comprising the same. Therefore, in certain aspects, the disclosure provides methods comprising biomarker genes for identifying or classifying a subject’s response to a cancer treatment comprising a RAS(ON) inhibitor compound.

[0006] Background

[0007] Cancer remains one of the most-deadly threats to human health. In the U.S., cancer affects nearly 1 .3 million new patients each year, and is the second leading cause of death after heart disease, accounting for approximately one in four deaths.

[0008] Oncogenic mutations in RAS (KRAS, HRAS, and NRAS) proto-oncogenes drive up to 30% of human cancers, accounting for more than 200,000 new cancer cases in the US each year, most notably of non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and pancreatic ductal adenocarcinoma (PDAC). Most oncogenic RAS mutations are gain-of-function (GOF) missense alterations at hotspot codons 12, 13, or 61 that result in an impairment of GTP hydrolysis and / or acceleration of GDP-to-GTP nucleotide exchange by these small GTPases, such that the normally tightly regulated cellular equilibrium of a RAS protein shifts predominantly towards the active, GTP-bound (RAS(ON)) state. This shift drives increased oncogenic flux via activation of downstream effectors and signaling pathways linked to cell proliferation and survival. KRAS is most frequently mutated in PDAC (92% of patients), followed by CRC (49%) and NSCLC (29%), predominantly at codon 12 in these three indications. Until the recent development of direct inhibitors of KRASG12C(OFF), RAS was considered undruggable.

[0009] KRASG12C(OFF) inhibitors introduce allele-specific covalent modification of the cysteine residue of the KRASG12Cprotein in the GDP-bound inactive (RAS(OFF)) state. However, KRASG12Cinhibitors only cover a small fraction of all oncogenic RAS mutations, including the most prevalent codon 12 mutations, leaving a significant unmet medical need for inhibitors targeting most alterations in cancer. Mutant- selective inhibitors of KRASG12Dand pan-KRAS inhibitors could potentially expand the therapeutic landscape beyond KRASG12Cmutant cancers. However, most currently described inhibitors bind in the same pocket on mutant KRAS as the first-generation KRASG12Cinhibitors and selectively target the inactive, GDP-bound state of KRAS. Furthermore, resistance to KRASG12C(OFF) inhibitors inevitably and rapidly arises in most patients with numerous recurring mechanisms of escape, including the emergence of secondary RAS mutations, KRASG12Cswitch II binding pocket mutations, targeted amplification of the KRASG12Callele, and upstream receptor tyrosine kinase (RTK) activation, all of which can reactivate RAS signaling via increased levels of GTP-bound RAS. While clinical data on more recent inactive-state inhibitors are not yet available, it is anticipated that these will also be vulnerable to RAS-GTP-driven mechanisms of resistance, which could potentially be addressed through inhibition of the active, GTP- bound state of RAS in tumors cells.

[0010] While our knowledge continues to grow around resistance to KRASG12C(OFF) inhibitors, resistance to RAS(ON) inhibitors largely remains unknown. Moreover, there are no validated biomarkers PATENT

[0011] ATTORNEY DOCKET NO.: 51432-056WO2 guiding selection of subjects that are likely to benefit from a RAS(ON) inhibitor therapy or for those subjects who are not likely to respond. The present disclosure addresses this need and provides related advantages.

[0012] Summary

[0013] Compositions and methods are provided for identifying subjects likely or not likely to have a durable response to RAS(ON) inhibitor therapy (e.g., a RAS(ON) multi-selective inhibitor therapy or a RAS(ON) mutant-selective inhibitor therapy).

[0014] The present invention provides methods for identifying, predicting, classifying, and treating cancer in a subject in need thereof, based on the genotype or expression status of biomarker genes comprising RAS and CDKN2A. In particular, the disclosure relates to the use of such biomarkers to inform the use of RAS(ON) inhibitors, either as monotherapy or in combination with additional therapeutic agents, such as cell cycle inhibitors or DNA damage response (DDR) inhibitors.

[0015] In one aspect, the disclosure provides a method generally comprising: (a) determining a genotype of one or more biomarker genes comprising CDKN2A and RAS in a biological sample obtained from a subject afflicted with cancer; and (b) classifying the subject as likely or unlikely to have a durable response to a therapy comprising a RAS(ON) inhibitor based on the genotype of each of the one or more biomarker genes in the biological sample. In certain embodiments, the method further comprises obtaining the biological sample from the subject, such as a tumor sample. In some embodiments, the tumor genotype comprises a mutation in one or more of the biomarker genes, which may inactivate the gene(s). The genotype may be compared with a reference genotype and optionally reported as a score.

[0016] In some embodiments, determining the genotype comprises genomic profiling or measuring gene expression, such as detecting RNA or protein levels. In certain embodiments, the subject is classified as likely or unlikely to have a durable response to a RAS(ON) inhibitor, wherein the durable response comprises progression-free survival (PFS). In some embodiments, the cancer may be lung cancer, pancreatic cancer, or colorectal cancer, and in specific embodiments, the cancer is non-small cell lung cancer (NSCLC) or lung adenocarcinoma.

[0017] In another aspect, the disclosure provides methods of predicting duration of tumor growth inhibition in response to a therapy comprising a RAS(ON) inhibitor by detecting a RAS mutation and a CDKN2A mutation in a biological sample obtained from a subject afflicted with cancer. In related embodiments, the disclosure provides methods of predicting reduced or increased duration of tumor growth inhibition to a RAS(ON) inhibitor based on whether the tumor sample comprises a CDKN2A inactivating mutation, decreased CDKN2A copy number, or decreased CDKN2A mRNA or protein expression, or alternatively, comprises wild-type CDKN2A or normal expression of wild-type CDKN2A.

[0018] Also provided are methods of predicting resistance of tumor growth to inhibition by a RAS(ON) inhibitor based on the detection of both a RAS mutation and CDKN2A loss-of-function alterations or decreased expression.

[0019] In some embodiments, the disclosure provides methods of selecting a subject for treatment with a combination therapy comprising a RAS(ON) inhibitor and a cell cycle inhibitor or DNA damage response (DDR) inhibitor, based on the presence of a RAS mutation and a CDKN2A mutation, copy number loss, or reduced expression. In certain embodiments, a prospective patient population may be enriched for PATENT

[0020] ATTORNEY DOCKET NO.: 51432-056WO2 subjects likely to respond to RAS(ON) inhibitor therapy by performing one or more of the diagnostic methods described herein on two or more individual subjects within the population.

[0021] In another aspect, the disclosure provides methods of treating a tumor in a subject comprising a RAS mutation and CDKN2A inactivation or reduced expression, wherein the method generally comprises administering to the subject:

[0022] (a) a RAS(ON) inhibitor and

[0023] (b) a cell cycle inhibitor or a DDR inhibitor, in amounts effective to treat the tumor.

[0024] Inhibitors may be administered simultaneously or sequentially, and as a single formulation or separate formulations. The subject may be treatment-naive or have received prior cancer therapy.

[0025] The RAS(ON) inhibitor may be a RAS(ON) mutant-selective inhibitor (e.g., selective for RAS G12C, G12D, G12V, G13C, or Q61 H), such as elironrasib, zoldonrasib, RMC-5127, RMC-8839, or RMC- 0708, or a RAS(ON) multi-selective inhibitor such as daraxonrasib.

[0026] The cell cycle inhibitor may be selected from CDK inhibitors, Wee1 inhibitors, or CHK inhibitors. The DDR inhibitor may be an ATM, ATR, PARP, or DNA-PK inhibitor.

[0027] In additional aspects, methods are provided for identifying a subject for treatment with the combination of a RAS(ON) inhibitor and a cell cycle or DDR inhibitor, wherein the subject is identified based on the presence of a RAS mutation and a CDKN2A mutation, copy number loss, or reduced expression.

[0028] In one aspect, the present disclosure provides a method comprising: (a) determining a genotype of one or more biomarker genes of a biomarker panel comprising CDKN2A and RAS in a biological sample of a subject afflicted with cancer; (b) determining the genotype of one or more biomarkers genes in the biological sample; and (c) classifying a subject as likely or not likely to have a durable response to a therapy comprising a RAS(ON) inhibitor based on the genotype of each of the one or more biomarker genes in the biological sample. In certain embodiments, the biomarker panel consists of CDKN2A and RAS.

[0029] In certain embodiments, the genotype comprises a mutation in the one or more biomarker genes. In certain embodiments, the mutation inactivates the one or more biomarker genes.

[0030] In certain embodiments, the method further comprises comparing the genotype with a reference genotype. In certain embodiments, the reference is to an untreated population. In certain embodiments, the reference is to a treated population. In certain embodiments, the genotype is reported as a score.

[0031] In certain embodiments, determining the genotype comprises genomic profiling. In certain embodiments, determining the genotype comprises measuring gene expression. In certain embodiments, measuring gene expression comprises detection of ribonucleic acids (RNAs) or polypeptides.

[0032] In certain embodiments, the subject is classified as likely to have a durable response to a RAS(ON) inhibitor treatment. In certain embodiments, the subject is classified as not likely to have a durable response to a RAS(ON) inhibitor treatment.

[0033] In certain embodiments, the RAS(ON) inhibitor comprises a small molecule comprising a cyclophilin A binding motif and a RAS binding motif. In certain embodiments, the RAS(ON) inhibitor is a RAS(ON) multi-selective inhibitor. In certain embodiments, the RAS(ON) inhibitor is selected from the group consisting of RMC-6236 and RMC-7977. PATENT

[0034] ATTORNEY DOCKET NO.: 51432-056WO2

[0035] In certain embodiments, the methods of the present disclosure further comprise administering a RAS(ON) inhibitor therapy to the subject. In certain embodiments, the methods comprise administering to the subject a combination therapy comprising a RAS(ON) inhibitor and one or more additional therapeutic agents. In certain embodiments, the methods comprise administering to the subject a combination therapy comprising a RAS(ON) inhibitor and a second therapy. In certain embodiments, the second therapy is a cell cycle inhibitor. In certain embodiments, the cell cycle inhibitor is a CDK4 / 6 inhibitor. In certain embodiments, the second therapy is a DNA damage response (DDR) inhibitor.

[0036] In one aspect, the present disclosure provides a method of predicting resistance of tumor growth to inhibition by a therapy comprising a RAS(ON) inhibitor, comprising: (a) detecting in a tumor sample of a subject a genotype of one or more biomarker genes; (b) analyzing the genotype of the one or more biomarker genes in the tumor sample; and (c) predicting resistance of tumor cell growth to inhibition by the therapy comprising a RAS(ON) inhibitor, if the tumor sample comprises (i) an inactivating CDKN2A mutation, (ii) a decreased copy number CDKN2A, or (iii) a decreased expression of CDKN2A mRNA or protein.

[0037] In one aspect, the present disclosure provides a method of treating a cancer (e.g., a non-small cell lung cancer (NSCLC), a pancreatic cancer, or a colorectal cancer (CRC)) comprising the step of treating a subject with a combination therapy comprising a RAS(ON) inhibitor and a second therapy when a tumor sample obtained from the subject comprises (i) a RAS mutation and (ii) an inactivating CDKN2A mutation, a decreased copy number CDKN2A, or a decreased expression of CDKN2A mRNA or protein.

[0038] In one aspect, the present disclosure provides a method of enriching a prospective patient population for subjects likely to have a durable response to a RAS(ON) inhibitor therapy, for example, a RAS(ON) multi-selective inhibitor or RAS(ON) G12C-selective inhibitor therapy, the method comprising performing the methods described herein of predicting resistance and / or response of tumor growth to inhibition by a RAS(ON) inhibitor therapy on two or more individual subjects within the prospective patient population.

[0039] In one aspect, the present disclosure provides a method of treating a cancer, comprising treating a subject undergoing a therapy comprising a RAS(ON) inhibitor, the method comprising: administering a second therapy when a tumor sample obtained from the subject comprises (i) a RAS mutation and (ii) an inactivating CDKN2A mutation, a decreased copy number CDKN2A, or a decreased expression of CDKN2A mRNA or protein. In certain embodiments, the RAS(ON) inhibitor is RMC-6236 or RMC-7977. In certain embodiments, the second therapy comprises a cell cycle inhibitor or a DDR inhibitor. In certain embodiments, the RAS mutation is a RASG12Xmutation.

[0040] It is specifically contemplated that any limitation discussed with respect to one embodiment of the disclosure may apply to any other embodiment of the disclosure. Furthermore, any compound or composition disclosed herein may be used in any method of the disclosure, and any method of the disclosure may be used to utilize any compound or composition of the disclosure.

[0041] Brief Description of the Figures

[0042] FIG. 1A, FIG. 1B, and FIG. 1C shows broad spectrum antitumor activity of RAS(ON) multi- selective inhibitor, daraxonrasib (RMC-6236) in preclinical models of RAS-addicted cancers. FIG. 1A shows tumor response waterfall plots of KRASG12XNSCLC xenograft models upon daraxonrasib daily PATENT

[0043] ATTORNEY DOCKET NO.: 51432-056WO2 treatment at 25 mg / kg. FIG. 1B shows tumor response waterfall plots of KRASG12XPDAC xenograft models upon daraxonrasib daily treatment at 25 mg / kg. FIG. 1C shows tumor response waterfall plots of KRASG12XCRC xenograft models upon daraxonrasib daily treatment at 25 mg / kg. 29 NSCLC, 22 PDAC and 23 CRC xenograft models were included (n = 1-10 per model). Average % mean tumor volume change ± SEM from baseline at response calling date are shown. mRECIST criteria were used to call tumor response as indicated on right hand side of each waterfall plot. Oncoplots illustrating gene alterations and expression levels in critical genes linked to the clinopathologic characteristics of the indicated models are shown below each waterfall. Color coding represents dark-green for mutations and light-green for the absence of mutations. The symbol denotes that mRNA corresponding genes not expressed, defined as having a gene expression value of < 0.5 CPM. The top row specifically highlights the mutation codon at KRASG12.

[0044] FIG. 2 shows a Kaplan-Meier analysis of KRASG12XNSCLC models with and without CDKN2A expression loss upon daily treatment of vehicle control or daraxonrasib (RMC-6236) at 25 mg / kg for up to 90 days. 29 KRASG12XNSCLC models were included in the analysis. 10 models with CDKN2A loss (n = 44 in both control and daraxonrasib treatment group) and 19 models without CDKN2A loss (n = 91 in both control and daraxonrasib treatment group) were included. Log-rank (Mantel-Cox) test and Cox Proportional Hazards model were used to compare predictive effect of indicated co-modifier on PFS of daraxonrasib treated subjects. KRASG12XNSCLC models with CDKN2A loss exhibited significantly shorter PFS (log-rank ***p < 0.001 , Cox Proportional Hazard Ratio 3.60, 95% interval 1.88-6.89) compared with those without CDKN2A loss.

[0045] FIG. 3 shows a Kaplan-Meier analysis of KRASG12CNSCLC models with and without CDKN2A expression loss upon daily treatment of vehicle control or elironrasib (RMC-6291).

[0046] FIG. 4 shows a Kaplan-Meier analysis of KRASG12XNSCLC models with and without KEAP1 mutation, 5 KEAP1MUTmodels (n = 26 in control, n = 28 in daraxonrasib (RMC-6236) treatment group) and 24 KEAP1WTmodels (n = 109 in control, n = 107 in daraxonrasib treatment group) were included. Log-rank (Mantel-Cox) test and Cox Proportional Hazards model were used to compare predictive effect of indicated co-modifier on PFS of daraxonrasib treated subjects. KRASG12XNSCLC models with KEAP1MUTexhibited significantly shorter PFS (log-rank *p < 0.05, Cox Proportional Hazard Ratio 2.10, 95% interval 1.10-4.03) compared with KEAP1WTmodels.

[0047] FIG. 5 shows a Kaplan-Meier analysis of KRASG12XNSCLC models with and without SMARCA mutation, 8 SMARCA4MUTmodels (n = 33 in both control and daraxonrasib (RMC-6236) treatment group) and 21 SMARCA4WTmodels (n = 102 in both control and daraxonrasib treatment group) were included. Log-rank (Mantel-Cox) test and Cox Proportional Hazards model were used to compare predictive effect of indicated co-modifier on PFS of daraxonrasib treated subjects.

[0048] Detailed Description

[0049] Emerging clinical data in patients with RAS-mutant cancers treated with RAS(ON) inhibitors have demonstrated evidence of clinical activity, including tumor regressions. However, biomarkers capable of predicting or guiding the selection of subjects most likely to benefit from RAS(ON) inhibitor therapy have not yet been identified and remain poorly understood. PATENT

[0050] ATTORNEY DOCKET NO.: 51432-056WO2

[0051] Accordingly, the present disclosure is based, at least in part, on the unexpected discovery that the genotype of specific biomarker genes can be used to predict a subject’s response, such as the durability of response, to a RAS(ON) inhibitor therapy. In certain embodiments, the genotype is predictive of the duration of tumor growth inhibition in response to a RAS(ON) inhibitor. In certain other embodiments, the genotype is predictive of reduced progression-free survival following treatment with a RAS(ON) inhibitor. As such, the present disclosure provides advantageous methods for determining whether a subject afflicted with a tumor (e.g., a cancer) is a candidate for monotherapy or combination therapy comprising a RAS(ON) inhibitor.

[0052] The disclosure is further based, in part, on the discovery that the presence of a RAS mutation in combination with CDKN2A loss is sensitivity to combination therapy comprising a RAS(ON) inhibitor and a cell cycle inhibitor (e.g., a cyclin-dependent kinase (CDK) inhibitor). Additionally, tumors harboring a RAS mutation and CDKN2A loss are also sensitive to combination therapy comprising a RAS(ON) inhibitor and a DNA damage response (DDR) inhibitor. These discoveries have important implications for stratifying patients who are receiving RAS(ON) inhibitor therapy and identifying those who may benefit from the addition of a cell cycle inhibitor or DDR inhibitor to their treatment regimen.

[0053] In some aspects, the present disclosure provide a methods generally comprising: determining the genotype of one or more biomarker genes in a biological sample obtained from a subject; and identifying whether the subject is likely to be sensitive to a therapy comprising a RAS(ON) inhibitor based on the genotype.

[0054] In certain embodiments, the method further comprises administering a RAS(ON) inhibitor to the subject if the subject is identified as likely to be sensitive to the therapy.

[0055] In some embodiments, the biomarker genes useful in the methods described herein include CDKN2A and RAS. The genotype may indicate, for example, the presence of a RAS mutation and / or loss-of-function alteration in CDKN2A. Identification of such genotype(s) may support the administration of a RAS(ON) inhibitor either as monotherapy or in combination with a CDK inhibitor or DDR inhibitor.

[0056] General Methods

[0057] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell culturing, molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, third edition (Sambrook et al., 2001) Cold Spring Harbor Press; Oligonucleotide Synthesis (P. Herdewijn, ed., 2004); Animal Cell Culture (R. I. Freshney), ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir & C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Manual of Clinical Laboratory Immunology (B. Detrick, N. R. Rose, and J. D. Folds eds., 2006); Immunochemical Protocols (J. Pound, ed., 2003); Lab Manual in Biochemistry: Immunology and Biotechnology (A. Nigam and A. Ayyagari, eds. 2007); Immunology Methods Manual: The PATENT

[0058] ATTORNEY DOCKET NO.: 51432-056WO2

[0059] Comprehensive Sourcebook of Techniques (Ivan Lefkovits, ed., 1996); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, eds.,1988); and others.

[0060] Definitions

[0061] In this application, unless otherwise clear from context, (i) the term “a” means “one or more”; (ii) the term "or" is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or”; (iii) the terms “comprising” and “including” are understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) where ranges are provided, endpoints are included.

[0062] As used herein, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. In certain embodiments, the term “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of a stated value, unless otherwise stated or otherwise evident from the context (e.g., where such number would exceed 100% of a possible value).

[0063] As used herein, the term “administration” refers to the administration of a composition comprising a RAS(ON) inhibitor compound to a subject or system. Administration also includes administering a prodrug derivative or analog or pharmaceutically acceptable salt to the subject, which can form an equivalent amount of active compound within the subject’s body. Administration to an animal subject (e.g., to a human) may be by any appropriate route. For example, in some embodiments, administration may be bronchial (including by bronchial instillation), buccal, enteral, intradermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal or vitreal. In some embodiments, a composition comprising the RAS(ON) inhibitor compound is administered orally.

[0064] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series and any one or any and all combinations of the elements.

[0065] A "biomarker gene" can be any gene having a genotype and / or expression level that can be determined, measured and / or evaluated as an indicator of a biologic process, pathogenic process, or pharmacologic response to a therapeutic intervention. A biomarker gene useful to practice the presently disclosed methods can be used as an indicator to determine whether a subject having a tumor (e.g., cancer) will be sensitive or resistant to a therapy comprising a RAS(ON) inhibitor and / or for monitoring response to a treatment with a therapy comprising a RAS(ON) inhibitor. In certain embodiments, a biomarker gene is a tumor suppressor gene. In certain embodiments, a biomarker gene is a oncogenic driver gene. Sensitivity or resistance to a therapy with a RAS(ON) inhibitor can be determined by analyzing a nucleic acid molecule (DNA, mRNA, cDNA etc.) corresponding to a biomarker gene or the protein encoded by the biomarker gene. Biomarker genes can include any gene whose genotype and / or level of expression in a tissue or cell can be used to predict response to a RAS(ON) inhibitor therapy. The detection, and in some cases the level, of one or more biomarker genes of the present disclosure permits PATENT

[0066] ATTORNEY DOCKET NO.: 51432-056WO2 the classification of a subject as likely or unlikely to have a durable response (e.g., progression free survival) to a RAS(ON) inhibitor therapy.

[0067] The term “combination therapy” refers to a method of treatment including administering to a subject at least two active therapeutic agents (e.g., a RAS(ON) inhibitor compound and a cell cycle inhibitor or DNA damage response inhibitor), as one or more pharmaceutical compositions, as part of a therapeutic regimen. For example, a combination therapy may include administration of a single pharmaceutical composition including at least two therapeutic agents and one or more pharmaceutically acceptable carrier, excipient, diluent, or surfactant. A combination therapy may include administration of two or more pharmaceutical compositions, each composition including one or more therapeutic agent and one or more pharmaceutically acceptable carrier, excipient, diluent, or surfactant. The two or more agents may optionally be administered simultaneously (as a single or as separate compositions) or sequentially (as separate compositions). The therapeutic agents may be administered in an effective amount. The therapeutic agent may be administered in a therapeutically effective amount. In some embodiments, the effective amount of one or more of the therapeutic agents may be lower when used in a combination therapy than the therapeutic amount of the same therapeutic agent when it is used as a monotherapy, e.g., due to an additive or synergistic effect of combining the two or more therapeutics.

[0068] As used herein, the terms "determine", "determine the genotype of a biomarker gene", "determine the level of a biomarker gene", "determine the amount of a biomarker gene”, "determine the biomarker gene level", and the like are meant to encompass any technique that can be used to detect or measure the genotype, presence, or expression level of one or more biomarker genes. Such techniques can give qualitative or quantitative results. Biomarker gene levels can be determined by detecting the entire biomarker molecule or by detecting fragments or reaction products that are characteristic of the biomarker gene. The terms determining, measuring, or taking a measurement refer to a quantitative or qualitative determination of a property of an entity, for example, quantifying the amount or concentration of a molecule or the activity level of a molecule. Any known method of detecting or measuring the level of a biomarker can be used to practice the present disclosure, so long as the method detects the genotype, presence, absence, or expression level of the biomarker gene.

[0069] In certain embodiments, determining the genotype of a biomarker gene is performed at the nucleic acid level by performing RNA-seq, a reverse transcriptase polymerase chain reaction (RT-PCR) or a hybridization assay with oligonucleotides that are substantially complementary to portions of cDNA molecules of the at least one biomarker gene under conditions suitable for RNA-seq, RT-PCR or hybridization and obtaining expression levels of the at least one biomarker gene.

[0070] As used herein, the term “dosage form” refers to a physically discrete unit of a compound (e.g., the RAS(ON) inhibitor compound) for administration to a subject. Each unit contains a predetermined quantity of compound. In some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). Those of ordinary skill in the art appreciate that the total amount of a therapeutic composition or compound administered to a particular subject is determined by one or more attending physicians and may involve administration of multiple dosage forms. PATENT

[0071] ATTORNEY DOCKET NO.: 51432-056WO2

[0072] As used herein, the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic compound (e.g., the RAS(ON) inhibitor compound) has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen includes a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen includes a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen includes a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen includes a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen or “therapy”).

[0073] The term “disorder” is used in this disclosure to mean, and is used interchangeably with, the terms disease, condition, or illness, unless otherwise indicated.

[0074] The terms "inhibit," "block," and "suppress" are used interchangeably and refer to any statistically significant decrease in a biological activity, including full blocking of the activity. As used herein, the term “inhibitor” refers to a compound that prevents a biomolecule, (e.g., a protein, nucleic acid) from completing or initiating a reaction. An inhibitor can inhibit a reaction by competitive, uncompetitive, or noncompetitive means, for example. With respect to its binding mechanism, an inhibitor may be an irreversible inhibitor or a reversible inhibitor. Exemplary inhibitors include, but are not limited to, nucleic acids, DNA, RNA, shRNA, siRNA, proteins, protein mimetics, peptides, peptidomimetics, antibodies, small molecules, chemicals, analogs that mimic the binding site of an enzyme, receptor, or other protein. In some embodiments, the inhibitor is a small molecule, e.g., a low molecular weight organic compound, e.g., an organic compound having a molecular weight (MW) of less than 1200 Daltons (Da). In some embodiments, the MW is less than 1100 Da. In some embodiments, the MW is less than 1000 Da. In some embodiments, the MW is less than 900 Da. In some embodiments, the range of the MW of the small molecule is between 800 Da and 1200 Da. Small molecule inhibitors include cyclic and acyclic compounds. Small molecules inhibitors include natural products, derivatives, and analogs thereof. Small molecule inhibitors can include a covalent cross-linking group capable of forming a covalent cross-link, e.g., with an amino acid side-chain of a target protein.

[0075] The term “mutation” as used herein indicates any modification of a nucleic acid or polypeptide which results in an altered nucleic acid or polypeptide. The term “mutation” may include, for example, point mutations, deletions or insertions of single or multiple residues in a polynucleotide, which includes alterations arising within a protein-encoding region of a gene as well as alterations in regions outside of a protein-encoding sequence, such as, but not limited to, regulatory or promoter sequences, as well as amplifications or chromosomal breaks or translocations. In particular embodiments, the mutation results in an amino acid substitution in the encoded-protein.

[0076] As used herein “patient” or “subject” are used interchangeably and refer to a mammal, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include, but are not limited to, humans, PATENT

[0077] ATTORNEY DOCKET NO.: 51432-056WO2 domestic animals, farm animals, sports animals, and zoo animals including, for example, humans, nonhuman primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and cattle. In certain embodiments, the subject has been diagnosed with cancer. In certain embodiments, the subject is a human afflicted with a tumor (e.g., cancer) who has been diagnosed with a need for treatment for a tumor (e.g., cancer).

[0078] As used herein, the term “pharmaceutical composition” refers to a compound, such as a RAS(ON) inhibitor compound disclosed herein, or a pharmaceutically acceptable salt thereof, formulated together with a pharmaceutically acceptable excipient.

[0079] A “pharmaceutically acceptable excipient,” as used herein, refers to any inactive ingredient (for example, a vehicle capable of suspending or dissolving the active compound) having the properties of being nontoxic and noninflammatory in a subject. Typical excipients include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, or waters of hydration. Excipients include, but are not limited to: butylated optionally substituted hydroxyltoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, optionally substituted hydroxylpropyl cellulose, optionally substituted hydroxylpropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol. Those of ordinary skill in the art are familiar with a variety of agents and materials useful as excipients. See, e.g., Ansel, et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, et al., Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. In some embodiments, a composition includes at least two different pharmaceutically acceptable excipients.

[0080] The term “pharmaceutically acceptable salt,” as use herein, refers to those salts of the compounds described herein that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:119, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), WileyVCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting the free base group with a suitable organic acid.

[0081] The terms “RAS pathway” and “RAS / MAPK pathway” are used interchangeably herein to refer to a signal transduction cascade downstream of various cell surface growth factor receptors in which activation of RAS (and its various isoforms and allotypes) is a central event that drives a variety of cellular effector events that determine the proliferation, activation, differentiation, mobilization, and other functional properties of the cell. For example, SHP2 conveys positive signals from growth factor receptors PATENT

[0082] ATTORNEY DOCKET NO.: 51432-056WO2 to the RAS activation / deactivation cycle, which is modulated by guanine nucleotide exchange factors (GEFs, such as SOS1) that load GTP onto RAS to produce functionally active GTP-bound RAS as well as GTP-accelerating proteins (GAPs, such as NF1) that facilitate termination of the signals by conversion of GTP to GDP. GTP-bound RAS produced by this cycle conveys essential positive signals to a series of serine / threonine kinases including RAF and MAP kinases, from which emanate additional signals to various cellular effector functions.

[0083] The terms “RAS inhibitor” and “inhibitor of [a] RAS” are used interchangeably to refer to any inhibitor that targets, that is, selectively binds to or inhibits a RAS protein. A RAS inhibitor may be RO7673396, for example.

[0084] As used herein, the term “RAS(ON) inhibitor” refers to an inhibitor that targets, that is, selectively binds to or inhibits, the GTP-bound, active state of RAS (e.g., selective over the GDP-bound, inactive state of RAS). Inhibition of the GTP-bound, active state of RAS includes, for example, the inhibition of oncogenic signaling from the GTP-bound, active state of RAS. In some embodiments, the RAS(ON) inhibitor is an inhibitor that selectively binds to and inhibits the GTP-bound, active state of RAS. In certain embodiments, RAS(ON) inhibitors may also bind to or inhibit the GDP-bound, inactive state of RAS (e.g., with a lower affinity or inhibition constant than for the GTP-bound, active state of RAS). In certain embodiments, a RAS(ON) inhibitor useful in the present disclosure may form a high affinity three- component complex, or conjugate, between a synthetic ligand and two intracellular proteins which do not interact under normal physiological conditions: the target protein of interest (e.g., RAS), and a widely expressed cytosolic chaperone (presenter protein) in the cell (e.g., cyclophilin A). More specifically, in some embodiments, the inhibitors of RAS described herein induce a new binding pocket in RAS by driving formation of a high affinity tri-complex, or conjugate, between the RAS protein and the widely expressed cytosolic chaperone, cyclophilin A (CYPA). A RAS(ON) inhibitor may be an antibody-drug conjugate. See also doi.org / 10.1021 / acs.jmedchem.4c02929.

[0085] As used herein, the term “RAS(OFF) inhibitor” refers to an inhibitor that targets, that is, selectively binds to or inhibits, the GDP-bound, inactive state of RAS (e.g., selective over the GTP-bound, active state of RAS). RAS(OFF) inhibitors are known in the art and described. Non-limiting examples of RAS(OFF) inhibitors include A2A-03, ABREV01 , ABT-200, ADT-030, ADT-1004, AN9025, BBP-454, BGB-53038, BI-2865, BI-2493, Bl 3706674, ERAS-4, ERAS-254, ERAS-4001 , HB-700 (G12X+G13D), JAB-23400, OC211 , PF-07934040, QTX3034, RSC-1255, YL-17231 , ZG2001 , PF-07985045, ADT-007, SIL204, and HZ-V068. Non-limiting examples of RASG12C(OFF) inhibitors include adagrasib (MRTX849), divarasib (RG6330 / GDC-6036), fulzerasib (IBI351 / GFH925), garsorasib (D-1553), glecirasib (JAB- 21822), olomorasib (LY3537982), opnurasib (JDQ443), sotorasib (AMG 510), ARS-853, ARS-1620, Bl 1823911 , BPI-421286, D3S-001 , GEC255, HBI-2438, HS-10370, JAB-21000, JAB-21822, JMKX001899, JNJ-74699157 (ARS-3248), MK-1084, YL-15293, SK-17, and BI-0474. Non-limiting examples of RASG12D(OFF) inhibitors include ASP3082, BPI-501836, ERAS-4693, ERAS-5024, HBW-012-D, HBW- 012-E, HBW-012336, HRS-4642, JAB-22000, KD-8, TSN1611 , LY3962673, MRTX282, MRTX1133, Q2a, SHR1127, TH-Z827, TH-Z835, TSN1611 , VRTX153, DN022150, GDC-7035, AZD0022, RNK08954, INCB186748, AST2169, and QLC1101. Non-limiting examples of RASG12V(OFF) inhibitors include JAB- 23000 and QTX3544. PATENT

[0086] ATTORNEY DOCKET NO.: 51432-056WO2

[0087] As used herein, the terms “RAS(ON) multi-selective inhibitor,” “RASMULTI inhibitor,” “RASMULTI(ON) inhibitor,” and “RAS(MULTI) inhibitor” refer to a RAS inhibitor of at least three RAS isoforms, including wild-type and / or variants with missense mutations at one of the following positions: 12, 13, 59, 61 , or 146. In some embodiments, a RAS(ON) multi-selective inhibitor (e.g., daraxonrasib or RMC-6236) refers to a RAS inhibitor of at least three RAS variants with missense mutations at one of the following positions: 12, 13, and 61. Exemplary RAS(ON) multi-selective inhibitors useful in combinations according to the present disclosure can be found in any one of the following patent applications: WO 2025051241 , WO 2025045233, WO 2024249299, WO 2024222864, WO 2024206858, WO 2024169914, WO 2024153208, WO 2024149214, WO 2024104364, WO 2024067857, WO 2024060966, WO 2024017859, WO 2024008834, WO 2023240263, WO 2023025832, WO 2022060836, WO 2021091956, CN119350371 , ON 117903169, ON 117720556, ON 117720555, ON 117720554, ON 117534687, ON 117534685, and ON 117534684, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein. In some embodiments, the RAS(ON) multi-selective inhibitor is one or more of daraxonrasib (RMC-6236), RMC-7977, RM-034, GFH547, ERAS-0015, Bl- 2852, BPI-572270, RCZY-690, RCZY-680 and compound 6A of WO 2024 / 067857.

[0088] As used herein, the terms “RAS(ON) mutant-selective inhibitor” refers to a RAS inhibitor selective for a RAS(ON) variant with missense mutation at one of the following positions: 12, 13, or 61. Non-limiting examples of RAS(ON) mutant-selective inhibitors include RAS(ON) G12C-selective inhibitors (e.g., elironrasib or RMC-6291), RAS(ON) G12D-selective inhibitors (e.g., zoldonrasib or RMC-9805), RAS(ON) Q61 H-selective inhibitors (e.g., RMC-0708), RAS(ON) G12V-selective inhibitors (e.g. RMC-5127), and RAS(ON) G13D-selective inhibitors. RAS(ON) mutant-selective inhibitors useful according to the methods of the present disclosure, can be found in any one of the following patent applications: WO 2024249299, WO 2024211663, WO 2024211712, WO 2024208934, WO 2024149819, WO 2024008610, WO 2024102421 , WO 2023240263, WO 2023133543, WO 2023015559, WO 2023086341 , WO 2023208005, WO 2023232776, WO 2023086341 , WO 2023060253, WO 2023015559, WO 2022235870, WO 2022235864, WO 2021091967, WO 2021091982, WO 2021108683, WO 2020132597, International Patent Application Numbers PCT / US2025 / 015061 and PCT / US2024 / 50922, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein.

[0089] A “therapeutic agent” is any substance, e.g., a compound or composition, capable of treating a disease or disorder. In some embodiments, therapeutic agents that are useful in connection with the present disclosure including RAS inhibitors and cancer chemotherapeutics. Many such therapeutic agents are known in the art and are disclosed herein.

[0090] The term “therapeutically effective amount” means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence or severity of, or delays onset of, one or more symptoms of the disease, disorder, or condition. Those of ordinary skill in the art will appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. It is specifically understood that particular PATENT

[0091] ATTORNEY DOCKET NO.: 51432-056WO2 subjects may, in fact, be “refractory” to a “therapeutically effective amount.” In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder, or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount may be formulated or administered in a single dose. In some embodiments, a therapeutically effective amount may be formulated or administered in a plurality of doses, for example, as part of a dosing regimen.

[0092] The term “treatment” (also “treat” or “treating”), in its broadest sense, refers to any administration of a substance (e.g., a RAS(ON) inhibitor compound) that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of, or reduces incidence of one or more symptoms, features, or causes of a particular disease, disorder, or condition. In some embodiments, such treatment may be administered to a subject who is diagnosed with the disease, disorder or condition but does not exhibit signs of the relevant disease, disorder, or condition or of a subject who exhibits only early signs of the disease, disorder, or condition. Alternatively, or additionally, in some embodiments, treatment may be administered to a subject who exhibits one or more established signs of the relevant disease, disorder or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, or condition. In any treatment method herein, a patient or subject may be in need of such treatment.

[0093] The term “wild-type” refers to an entity having a structure or activity as found in nature in a “normal” (as contrasted with mutant, diseased, altered, etc.) state or context. Those of ordinary skill in the art will appreciate that wild-type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0094] I. Compositions

[0095] Provided herein are compositions that inhibit RAS (e.g., RAS(ON) inhibitor compounds) and uses thereof. Also provided are pharmaceutical compositions including one or more such compounds, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. RAS(ON) inhibitor compounds may be used in methods of modulating RAS (e.g., in a subject or in a cell) and methods of treating cancer, as described herein. The present disclosure provides, inter alia, compositions, methods, and kits for treating RAS mutant cancer comprising a RAS(ON) inhibitor compound alone or in combination with a cell cycle inhibitor or DNA damage response inhibitor based on the biomarkers of the subject.

[0096] (a) RAS(ON) inhibitors

[0097] Compositions of the present disclosure may include one or more RAS(ON) inhibitor compounds. A RAS(ON) inhibitor compound of the present disclosure forms a high affinity tri-complex with two intracellular proteins which do not interact under normal physiological conditions: RAS and a widely expressed cytosolic chaperone in the cell, cyclophilin A (CypA). RAS(ON) inhibitors disclosed herein may be administered or formulated in combination with an additional therapeutic agent described herein.

[0098] In some embodiments, the present disclosure provides non-covalent binding of RAS by a RAS(ON) multi-selective inhibitor. PATENT

[0099] ATTORNEY DOCKET NO.: 51432-056WO2

[0100] One or more RAS(ON) multi-selective inhibitors useful according to the present disclosure can be found in any of the following patent applications: WO 2025051241 , WO 2025045233, WO 2024249299, WO 2024222864, WO 2024206858, WO 2024169914, WO 2024153208, WO 2024149214, WO 2024104364, WO 2024067857, WO 2024060966, WO 2024017859, WO 2024008834, WO 2023240263, WO 2023025832, WO 2022060836, WO 2021091956, CN119350371 , ON 117903169, ON 117720556, ON 117720555, ON 117720554, ON 117534687, ON 117534685, and ON 117534684, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein. In some embodiments, the RAS(ON) multi-selective inhibitor is one or more of daraxonrasib (RMC-6236), RMC-7977, RM-034, GFH547, ERAS-0015, BI-2852, BPI-572270, RCZY-690, RCZY-680 and compound 6A of WO 2024067857. In some embodiments, the RAS(ON) multi-selective inhibitor useful according to the present disclosure is daraxonrasib (RMC-6236)

[0101] The RAS(ON) multi-selective compounds useful according to the present disclosure exhibit inhibitory activities across a variety of RAS mutants. In some embodiments, a RAS(ON) multi-selective compound inhibits wild type RAS. In some embodiments, a RAS(ON) multi-selective compound inhibits wild type KRAS. In some embodiments, a RAS(ON) multi-selective compound inhibits a RAS mutant with one or more mutations at G12X, G13X, and / or Q61X, wherein X represents any naturally occurring amino acid residue. In certain instances, X is A, C, D, V, S, R, H, K, or L amino acid residue.

[0102] In certain embodiments, a RAS(ON) multi-selective compound inhibits a RAS mutant with one or more mutations at G12X, wherein X represents any naturally occurring amino acid residue. In certain instances, X is A, C, D, V, S or R amino acid residue.

[0103] In other embodiments, a RAS(ON) multi-selective compound inhibits a RAS mutant with one or more mutations at G13X, wherein X is any naturally occurring amino acid residue. In certain instances, X is A, C, D, V, S or R amino acid residue.

[0104] In other embodiments, a RAS(ON) multi-selective compound inhibits a RAS mutant with one or more mutations at Q61X, wherein X is any naturally occurring amino acid residue. In certain instances, X is A, C, D, V, S, R, H, K, or L amino acid residue. In other instances, X is H, K, R, or L amino acid residue.

[0105] A variety of RAS proteins may be inhibited by a RAS(ON) multi-selective compound (e.g., KRAS, NRAS, HRAS, and mutants thereof at positions 12, 13 and 61 , such as G12A, G12C, G12D, G12V, G12S, G12R, G13C, G13D, Q61 H, Q61 K, Q61 R and Q61 L, and others described herein, or a combination thereof). In some embodiments, a RAS(ON) multi-selective compound inhibits a G12A, G12C, G12D, G12R, G12S, G12V, or Q61 H mutant of RAS, or a combination thereof.

[0106] Compositions and methods described herein may include one or more RAS(ON) mutant-selective inhibitors. Numerous RAS(ON) mutant-selective inhibitors have been disclosed. PATENT

[0107] ATTORNEY DOCKET NO.: 51432-056WO2

[0108] In some embodiments, the RAS(ON) mutant-selective inhibitor is a RAS(ON) G12C-selective inhibitor. In some embodiments, the RAS(ON) mutant-selective inhibitor is a RAS(ON) G12D-selective inhibitor. In some embodiments, the RAS(ON) mutant-selective inhibitor is a RAS(ON) G13C-selective inhibitor. In some embodiments, the RAS(ON) mutant-selective inhibitor is a RAS(ON) Q61 H-selective inhibitor. In some embodiments, the RAS(ON) mutant-selective inhibitor is a RAS(ON) G12V-selective inhibitor. In some embodiments, the RAS(ON) mutant-selective inhibitor is a RAS(ON) G13D-selective inhibitor. RAS(ON) mutant-selective inhibitors useful according to the methods of the present disclosure can be found in any one of the following patent applications: WO 2024249299, WO 2024211663, WO 2024211712, WO 2024208934, WO 2024149819, WO 2024008610, WO 2024102421 , WO 2023240263, WO 2023133543, WO 2023015559, WO 2023086341 , WO 2023208005, WO 2023232776, WO 2023086341 , WO 2023060253, WO 2023015559, WO 2022235870, WO 2022235864, WO 2021091967, WO 2021091982, WO 2021108683, WO 2020132597, International Patent Application Numbers PCT / US2025 / 015061 and PCT / US2024 / 50922, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein.

[0109] In some embodiments, the RAS(ON) mutant-selective inhibitor useful according to the present disclosure is zoldonrasib (RMC-9805), a G12D-selective inhibitor

[0110] In some embodiments, the RAS(ON) mutant-selective inhibitor is elironrasib (RMC-6291), a

[0111] G12C-selective inhibitor

[0112] In some embodiments, the RAS(ON) mutant-selective inhibitor is RMC-5127, a G12V-selective PATENT

[0113] ATTORNEY DOCKET NO.: 51432-056WO2

[0114] In some embodiments, the RAS(ON) mutant-selective inhibitor is RMC-8839, a G13C-selective inhibitor. In some embodiments, the RAS(ON) mutant-selective inhibitor is RMC-0708, a Q61 H-selective inhibitor.

[0115] The RAS(ON) inhibitor compounds described herein may be made from commercially available starting materials or synthesized using known organic, inorganic, or enzymatic processes. By way of example, the RAS(ON) compounds can be synthesized using the methods described in WO 2022060836, WO 2021091956, or WO 2021091982, together with synthetic methods known in the art of synthetic organic chemistry, or variations thereon as appreciated by those skilled in the art.

[0116] (b) Cell Cycle Inhibitors

[0117] Compositions and methods described herein may include a cell cycle inhibitor. A cell cycle inhibitor may be administered or formulated in combination with a RAS(ON) inhibitor described herein. A cell cycle inhibitor may be administered or formulated in combination with a RAS(ON) inhibitor described herein. i) CDK inhibitors

[0118] In certain embodiments, a cell cycle inhibitor is a cyclin-dependent kinase (CDK) inhibitor. Cyclin- dependent kinases are a family of protein kinases that regulate cell division and proliferation. Cell cycle progression is controlled by cyclins and their associated cyclin-dependent kinases, such as CDK1 , CDK2, CDK3, CDK4 and CDK6, while other CDKs such as CDK7, CDK8 and CDK9 are critical to transcription. CDK binding to cyclins forms heterodimeric complexes that phosphorylate their substrates on serine and threonine residues, which in turn initiates events required for cell-cycle transcription and progression. In some embodiments, a CDK inhibitor is a CDK2 inhibitor. In some embodiments, a CDK inhibitor is a CDK4 / 6 inhibitor. In some embodiments, a CDK inhibitor is a CDK7 inhibitor. In some embodiments, a CDK inhibitor is a CDK9 inhibitor. In some embodiments, a CDK inhibitor is one or more palbociclib > . In some embodiments, a CDK inhibitor is one or more of tagtociclib (PF-07104091) PATENT

[0119] ATTORNEY DOCKET NO.: 51432-056WO2 dinaciclib

[0120] AZD4573

[0121] In some embodiments, reference to the term CDK inhibitor includes any such CDK inhibitor disclosed in any one of the following patent applications: WO 2025040170, WO 2025060620, WO 2024238574, WO 2024027825, WO 2024048541 , WO 2022166793, WO 2022187611 , WO 2022130304, WO 2021227906, WO 2021057867, WO 2020207260, WO 2020138370, WO 2020125513, WO 2020093011 , WO 2020148635, WO 2020215156, WO 2020052627, WO 2017177837, WO 2017162215, WO 2017177836, WO 2017172826, WO 2016193939, WO 2016014904, WO 2016015598, WO 2016015605, WO 2015181737, WO 2012061156 A1 , WO 2012038411 , WO 2010020675, WO 2010125004, WO 2007139732, WO 2006024945, ON 114478529, ON 108794496, ON 105294737, CN107652284, KR 20180106188, and US 2017152269, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference. ii) Wee1 inhibitors

[0122] In some embodiments, compositions and methods described herein may include one or more Wee1 inhibitors. A Wee1 inhibitor may be administered or formulated in combination with a RAS(ON) inhibitor and / or any additional therapeutic agent described herein. Wee1 is a kinase that plays a critical role in regulating the cell cycle by inhibiting the activity of cyclin-dependent kinases (CDKs) and preventing the progression of cells through the G2 / M checkpoint. Wee1 is overexpressed in several cancer types and has been implicated in tumor growth and survival. In some embodiments, a Wee1 inhibitor is one or more of imp7068, adavosertib 096 . In some embodiments, reference to the term Wee1 inhibitor includes any such Wee1 inhibitor disclosed in any one of the following patent applications: WO 2022011391 , WO 2022247641 , WO 2021043152, WO 2020221358, WO 2020083404, WO 2020192581 , WO 2019085933,

[0123] WO 2018133829, WO 2015115355, WO 2015183776, WO 2014085216, and ON 114831993, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference. iii) CHK inhibitors PATENT

[0124] ATTORNEY DOCKET NO.: 51432-056WO2

[0125] In some embodiments, compositions methods described herein may include one or more checkpoint kinase (CHK) inhibitors. A CHK inhibitor may be administered or formulated in combination with a RAS(ON) inhibitor and / or any additional therapeutic agent described herein. CHK1 kinase is a critical regulator of the cell cycle and the DNA damage response pathway. In some embodiments, the CHK inhibitor is a CHK1 inhibitor. In some embodiments, a CHK inhibitor is a CHK2 inhibitor. In some embodiments, a CHK1 inhibitor is one or more BBI-355 rabusertib , BEBT-260, and PEP07. In some embodiments, reference to the term CHK1 inhibitor includes any such CHK1 inhibitor disclosed in any one of the following patent applications: WO 2024196923, WO 2024211271 , WO 2024211270, WO 2024118564, WO 2023230477, WO 2022251502, WO 2021113661 , WO 2021104461 , WO 2019012030, WO 2010118390, WO 2008067027, WO 2002070494, CN119661557, and TW202126818, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference.

[0126] (c) DNA Damage Response (DDR) Inhibitors

[0127] Compositions and methods described herein may include a DDR inhibitor. A DDR inhibitor may be administered or formulated in combination with a RAS(ON) inhibitor described herein. The DDR pathway is a critical cellular pathway that is activated in response to DNA damage and is essential for maintaining genomic stability, thereby preventing the development of cancer. However, cancer cells often have defects in the DDR pathway, which makes them more sensitive to DDR inhibitors. DDR inhibitors have shown promise in preclinical studies as potential cancer therapeutics, particularly in combination with other agents. i) ATM inhibitors

[0128] In some embodiments, compositions and methods described herein may include one or more ataxia telangiectasia mutated (ATM) inhibitors. An ATM inhibitor may be administered or formulated in combination with a RAS(ON) inhibitor and / or any additional therapeutic agent described herein. ATM plays a role in regulating the replication stress response and maintaining genomic stability. In some PATENT

[0129] ATTORNEY DOCKET NO.: 51432-056WO2 embodiments, an ATM inhibitor is one or more lartesertib , AZD1390 YH2051 , and VE-821 some embodiments, reference to the term ATM inhibitor includes any such ATM inhibitor disclosed in any one of the following patent applications: WO 2024189299, WO 2022058351 , WO 2021197339, WO 2021098734, WO 2021260580, WO 2020193660, WO 2020063855, WO 2016155884, WO 2007026157, WO 2006085067, US 2016113935, ON 116440082, ON 117180432 and ON 115105596 each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference. ii) ATR inhibitors

[0130] In some embodiments, compositions and methods described herein may include one or more ataxia telangiectasia and Rad3-related (ATR) inhibitors. An ATR inhibitor may be administered or formulated in combination with a RAS(ON) inhibitor and / or any additional therapeutic agent described herein. In some embodiments, an ATR inhibitor is one or more berzosertib, gartisertib, camonsertib, to the term ATR inhibitor includes any such ATR inhibitor disclosed in any one of the following patent applications: WO 2025019344, WO 2025019346, WO 2023138343, WO 2023126823, WO 2023109883, WO 2023016529, WO 2022237875, WO 2022268025, WO 2021012049, WO 2021023272, WO PATENT

[0131] ATTORNEY DOCKET NO.: 51432-056WO2

[0132] 2021260579, WO 2021228758, WO 2019050889, WO 2019154365, WO 2019036641 , WO 2019133711 , WO 2017059357, WO 2013049859, WO 2007046426, WO 2007015632, and CN113797341 , each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference. iii) PARP inhibitors

[0133] In some embodiments, compositions and methods described herein may include one or more Poly(ADP-ribose) polymerase (PARP) inhibitors. A PARP inhibitor may be administered or formulated in combination with a RAS(ON) inhibitor and / or any additional therapeutic agent described herein. There are 17 PARP (aka tankyrase) family members that have been identified. PARP enzymes play a critical role in DNA damage repair, particularly in the repair of single-strand DNA breaks. PARP inhibitors block the activity of PARP enzymes, leading to the accumulation of DNA damage and ultimately cell death. In some embodiments, a PARP inhibitor is one or more olaparib , rucaparib , , and veliparib (ABT-888) some embodiments, reference to the term PARP inhibitor includes any such PARP inhibitor disclosed in any one of the following patent applications: WO 2025024581 , WO 2025037273, WO 2025061057, WO 2024256377, WO 2024255782, WO 2023051812, WO 2023051807, WO 2023051716, WO 2023278592, WO 2022228387, WO 2022022664, WO 2022000946, WO 2022222921 , WO 2021163530, WO 2020122034, WO 2020239097, WO 2020142583, WO 2020156577, WO 2020098774, WO 2020196712, WO 2019200382, WO 2018125961 , WO 2018205938, WO 2018192576, WO 2018218025, WO 2017032289, WO 2017177838, WO 2017029601 , WO 2017088723, WO 2016155655, WO 2015154630, WO 2013097225, WO 2012130166, WO 2011006794, WO 2009046205, WO 2009063244, WO 2008084261 , WO 2007138351 , WO 2006110816, WO 2005053662, WO 2005012524, CN113698356, ON 113603647, ON 115073544, ON 108938634, ON 104887680, ON 110343088, CN108976236, ON 117069731 , ON 119185316, ON 119112794, and ON 107629071 , each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference. vi) DNA-PK inhibitors

[0134] In some embodiments, compositions and methods described herein may include one or more DNA-dependent protein kinase (DNA-PK) inhibitors. A DNA-PK inhibitor may be administered or formulated in combination with a RAS(ON) inhibitor and / or any additional therapeutic agent described herein. DNA-PK is a serine / threonine protein kinase that plays a crucial role in DNA repair and maintenance of genome stability. In some embodiments, a DNA-PK inhibitor is one or more NU7441 PATENT

[0135] ATTORNEY DOCKET NO.: 51432-056WO2 , , rm DNA-

[0136] PK inhibitor includes any such DNA-PK inhibitor disclosed in any one of the following patent applications: WO 2025023957, WO 2023220418, WO 2023215991 , WO 2023165603, WO 2022187965, WO 2021 197159, WO 2021260583, WO 2021204111 , WO 2021104277, WO 2021098813, WO 2021022078, WO 2020259613, WO 2019143678, WO 2019143675, WO 2019201283, WO 2015058031 , WO 2014159690, WO 2012028233, WO 2009010761 , WO 2006032869, WO 2006109084, ON 112574179, ON 112300132, ON 115322209, and ON 112300126, each of which is incorporated herein by reference in its entirety, including the compound structures disclosed therein which are specifically incorporated herein by reference.

[0137] (d) Pharmaceutical Compositions

[0138] The disclosure provides pharmaceutical compositions including a therapeutic agent disclosed herein (e.g., a RAS(ON) inhibitor, Cell cycle inhibitor, and / or DDR inhibitor), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, as well as methods of using the compounds of the disclosure to prepare such compositions.

[0139] In some embodiments, a compound is present in a pharmaceutical composition in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0140] Compounds described herein, whether expressly stated or not, may be provided or utilized in salt form, e.g., a pharmaceutically acceptable salt form, unless expressly stated to the contrary. The term “pharmaceutically acceptable salt,” as use herein, refers to those salts of the compounds described herein that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical PATENT

[0141] ATTORNEY DOCKET NO.: 51432-056WO2

[0142] Sciences 66:1 19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting the free base group with a suitable organic acid.

[0143] The compounds of the disclosure may have ionizable groups so as to be capable of preparation as pharmaceutically acceptable salts. These salts may be acid addition salts involving inorganic or organic acids or the salts may, in the case of acidic forms of the compounds of the disclosure, be prepared from inorganic or organic bases. In some embodiments, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well-known in the art, such as hydrochloric, sulfuric, hydrobromic, acetic, lactic, citric, or tartaric acids for forming acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, and the like for forming basic salts. Methods for preparation of the appropriate salts are well-established in the art.

[0144] Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-optionally substituted hydroxyl-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine and the like.

[0145] For use as treatment of subjects, the compounds of the disclosure, or a pharmaceutically acceptable salt thereof, can be formulated as pharmaceutical or veterinary compositions. Depending on the subject to be treated, the mode of administration, and the type of treatment desired, e.g., prevention, prophylaxis, or therapy, the compounds, or a pharmaceutically acceptable salt thereof, are formulated in ways consonant with these parameters. A summary of such techniques may be found in Remington: The Science and Practice of Pharmacy, 21stEdition, Lippincott Williams & Wilkins, (2005); and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York, each of which is incorporated herein by reference.

[0146] Compositions can be prepared according to conventional mixing, granulating or coating methods, respectively, and the present pharmaceutical compositions can contain from about 0.1% to about 99%, from about 5% to about 90%, or from about 1% to about 20% of a compound of the present disclosure, or pharmaceutically acceptable salt thereof, by weight or volume. In some embodiments, compounds, or a pharmaceutically acceptable salt thereof, described herein may be present in amounts totaling 1-95% by weight of the total weight of a composition, such as a pharmaceutical composition.

[0147] The composition may be provided in a dosage form that is suitable for intraarticular, oral, parenteral (e.g., intravenous, intramuscular), rectal, cutaneous, subcutaneous, topical, transdermal, PATENT

[0148] ATTORNEY DOCKET NO.: 51432-056WO2 sublingual, nasal, vaginal, intravesicular, intraurethral, intrathecal, epidural, aural, or ocular administration, or by injection, inhalation, or direct contact with the nasal, genitourinary, reproductive or oral mucosa. Thus, the pharmaceutical composition may be in the form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, preparations suitable for iontophoretic delivery, or aerosols. The compositions may be formulated according to conventional pharmaceutical practice.

[0149] Formulations may be prepared in a manner suitable for systemic administration or topical or local administration. Systemic formulations include those designed for injection (e.g., intramuscular, intravenous or subcutaneous injection) or may be prepared for transdermal, transmucosal, or oral administration. A formulation will generally include a diluent as well as, in some cases, adjuvants, buffers, preservatives and the like. Compounds, or a pharmaceutically acceptable salt thereof, can be administered also in liposomal compositions or as microemulsions.

[0150] For injection, formulations can be prepared in conventional forms as liquid solutions or suspensions or as solid forms suitable for solution or suspension in liquid prior to injection or as emulsions. Suitable excipients include, for example, water, saline, dextrose, glycerol and the like. Such compositions may also contain amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as, for example, sodium acetate, sorbitan monolaurate, and so forth.

[0151] Various sustained release systems for drugs have also been devised. See, for example, U.S. Patent No. 5,624,677.

[0152] Systemic administration may also include relatively noninvasive methods such as the use of suppositories, transdermal patches, transmucosal delivery and intranasal administration. Oral administration is also suitable for compounds of the disclosure, or a pharmaceutically acceptable salt thereof. Suitable forms include syrups, capsules, and tablets, as is understood in the art.

[0153] Each compound, or a pharmaceutically acceptable salt thereof, as described herein, may be formulated in a variety of ways that are known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately. Other modalities of combination therapy are described herein.

[0154] The individually or separately formulated agents can be packaged together as a kit. Non-limiting examples include, but are not limited to, kits that contain, e.g., two pills, a pill and a powder, a suppository and a liquid in a vial, two topical creams, etc. The kit can include optional components that aid in the administration of the unit dose to subjects, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, the unit dose kit can contain instructions for preparation and administration of the compositions. The kit may be manufactured as a single use unit dose for one subject, multiple uses for a particular subject (at a constant dose or in which the individual compounds, or a pharmaceutically acceptable salt thereof, may vary in potency as therapy progresses); or the kit may contain multiple doses suitable for administration to multiple subjects (“bulk packaging”). The kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.

[0155] Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or PATENT

[0156] ATTORNEY DOCKET NO.: 51432-056WO2 fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, optionally substituted hydroxylpropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like.

[0157] Two or more compounds may be mixed together in a tablet, capsule, or other vehicle, or may be partitioned. In one example, the first compound is contained on the inside of the tablet, and the second compound is on the outside, such that a substantial portion of the second compound is released prior to the release of the first compound.

[0158] Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders, granulates, and pellets may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

[0159] Dissolution or diffusion-controlled release can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of compounds, or by incorporating the compound, or a pharmaceutically acceptable salt thereof, into an appropriate matrix. A controlled release coating may include one or more of the coating substances mentioned above or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-optionally substituted hydroxylmethacrylate, methacrylate hydrogels, 1 ,3 butylene glycol, ethylene glycol methacrylate, or polyethylene glycols. In a controlled release matrix formulation, the matrix material may also include, e.g., hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, or halogenated fluorocarbon.

[0160] The liquid forms in which the compounds, or a pharmaceutically acceptable salt thereof, and compositions of the present disclosure can be incorporated for administration orally include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

[0161] Generally, when administered to a human, the oral dosage of any of the compounds of the disclosure, or a pharmaceutically acceptable salt thereof, will depend on the nature of the compound, and can readily be determined by one skilled in the art. A dosage may be, for example, about 0.001 mg to PATENT

[0162] ATTORNEY DOCKET NO.: 51432-056WO2 about 2000 mg per day, about 1 mg to about 1000 mg per day, about 5 mg to about 500 mg per day, about 100 mg to about 1500 mg per day, about 500 mg to about 1500 mg per day, about 500 mg to about 2000 mg per day, or any range derivable therein.

[0163] In some embodiments, the pharmaceutical composition may further comprise an additional compound having therapeutic activity. Depending on the mode of administration, compounds, or a pharmaceutically acceptable salt thereof, will be formulated into suitable compositions to permit facile delivery. Each compound, or a pharmaceutically acceptable salt thereof, of a combination therapy may be formulated in a variety of ways that are known in the art. For example, the first and second agents of the combination therapy may be formulated together or separately. Desirably, the first and second agents are formulated together for the simultaneous or near simultaneous administration of the agents.

[0164] It will be appreciated that the compounds and pharmaceutical compositions of the present disclosure can be formulated and employed in combination therapies, that is, the compounds and pharmaceutical compositions can be formulated with or administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder, or they may achieve different effects (e.g., control of any adverse effects).

[0165] Administration of each drug in a combination therapy, as described herein, can, independently, be one to four times daily for one day to one year, and may even be for the life of the subject. Chronic, long-term administration may be indicated.

[0166] II. Methods of Use

[0167] In some aspects, the present disclosure provides methods of identifying, classifying, or treating subjects with RAS mutant cancers including cancers comprising a CDKN2A mutation. In various embodiments, the treatment methods generally comprise administering to the subject in need thereof a therapeutically effective amount of a RAS(ON) inhibitor compound, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound in combination with a cell cycle inhibitor or DNA damage response inhibitor. Suitable compositions comprising a RAS(ON) inhibitor compounds, a cell cycle inhibitor, and / or a DNA damage response inhibitor useful in the methods of the disclosure are described in section I and incorporated into this section by reference.

[0168] In some embodiments, particular biomarker genes can be used to predict a subject's response to a RAS(ON) inhibitor therapy. In certain embodiments, the genotype is predictive of sensitivity to a RAS(ON) inhibitor therapy. In certain embodiments, the genotype is predictive of resistance to a RAS(ON) inhibitor therapy. The present disclosure provides advantageous methods for determining whether a subject afflicted with a tumor (e.g., cancer) is a candidate for a combination therapy comprising a RAS(ON) inhibitor and an additional therapeutic agent (e.g., a cell cycle inhibitor and / or DDR inhibitor).

[0169] In one aspect, the present disclosure provides a method of treating a subject afflicted with a tumor resistant to a RAS(ON) inhibitor therapy comprising administering to the subject an amount of a RAS(ON) inhibitor and an amount of a cell cycle inhibitor that together are effective to treat the tumor. In another aspect, the present disclosure provides a method of treating a subject afflicted with a tumor PATENT

[0170] ATTORNEY DOCKET NO.: 51432-056WO2 resistant to a RAS(ON) inhibitor therapy comprising administering to the subject an amount of a RAS(ON) inhibitor and an amount of a DNA damage response inhibitor, effective to treat the tumor. In certain embodiments, the tumor has a RAS mutation and an inactivating CDKN2A mutation, a decreased copy number of CDKN2A, or a decreased expression of CDKN2A mRNA or protein.

[0171] In one aspect, the present disclosure provides a method of identifying a subject afflicted with cancer for treatment with a RAS(ON) inhibitor and a cell cycle inhibitor, the method comprising: determining the RAS and CDKN2A mutation status from a biological sample previously obtained from the subject; and identifying the subject as a candidate for treatment with the RAS(ON) inhibitor and cell cycle inhibitor combination when the cancer comprises a RAS mutation and an inactivating CDKN2A mutation, a decreased copy number of CDKN2A, or a decreased expression of CDKN2A mRNA or protein.

[0172] In one aspect, the present disclosure provides a method identifying a subject afflicted with cancer for treatment with a RAS(ON) inhibitor and a DNA damage response inhibitor, the method comprising: determining the RAS and CDKN2A mutation status from a biological sample previously obtained from the subject; and identifying the subject as a candidate for treatment with the RAS(ON) inhibitor and cell cycle inhibitor combination when the cancer comprises a RAS mutation and an inactivating CDKN2A mutation, a decreased copy number of CDKN2A, or a decreased expression of CDKN2A mRNA or protein.

[0173] In one aspect, the present disclosure provides a method comprising: (a) determining the genotype of one or more biomarker genes in a biological sample of a subject; (b) identifying whether the subject is likely or unlikely to have a durable response to a therapy comprising a RAS(ON) inhibitor based on the genotype. In certain embodiments, the methods further comprise administering to the subject optionally in need thereof the RAS(ON) inhibitor if the subject is identified as likely to have a durable response to a therapy comprising a RAS(ON) inhibitor. In certain embodiments, the methods further comprise administering to the subject optionally in need thereof the RAS(ON) inhibitor and cell cycle inhibitor if the subject is identified as unlikely to have a durable response to a therapy comprising a RAS(ON) inhibitor therapy. In certain embodiments, the methods further comprise administering to the subject optionally in need thereof the RAS(ON) inhibitor and DDR inhibitor if the subject is identified as unlikely to have a durable response to a therapy comprising a RAS(ON) inhibitor therapy. In certain embodiments, the biomarker genes useful in the methods of the present disclosure include CDKN2A, and RAS.

[0174] In one aspect, the present disclosure provides a method of treating a tumor in a subject optionally in need thereof comprising an amount of a RAS(ON) inhibitor and an amount of a cell cycle inhibitor, effective to treat the tumor. In another aspect, the present disclosure provides a method of treating a tumor in a subject optionally in need thereof comprising administering to the subject an amount of a RAS(ON) inhibitor and an amount of a DDR inhibitor, effective to treat the tumor. In some embodiments, the methods include treating a tumor unlikely to have a durable response (e.g., progression free survival or prolonged progression free survival) to a RAS(ON) inhibitor therapy comprising administering to a tumor so-identified an amount of a RAS(ON) inhibitor and an amount of a cell cycle inhibitor, effective to treat the tumor. In some embodiments, the methods include treating a tumor unlikely to have a durable response (e.g., progression free survival or prolonged progression free survival) to a RAS(ON) inhibitor therapy comprising administering to a tumor so-identified an amount of a RAS(ON) inhibitor and an amount of a DDR inhibitor, effective to treat the tumor. In some embodiments, the methods include PATENT

[0175] ATTORNEY DOCKET NO.: 51432-056WO2 treating a tumor in a subject optionally in need thereof, wherein the tumor comprises a RAS mutation (e.g., a RASG12Xmutation, a RASG13Xmutation, or a RASQ61Xmutation) and a CDKN2A mutation (e.g., an inactivating mutation or decreased or absent protein expression), comprising administering to the subject an amount of a RAS(ON) inhibitor and an amount of a cell cycle inhibitor effective to treat the tumor. In some embodiments, the methods include treating a tumor in a subject, wherein the tumor comprises a RAS mutation (e.g., a RASG12Xmutation, a RASG13Xmutation, or a RASQ61Xmutation) and a CDKN2A mutation (e.g., an inactivating mutation or decreased or absent protein expression), comprising administering to the subject an amount of a RAS(ON) inhibitor and an amount of a DDR inhibitor effective to treat the tumor.

[0176] In one aspect, the present disclosure provides a method comprising: (a) determining a genotype of one or more biomarker genes comprising CDKN2A and RAS in a biological sample of a subject afflicted with cancer; (b) determining the genotype of one or more biomarkers genes in the biological sample; and (c) classifying a subject as likely or not likely to have a durable response to a therapy comprising a RAS(ON) inhibitor based on the genotype of each of the one or more biomarker genes in the biological sample. In certain embodiments, the biomarker panel consists of CDKN2A and RAS.

[0177] In certain embodiments, the genotype comprises a mutation in the one or more biomarker genes. In certain embodiments, the mutation inactivates the one or more biomarker genes.

[0178] In certain embodiments, the method further comprises comparing the genotype with a reference genotype. In certain embodiments, the reference is to an untreated population. In certain embodiments, the reference is to a treated population. In certain embodiments, the genotype is reported as a score.

[0179] In certain embodiments, determining the genotype comprises genomic profiling. In certain embodiments, determining the genotype comprises measuring gene expression. In certain embodiments, measuring gene expression comprises detection of ribonucleic acids (RNAs) or polypeptides.

[0180] In certain embodiments, the subject is classified as likely to have a durable response to a RAS(ON) inhibitor treatment. In certain embodiments, the subject is classified as not likely to have a durable response to a RAS(ON) inhibitor treatment.

[0181] In certain embodiments, the methods of the present disclosure further comprise administering a RAS(ON) inhibitor therapy to the subject optionally in need thereof. In certain embodiments, the methods comprise administering to the subject optionally in need thereof a combination therapy comprising a RAS(ON) inhibitor and one or more additional therapeutic agents. In certain embodiments, the methods comprise administering to the subject a combination therapy comprising a RAS(ON) inhibitor and a second therapy. In certain embodiments, the second therapy is a cell cycle inhibitor. In certain embodiments, the cell cycle inhibitor is a CDK4 / 6 inhibitor. In certain embodiments, the second therapy is a DNA damage response (DDR) inhibitor

[0182] In one aspect, the present disclosure provides a method of predicting resistance to tumor growth inhibition in response to a therapy comprising a RAS(ON) inhibitor, comprising: (a) detecting in a tumor sample previously obtained from a subject a genotype of one or more biomarker genes; (b) analyzing the genotype of the one or more biomarker genes in the tumor sample; and (c) predicting resistance to tumor cell growth inhibition in response to the therapy comprising a RAS(ON) inhibitor, if the tumor sample comprises (i) an inactivating CDKN2A mutation, (ii) a decreased copy number CDKN2A, or (iii) a decreased expression of CDKN2A mRNA or protein. PATENT

[0183] ATTORNEY DOCKET NO.: 51432-056WO2

[0184] In one aspect, the present disclosure provides a method of treating a cancer (e.g., a non-small cell lung cancer (NSCLC), a pancreatic cancer, or a colorectal cancer (CRC)) comprising the step of treating a subject optionally in need thereof with a combination therapy comprising a RAS(ON) inhibitor and a second therapy when a tumor sample obtained from the subject comprises (i) a RAS mutation and (ii) an inactivating CDKN2A mutation, a decreased copy number CDKN2A, or a decreased expression of CDKN2A mRNA or protein.

[0185] In one aspect, the present disclosure provides a method of enriching a prospective patient population for subjects likely to have a durable response to a RAS(ON) inhibitor therapy, the method comprising performing the methods described herein of predicting resistance and / or response of tumor growth inhibition by a RAS(ON) inhibitor therapy on two or more individual subjects within the prospective patient population.

[0186] In one aspect, the present disclosure provides a method of treating a cancer, comprising treating a subject optionally in need thereof undergoing a cancer therapy, comprising a RAS(ON) inhibitor and a second therapy when a tumor sample obtained from the subject comprises (i) a RAS mutation and (ii) an inactivating CDKN2A mutation, a decreased copy number CDKN2A, or a decreased expression of CDKN2A mRNA or protein. In certain embodiments, the RAS(ON) inhibitor is daraxonrasib or RMC-7977. In certain embodiments, the second therapy comprises a cell cycle inhibitor or a DDR inhibitor. In certain embodiments, the RAS mutation is a RASG12Xmutation.

[0187] Accordingly, the present disclosure provides methods of treating cancer in a subject in need thereof. In some embodiments, the cancer is colorectal cancer, non-small cell lung cancer, small-cell lung cancer, pancreatic cancer, appendiceal cancer, melanoma, acute myeloid leukemia, small bowel cancer, ampullary cancer, germ cell cancer, cervical cancer, cancer of unknown primary origin, endometrial cancer, esophagogastric cancer, Gl neuroendocrine cancer, ovarian cancer, sex cord stromal tumor cancer, hepatobiliary cancer, or bladder cancer. In some embodiments, the cancer is appendiceal, endometrial or melanoma.

[0188] In some embodiments, the compounds of the disclosure or pharmaceutically acceptable salts thereof, pharmaceutical compositions comprising such compounds or salts, and methods provided herein may be used for the treatment of a wide variety of cancers including tumors such as lung, prostate, breast, brain, skin, cervical carcinomas, testicular carcinomas, etc. More particularly, cancers that may be treated by the compounds or salts thereof, pharmaceutical compositions comprising such compounds or salts, and methods of the invention include, but are not limited to tumor types such as astrocytic, breast, cervical, colorectal, endometrial, esophageal, gastric, head and neck, hepatocellular, laryngeal, lung, oral, ovarian, prostate, and thyroid carcinomas and sarcomas. Other cancers include, for example:

[0189] Cardiac, for example: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma, and teratoma;

[0190] Lung, for example: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;

[0191] Gastrointestinal, for example: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small PATENT

[0192] ATTORNEY DOCKET NO.: 51432-056WO2 bowel (adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma);

[0193] Genitourinary tract, for example: kidney (adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma);

[0194] Liver, for example: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma;

[0195] Biliary tract, for example: gall bladder carcinoma, ampullary carcinoma, cholangiocarcinoma;

[0196] Bone, for example: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumors;

[0197] Nervous system, for example: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, neurofibromatosis type 1 , meningioma, glioma, sarcoma);

[0198] Gynecological, for example: uterus (endometrial carcinoma, uterine carcinoma, uterine corpus endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma);

[0199] Hematologic, for example: blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases (e.g., myelofibrosis and myeloproliferative neoplasms), multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma);

[0200] Skin, for example: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands, for example: neuroblastoma.

[0201] In each of the preceding embodiments, the cancer comprises a RAS mutant protein. Accordingly, in some embodiments, a method of the present disclosure is employed in a method of treating a subject having a cancer comprising a RAS mutant (e.g., a mutant KRAS, HRAS or NRAS). Accordingly, in some embodiments, the mutant RAS is a RAS G12X mutation, wherein X represents any naturally occurring amino acid residue. In certain instances, X is A, C, D, V, S or R amino acid residue. In certain embodiments, the mutant RAS is a G13X mutation, wherein X is any naturally occurring amino acid PATENT

[0202] ATTORNEY DOCKET NO.: 51432-056WO2 residue. In certain instances, X is A, C, D, V, S or R amino acid residue. In certain embodiments, the mutant RAS is a Q61X mutation, wherein X is any naturally occurring amino acid residue. In certain instances, X is A, C, D, V, S, R, H, K, or L amino acid residue. In other instances, X is H, K, R, or L amino acid residue. In some embodiments, the cancer comprises a RAS mutation, such as a RAS mutation described herein. In some embodiments, a mutation is selected from:

[0203] (a) the following KRAS mutants: G12D, G12V, G12C, G13D, G12R, G12A, Q61 H, G12S, A146T, G13C, Q61 L, Q61 R, K117N, A146V, G12F, Q61 K, L19F, Q22K, V14I, A59T, A146P, G13R, G12L, or G13V, and combinations thereof;

[0204] (b) the following HRAS mutants: Q61R, G13R, Q61 K, G12S, Q61 L, G12D, G13V, G13D, G12C, K117N, A59T, G12V, G13C, Q61 H, G13S, A18V, D119N, G13N, A146T, A66T, G12A, A146V, G12N, or G12R, and combinations thereof; and

[0205] (c) the following NRAS mutants: Q61R, Q61 K, G12D, Q61 L, Q61 H, G13R, G13D, G12S, G12C, G12V, G12A, G13V, G12R, P185S, G13C, A146T, G60E, Q61 P, A59D, E132K, E49K, T50I, A146V, or A59T, and combinations thereof; or a combination of any of the foregoing.

[0206] The cancer may, for example, be pancreatic cancer, colorectal cancer, non-small cell lung cancer, acute myeloid leukemia, multiple myeloma, thyroid gland adenocarcinoma, a myelodysplastic syndrome, or squamous cell lung carcinoma. In some embodiments, the cancer comprises a RAS mutation (e.g., KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12S, KRAS G12V, and KRAS Q61 K). In some embodiments, the RAS mutation is at a position selected from the group consisting of G12A, G12C, G12D, G12R, G12S, G12V, and Q61 H, or a combination thereof. In some embodiments, the RAS mutation is at a position selected from the group consisting of G12A, G12C, G12S, and G12V, or a combination thereof. In some embodiments, the RAS mutation is at a position selected from the group consisting of G12C and G12V, or a combination thereof.

[0207] In some embodiments, the cancer comprises a RAS G12C mutation, and the cancer is lung cancer, colorectal cancer or pancreatic cancer. In some embodiments, the cancer comprises a RAS G12D amplification, and the cancer is lung cancer, colorectal cancer or pancreatic cancer. In some embodiments, the cancer comprises a RAS G12V mutation, and the cancer is lung cancer, colorectal cancer or pancreatic cancer. In some embodiments, the cancer comprises a RAS G13C mutation, and the cancer is lung cancer, colorectal cancer or pancreatic cancer.

[0208] Also provided is a method of inhibiting a RAS protein in a cell, the method comprising contacting the cell with an effective amount of a RAS(ON) inhibitor compound, or a pharmaceutically acceptable salt thereof, optionally in combination with a cell cycle inhibitor or DDR inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same. A method of inhibiting RAF-RAS binding, the method comprising contacting the cell with an effective amount of a RAS(ON) inhibitor compound, or a pharmaceutically acceptable salt thereof, optionally in combination with a cell cycle inhibitor or DDR inhibitor, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, is also provided. The cell may be a cancer cell. The cancer cell may be of any type of cancer described herein. The cell may be in vivo or in vitro.

[0209] In certain embodiments, the methods described herein include determining the genotype of one or more biomarker genes such as CDKN2A and RAS. PATENT

[0210] ATTORNEY DOCKET NO.: 51432-056WO2

[0211] In certain embodiments, the one or more biomarker genes comprise CDKN2A.

[0212] As exemplified herein, CDKN2A-deficiency drives selective resistance to a RAS(ON) inhibitor monotherapy therapy in vivo. The discovery that CDKN2A-deficiency drives selective resistance to a RAS(ON) inhibitor therapy, while this resistance can be effectively overcome comprising a RAS(ON) inhibitor therapy plus a cell cycle inhibitor therapy combination or a RAS(ON) inhibitor therapy plus a DDR inhibitor therapy combination has important implications for the identification of subjects undergoing a RAS(ON) inhibitor therapy, who can benefit from the addition of a cell cycle inhibitor therapy or DDR inhibitor therapy to their treatment regimen.

[0213] In one aspect, the present disclosure provides a method of predicting duration of response of tumor growth inhibition by a RAS(ON) inhibitor therapy, the method comprising: (a) determining in a tumor sample of a subject a genotype of one or more biomarker genes; (b) analyzing the genotype of the one or more biomarker genes in the tumor sample; and (c) predicting a decrease in duration of response of tumor cell growth inhibition by the therapy comprising a RAS(ON) inhibitor, if the tumor sample comprises (i) an inactivating CDKN2A mutation, (ii) a decreased copy number of CDKN2A, (iii) decreased expression of CDKN2A mRNA or protein, or predicting an increase in duration of tumor cell growth inhibition by a therapy comprising a RAS(ON) inhibitor, if the tumor sample comprises wild-type and / or normal expression of CDKN2A mRNA or protein.

[0214] In one aspect, the present disclosure provides a method of treating non-small cell lung cancer (NSCLC), comprising treating a subject with a RAS(ON) inhibitor and a cell cycle inhibitor when a tumor sample obtained from the subject comprises (i) a RAS mutation, and (ii) an inactivating CDKN2A mutation, (iii) a decreased copy number of CDKN2A, or (iii) decreased expression of CDKN2A mRNA or protein.

[0215] In one aspect, the present disclosure provides a method of treating non-small cell lung cancer (NSCLC), comprising treating a subject with a RAS(ON) inhibitor and a DDR inhibitor when a tumor sample obtained from the subject comprises (i) a RAS mutation, and (ii) an inactivating CDKN2A mutation, (iii) a decreased copy number of CDKN2A, or (iii) decreased expression of CDKN2A mRNA or protein.

[0216] The present disclosure provides, in part, methods for accurately classifying a subject afflicted with cancer as likely or unlikely to have a durable response to a therapy comprising a RAS(ON) inhibitor. The methods comprise obtaining a tumor sample of the subject and determining a genotype of one or more biomarker genes. In certain embodiments, the biological sample (for example, tumor sample) comprises polypeptides encoded by the one or more biomarker genes. Alternatively, the biological sample can comprise mRNA molecules or genomic DNA corresponding to the one or more biomarker genes. In certain embodiments, the methods involve obtaining a tumor sample of the subject and contacting the tumor sample with a reagent capable of determining the genotype by detecting, for example, a polypeptide or a nucleic acid that encodes the biomarker gene or fragments thereof.

[0217] In certain embodiments, the present disclosure provides methods for selecting a subject for treatment with a combination therapy comprising a RAS(ON) inhibitor and a cell cycle inhibitor or DDR inhibitor if it is likely that the subject will respond to the combination therapy, wherein the likelihood of response is determined by performing any of the disclosed methods for predicting resistance, sensitivity, or response of tumor growth to inhibition by the therapy on a biological sample obtained from the subject. PATENT

[0218] ATTORNEY DOCKET NO.: 51432-056WO2

[0219] In certain embodiments, the determination is based on results of an existing biological sample of the subject.

[0220] A "biomarker gene" can be any gene having a genotype and / or expression level that can be determined, measured and / or evaluated as an indicator of a biologic process, pathogenic process, or pharmacologic response to a therapeutic intervention. A biomarker gene useful to practice the presently disclosed methods can be used as an indicator to determine whether a subject having a tumor (e.g., cancer) will be sensitive or resistant to a therapy comprising a RAS(ON) inhibitor and / or for monitoring response to a treatment with a therapy comprising a RAS(ON) inhibitor. In certain embodiments, a biomarker gene is an oncogene and / or tumor suppressor gene. Sensitivity or resistance to a therapy with a RAS(ON) inhibitor can be determined by analyzing a nucleic acid molecule (DNA, mRNA, cDNA etc.) corresponding to a biomarker gene or the protein encoded by the biomarker gene. Biomarker genes can include any gene whose genotype and / or level of expression in a tissue or cell can be used to predict response to a RAS(ON) inhibitor therapy. The detection, and in some cases the level, of one or more biomarker genes of the present disclosure permits the classification of a subject as sensitive or resistant to a RAS(ON) inhibitor therapy.

[0221] As used herein, the terms "determine," "determine the genotype of a biomarker gene," "determine the level of a biomarker gene," "determine the amount of a biomarker gene," "determine the biomarker gene level," and the like are meant to encompass any technique that can be used to detect or measure the genotype, presence, or expression level of one or more biomarker genes. Such techniques can give qualitative or quantitative results. Biomarker gene levels can be determined by detecting the entire biomarker molecule or by detecting fragments or reaction products that are characteristic of the biomarker gene. The terms determining, measuring, or taking a measurement refer to a quantitative or qualitative determination of a property of an entity, for example, quantifying the amount or concentration of a molecule or the activity level of a molecule. Any known method of detecting or measuring the level of a biomarker can be used to practice the present disclosure, so long as the method detects the genotype, presence, absence, or expression level of the biomarker gene.

[0222] In certain embodiments, determining the genotype of a biomarker gene is performed at the nucleic acid level by performing RNA-seq, a reverse transcriptase polymerase chain reaction (RT-PCR) or a hybridization assay with oligonucleotides that are substantially complementary to portions of cDNA molecules of the at least one biomarker gene under conditions suitable for RNA-seq, RT-PCR or hybridization and obtaining expression levels of the at least one biomarker gene.

[0223] The term "classifying" includes associating a sample with a response to a RAS(ON) inhibitor therapy. In certain instances, "classifying" is based on statistical evidence, empirical evidence, or both. In certain embodiments, the methods of classifying utilize a training set of samples having known genotypes. Once established, the training data set can serve as a basis, model, or template against which the features of an unknown sample are compared, to classify the sample.

[0224] The term "control" refers to any reference standard suitable to provide a comparison to the expression products in the test sample. A control can comprise a reference standard expression product level or genotype score from any suitable source, including but not limited to housekeeping genes, an expression product level range from normal tissue (or other previously analyzed control sample), a previously determined expression product level range within a test sample of a group of patients, or a set PATENT

[0225] ATTORNEY DOCKET NO.: 51432-056WO2 of patients with a certain outcome or receiving a certain therapy. It will be understood by those of ordinary skill in the art that such control samples and reference standard expression product levels can be used in combination as controls in the methods of the present disclosure. In various embodiments, the biomarker gene expression can be compared to a reference. A "reference" can be any value derived by art known methods for establishing a reference. Moreover, a measure of response to a certain therapy can be compared to a reference, e.g., measure of response in a patient or a set of patients receiving a certain receiving a certain therapy. These patients classified as sensitive to a certain therapy or patients classified as resistant to a certain therapy can be used as a reference or control.

[0226] A predetermined reference value is a value decided or obtained, usually beforehand, as a control. The concept of a control is well-established in the field, and can be determined, in a non-limiting example, empirically from, e.g., non-afflicted subjects (versus afflicted subjects, including afflicted subjects having different grades of the relevant affliction), or a non-afflicted sample, e.g., a tumor that is not resistant to RAS(ON) inhibitors and may be normalized as desired (in non-limiting examples, for volume, mass, age, location, gender) to negate the effect of one or more variables.

[0227] The term "expression" as used herein, refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. The term also encompasses translation of RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post- translational modifications. The expressed protein can be within the cytoplasm of a host cell, into the extracellular milieu such as the growth medium of a cell culture or anchored to the cell membrane.

[0228] The term "gene product" as used herein, refers to RNA transcribed from a gene and to one or more proteins, polypeptides of fragments thereof that are the product of translation of the RNA transcribed from the gene, and further encompasses all naturally occurring post-transcriptional and post- translational modifications. The expressed protein can be within the cytoplasm of a host cell, into the extracellular milieu such as the growth medium of a cell culture or anchored to the cell membrane.

[0229] The terms "expression level" and "level of expression" as used herein refers to information regarding the relative or absolute level of expression of one or more biomarker genes in a cell or group of cells. The level of expression of a biomarker gene can be determined based on the level of RNA, such as mRNA, encoded by the gene. Alternatively, the level of expression can be determined based on the level of a polypeptide or fragment thereof encoded by the biomarker gene. Gene expression data can be acquired for an individual cell, or for a group of cells such as a tumor or biopsy sample. Gene expression data and gene expression levels can be stored on computer readable media, for example, the computer readable medium used in conjunction with a microarray or chip reading device. Such gene expression data can be manipulated to generate gene expression signatures.

[0230] The expression level of a biomarker gene can be determined using a reagent such as a probe, primer, or antibody and / or a method performed on a biological sample, for example a tumor sample of the subject, for ascertaining or measuring quantitatively, semi-quantitatively or qualitatively the amount of a of a polypeptide or mRNA (or cDNA derived therefrom) corresponding to one or more biomarker genes. For example, a level of a biomarker gene can be determined by a number of methods including for example immunoassays including for example immunohistochemistry, ELISA, Western blot, immunoprecipitation and the like, where a detection agent such as an antibody for example, a labeled antibody, specifically binds the encoded polypeptide and permits relative or absolute ascertaining of the amount of polypeptide PATENT

[0231] ATTORNEY DOCKET NO.: 51432-056WO2 encoded by the biomarker gene, hybridization and PCR protocols where a probe or primer or primer set are used to ascertain the amount of nucleic acid corresponding to the biomarker gene, including for example probe based and amplification based methods including for example microarray analysis, RT- PCR such as quantitative RT-PCR (qRT-PCR), gRT-PCR, serial analysis of gene expression (SAGE), Northern Blot, digital molecular barcoding technology, for example Nanostring Counter Analysis, and TaqMan quantitative PCR assays.

[0232] Other methods of mRNA detection and quantification can be applied, such as mRNA in situ hybridization in formalin-fixed, paraffin-embedded (FFPE) tissue samples or cells, which uses probe sets for each mRNA that bind specifically to an amplification system to amplify the hybridization signals; these amplified signals can be visualized using a standard fluorescence microscope or imaging system.

[0233] TaqMan probe-based gene expression analysis (PCR- based) can also be used for measuring biomarker gene expression levels in tissue samples, including mRNA levels in FFPE samples. TaqMan probe-based assays utilize a probe that hybridizes specifically to the mRNA target. This probe contains a quencher dye and a reporter dye (fluorescent molecule) attached to each end, and fluorescence is emitted only when specific hybridization to the mRNA target occurs. During the amplification step, the exonuclease activity of the polymerase enzyme causes the quencher and the reporter dyes to be detached from the probe, and fluorescence emission can occur. This fluorescence emission is recorded and signals are measured by a detection system; these signal intensities are used to calculate the abundance of a given transcript (gene expression) in a sample.

[0234] As used herein, a "nucleic acid" can generally refer to a polynucleotide sequence, or fragment thereof. A nucleic acid can comprise nucleotides. A nucleic acid can be exogenous or endogenous to a cell. A nucleic acid can exist in a cell-free environment. A nucleic acid can be a gene or fragment thereof. A nucleic acid can be DNA nucleic acid, can be RNA nucleic acid, or can comprise one or more analogs (for example, altered backbone, sugar, or nucleobase). "Nucleic acid", "polynucleotide, "target polynucleotide", and "target nucleic acid" can be used interchangeably.

[0235] As used herein, the term "mRNA" or sometimes refer by "mRNA transcripts" include but is not limited to pre-mRNA transcript(s), transcript processing intermediates, mature mRNA(s) ready for translation and transcripts of the gene or genes, or nucleic acids derived from the mRNA transcript(s). Transcript processing can include splicing, editing and degradation. As used herein, a nucleic acid derived from an mRNA transcript refers to a nucleic acid for whose synthesis the mRNA transcript or a subsequence thereof has ultimately served as a template. Thus, a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, etc., are all derived from the mRNA transcript and detection of such derived products is indicative of the presence and / or abundance of the original transcript in a sample. Thus, mRNA derived samples include, but are not limited to, mRNA transcripts of the gene or genes, cDNA reverse transcribed from the mRNA, cRNA transcribed from the cDNA, DNA amplified from the genes, RNA transcribed from amplified DNA, and the like.

[0236] As used herein "sequencing" a nucleic acid molecule means determining the identity of at least one nucleotide in the molecule. In certain embodiments, the identity of less than all of the nucleotides in a molecule are determined. In certain embodiments, the identity of a majority or all of the nucleotides in the molecule is determined. PATENT

[0237] ATTORNEY DOCKET NO.: 51432-056WO2

[0238] As used herein, the term "biological sample" refers to any sample obtained from a subject. A biological sample can be obtained from a subject prior to or subsequent to a diagnosis, at one or more time points prior to or following treatment or therapy, at one or more time points during which there is no treatment or therapy, or can be collected from a healthy subject. The biological sample can be a tissue sample or a fluid sample. In certain embodiments, the biological sample includes a tissue sample, a biopsy sample, a tumor aspirate, a bone marrow aspirate or a blood sample (or a fraction thereof, such as blood or serum). In certain embodiments, the biological sample includes a tumor cell or cancer cell, for example a circulating tumor cell present in a fluid sample, for example, blood or a fraction thereof. In certain embodiments, the biological sample includes a cell free nucleic acid present in a fluid sample, for example, blood or a fraction thereof. In one embodiment, the biological sample comprises a cell lysate (or lysate fraction) or cell extract; or a solution containing one or more molecules derived from a cell or cellular material (for example a polypeptide or nucleic acid). The cell lysate can include proteins, nuclear and / or mitochondrial fractions. In certain embodiments, the cell lysate includes a cytosolic fraction. In certain embodiments, the cell lysate includes a nuclear / mitochondrial fraction and a cytosolic fraction.

[0239] The source of a biological sample can be solid tissue as from a fresh, frozen and / or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; or cells from any time in gestation or development of the subject. The biological sample can contain compounds that are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like. The biological sample can be preserved as a frozen sample or as formaldehyde- or paraformaldehyde-fixed paraffin* embedded (FFPE) tissue preparation. For example, the sample can be embedded in a matrix, for example, an FFPE block or a frozen sample. However, other tissue and sample types are amenable for use herein. In one embodiment, the other tissue and sample types can be fresh frozen tissue, wash fluids, or cell pellets, or the like. A biological sample can be a tumor sample, which contains nucleic acid molecules from a tumor or cancer. A biological sample that is a tumor sample can be DNA, for example, genomic DNA, or cDNA derived from RNA. In one embodiment, the tumor nucleic acid sample is purified or isolated (for example, it is removed from its natural state). In one embodiment, the sample is a tissue (for example, a tumor biopsy), a ctDNA or cell free nucleic acid.

[0240] In certain embodiments, a tumor sample is isolated from a human subject. In certain embodiments, the analysis is performed on a tumor biopsy embedded in paraffin wax. In one embodiment, the sample can be a fresh frozen tissue sample. In certain embodiments, the sample is a bodily fluid obtained from the subject. The bodily fluid can be blood or fractions thereof (specifically, serum, plasma), urine, saliva, sputum or cerebrospinal fluid (CSF). The sample can contain cellular as well as extracellular sources of nucleic acid. The extracellular sources can be cell-free nucleic acids and / or exosomes. The methods described herein, including the RT-PCR methods, are sensitive, precise and have multi-analyte capability for use with paraffin embedded samples. See, for example, Cronin et al., Am. J Pathol. 164(1):35-42 (2004).

[0241] "Likely to" or "increased likelihood," as used herein, refers to an increased probability that an event will occur. Thus, in certain embodiments, a subject that is likely to respond to a treatment comprising a RAS(ON) inhibitor described herein has an increased probability of responding to a treatment comprising the RAS(ON) inhibitor relative to a reference subject or group of subjects. PATENT

[0242] ATTORNEY DOCKET NO.: 51432-056WO2

[0243] "Unlikely to" refers to a decreased probability that an event, item, object, thing or person will occur with respect to a reference. Thus, a subject that is unlikely to respond to a treatment comprising a RAS(ON) inhibitor described herein has a decreased probability of responding to a treatment comprising the RAS(ON) inhibitor relative to a reference subject or group of subjects.

[0244] The biomarkers described above allow for a determination if a subject is likely to or unlikely to have a durable response (e.g., long progression free survival or length of tumor growth inhibition), that can be used by the responsible medical staff to select a treatment for the subject. For example, in case of a positive prognosis, such as an indication that a long progression free survival can be expected, the treating medical staff can treat with a RAS(ON) inhibitor, whereas in case of a negative prognosis, the medical staff can use the information to adjust or change the treatment prescribed in order to initiate a combination treatment, such as a RAS(ON) inhibitor therapy plus a cell cycle inhibitor therapy or a RAS(ON) inhibitor therapy plus a DDR inhibitor therapy, that may be better for the patient in question.

[0245] Several criteria and definitions published in the literature can be used to determine the effect of one or more treatments on tumors in a subject suffering from cancer. Based on these criteria, tumors are defined as “responsive,” “stable,” or “progressive” when they improve, remain the same, or worsen during treatment, respectively. The amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, and / or weight of the tumor.

[0246] Examples of the commonly used criteria published in the literature include Response Evaluation Criteria in Solid Tumors (RECIST), Modified Response Evaluation Criteria in Solid Tumors (mRECIST), PET Response Criteria in Solid Tumors (PERCIST), Choi Criteria, Lugano Response Criteria, European Association for the Study of the Liver (EASL) Criteria, Response Evaluation Criteria in the Cancer of the Liver (RECICL), and WHO Criteria in Tumor Response.

[0247] As used herein, "progression free survival" or “PFS” is the time from treatment to the date of the first confirmed disease progression per RECIST 1.1 criteria.

[0248] “RECIST” shall mean an acronym that stands for “Response Evaluation Criteria in Solid Tumors” and is a set of published rules that define when cancer patients improve (“respond”), stay the same (“stable”) or worsen (“progression”) during treatments. Response as defined by RECIST criteria have been published, for example, a Journal of the National Cancer Institute, Vol. 92, No. 3, Feb. 2, 2000 and RECIST criteria can include other similar published definitions and rule sets. One skilled in the art would understand definitions that go with RECIST criteria, as used herein, such as “Partial Response (PR),” “Complete Response (CR),” “Stable Disease (SD)” and “Progressive Disease (PD).”

[0249] As used herein, "survival" refers to the subject remaining alive, and includes overall survival as well as progression free survival.

[0250] As used herein, "reducing the tumor," means reducing the size, volume, or weight of the tumor, reducing the number of metastases, reducing the size or weight of a metastasis, or combinations thereof. In certain embodiments, a metastasis is cutaneous or subcutaneous. Thus, in certain embodiments, administration of the immune checkpoint inhibitor reduces the size or volume of the tumor by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98% or at least about 99%, for example, relative to a control drug in a subject of the same genotype. In certain embodiments, administration of the RAS(ON) inhibitor therapy or combination PATENT

[0251] ATTORNEY DOCKET NO.: 51432-056WO2 therapy comprising the same, reduces the weight of the tumor by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98% or at least about 99%, for example, relative to a control drug in a subject of the same genotype. In certain embodiments, administration of the RAS(ON) inhibitor therapy or combination therapy comprising the same, reduces the size or volume of a metastasis by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98% or at least about 99%, for example, relative to a control drug in a subject of the same genotype. In certain embodiments, administration of the RAS(ON) inhibitor therapy or combination therapy comprising the same, reduces the number of metastases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98% or at least about 99% for example, relative to a control drug in a subject of the same genotype. In certain embodiments, combinations of these effects are achieved.

[0252] As used herein, the terms "cancer" or "tumor" refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist isolated within an animal, or can be non- tumorigenic, such as a leukemia cell. Cancers include, but are not limited to, B cell malignancies, for example, multiple myeloma, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, skin cancer, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematological tissues, and the like. Other nonlimiting examples of types of cancers applicable to the methods encompassed by the present disclosure include human sarcomas and carcinomas, for example, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheli osarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, for example, PATENT

[0253] ATTORNEY DOCKET NO.: 51432-056WO2 acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and nomHodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, the cancer is an epithelial cancer such as, but not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (for example, serous ovarian carcinoma), or breast carcinoma.

[0254] As used herein, "genomic profiling" means sequencing a part or all of the genome of a subject, such as to identify the nucleotide sequence of one or more genes in the subject, such as to identify genomic alterations (for example, mutations) in one or more biomarker genes that would identify the subject as a candidate to receive certain drugs or other therapeutic agents. Genomic profiling can be performed by a method described herein, such as by a next-generation sequencing method, or a massively parallel sequencing method.

[0255] The term "probe" refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example, a nucleotide transcript or protein encoded by or corresponding to a marker. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes can be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0256] As used herein, the term "genotype" refers to the alleles at one or more specific biomarker genes. The genotype of a biomarker gene can be determined by methods that include nucleic acids (RNA, cDNA, and DNA) and proteins, and variants and fragments thereof.

[0257] As used herein, the term "sensitive" in the context of a RAS(ON) inhibitor therapy, means that the RAS(ON) inhibitor therapy is effective at, in non-limiting examples, lengthening the time tumor growth inhibition or progression free survival, or reducing tumor burden, relative to the RAS(ON) inhibitor therapy in a subject having a cancer comprising a different biomarker signature (e.g., a subject having a cancer comprising a resistant biomarker signature).

[0258] As used herein, the term "resistant" in the context of a RAS(ON) inhibitor therapy, means that the RAS(ON) inhibitor therapy is less effective at, in non-limiting examples, lengthening the time tumor growth inhibition or progression free survival, or reducing tumor burden, relative to the RAS(ON) inhibitor therapy in a subject having a cancer comprising a different biomarker signature (e.g., a subject having a cancer comprising a sensitive biomarker signature).

[0259] In certain embodiments, the resistance and / or sensitivity profiles of one or more biomarker genes for a RAS(ON) inhibitor can be compared to the corresponding resistance and / or sensitivity scores for a standard of care therapy in order to determine whether a subject is likely to benefit from a RAS(ON) inhibitor therapy. For example, a RAS(ON) inhibitor therapy can be compared to standard of care (SoC) therapy for a particular cancer to determine which genotypes are sensitive or resistant relative to the SoC PATENT

[0260] ATTORNEY DOCKET NO.: 51432-056WO2 therapy using the described herein. Then, by excluding resistant patients and enrichment for sensitive patients within a prospective patient population, the performance of a RAS(ON) inhibitor relative to SoC can be improved.

[0261] As used herein, the term "polynucleotide," synonymously referred to as "nucleic acid molecule," "nucleotides" or "nucleic acids," refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA "Polynucleotides" include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and doublestranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.

[0262] A nucleic acid molecule corresponding to a biomarker gene of the present disclosure can be isolated using standard molecular biology techniques and the sequence information in the database records described herein. Using all or a portion of such nucleic acid sequences, nucleic acid molecules of the present disclosure can be isolated using standard hybridization and cloning techniques (for example, as described in Sambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0263] A nucleic acid molecule of the present disclosure can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.

[0264] As used herein, the terms "peptide," "polypeptide," or "protein" can refer to a molecule comprised of amino acids and can be recognized as a protein by those of ordinary skill in the art. The conventional one-letter or three-letter code for amino acid residues is used herein. The terms "peptide," "polypeptide," and "protein" can be used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0265] In the case of measuring protein levels to determine biomarker gene expression, any method known in the art is suitable provided it results in adequate specificity and sensitivity.

[0266] For example, protein levels can be measured by binding to an antibody or an antibody fragment specific for the protein and measuring the amount of antibody-bound protein. Antibodies can be labeled PATENT

[0267] ATTORNEY DOCKET NO.: 51432-056WO2 by radioactive, fluorescent or other detectable reagents to facilitate detection. Methods of detection include, without limitation, enzyme-linked immunosorbent assay (ELISA) and immunoblot techniques.

[0268] The presently disclosed methods detect mRNA, polypeptide, genomic DNA, or fragments thereof, in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of mRNA or a fragment thereof include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of polypeptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of biomarker genomic DNA or a fragment thereof include Southern hybridizations. Furthermore, in vivo techniques for detection of one or more polypeptides or fragments thereof include labeled antibodies. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0269] In some embodiments, the genotype, presence or level of at least one, two, three, four, five, six, seven, eight, nine, ten, fifty, sixty, or more biomarker genes of the disclosure are determined in the tumor sample. In some embodiments, the presently disclosed methods employ a statistical algorithm and / or empirical data (for example, the presence or level of one or biomarker genes described herein). In certain instances, a single learning statistical classifier system can be used to classify a sample. The use of a single learning statistical classifier system typically classifies the sample accurately with a sensitivity, specificity, positive predictive value, negative predictive value, and / or overall accuracy of at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0270] Other suitable statistical algorithms are well known to those of skill in the art. For example, learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex data sets (for example, panel of markers of interest) and making decisions based upon such data sets. In some embodiments, a single learning statistical classifier system such as a classification tree (for example, random forest) is used. In other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem. Examples of learning statistical classifier systems include, but are not limited to, those using inductive learning (for example, decision / classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), Probably Approximately Correct (PAC) learning, connectionist learning (for example, neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons such as multi-layer perceptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc.), reinforcement learning (for example, passive learning in a known environment such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement learning, and genetic algorithms and evolutionary programming. Other learning statistical classifier systems include support vector machines (for example, Kernel methods), multivariate adaptive regression splines (MARS), Levenberg-Marquardt algorithms, Gauss* Newton algorithms, mixtures of Gaussians, gradient descent algorithms, and learning vector quantization (LVQ). In certain embodiments, the method of the present disclosure further comprises sending the cancer classification results to a clinician, for example, an oncologist or hematologist. PATENT

[0271] ATTORNEY DOCKET NO.: 51432-056WO2

[0272] In certain embodiments, determining the genotype of one or more biomarker genes comprises genomic profiling to directly determine the genotype of the one or more biomarker genes. In certain embodiments, genomic profiling comprises contacting the biological sample with reagents, including, probes and / or primers, for sequencing the one or more biomarker genes or a portion thereof. In certain embodiments, probes or primers can be designed to detect a mutation in the one or more biomarker genes. In certain embodiments, the mutation is an inactivating mutation. In certain embodiments, the mutation results in decreased gene expression.

[0273] In certain embodiments, the methods include directly determining the genotype of one or more biomarker genes by genomic profiling to detect the presence or absence of a genetic alteration characterized by at least one alteration affecting the integrity of a gene encoding one or more biomarkers polypeptide, or the mis-expression of the biomarker (for example, mutations and / or splice variants). For example, such genetic alterations can be detected by ascertaining the existence of at least one of a deletion of one or more nucleotides from one or more biomarker genes; an addition of one or more nucleotides to one or more biomarker genes; a substitution of one or more nucleotides of one or more biomarker genes; a chromosomal rearrangement of one or more biomarker genes; an alteration in the level of a mRNA transcript of one or more biomarker genes; aberrant modification of one or more biomarker genes; such as of the methylation pattern of the genomic DNA; the presence of a non-wild type splicing pattern of a messenger RNA transcript of one or more biomarker genes; a non-wild type level of one or more biomarkers polypeptide; allelic loss of one or more biomarker genes, and inappropriate post- translational modification of one or more biomarkers polypeptide. As described herein, there are a large number of assays known in the art which can be used for detecting alterations in one or more biomarker genes.

[0274] In certain embodiments, detection of the alteration involves the use of a probe / primer in a polymerase chain reaction (PCR), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, for example, Landegran et al. (1988) Science 241 :1077- 1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91 :360-364), the latter of which can be particularly useful for detecting point mutations in one or more biomarker genes (see Abravaya et al. (1995) Nucleic Acids Res. 23:675- 682). This method can include collecting a sample of cells from a subject, isolating nucleic acid from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to one or more biomarker genes of the present disclosure, or fragments thereof, under conditions such that hybridization and amplification of the biomarker gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and / or LCR can be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

[0275] Alternative amplification methods include self-sustained sequence replication (Guatelli, J.C. et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q» Beta Replicase (Lizardi, P. M. et al. (1988) Bio- Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are PATENT

[0276] ATTORNEY DOCKET NO.: 51432-056WO2 especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

[0277] In certain embodiments, mutations in the presently disclosed one or more biomarker genes, or a fragment thereof, from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA Moreover, the use of sequence specific ribozymes can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[0278] In certain embodiments, genetic mutations in the presently disclosed one or more biomarker genes, or a fragment thereof, can be identified by hybridizing a nucleic acid to high density arrays containing hundreds or thousands of oligonucleotide probes (Cronin, M. T. et al. (1996) Hum. Mutat. 7:244-255; Kozal, M. J. et al. (1996) Nat. Med. 2:753-759).

[0279] In certain embodiments, any of a variety of sequencing methods known in the art can be used to directly sequence the presently disclosed one or more biomarker genes, or a fragment thereof, and detect mutations by comparing the sequence of the sample biomarker gene with the corresponding wild-type (control) sequence. Non-limiting examples of sequencing reactions include next-generation sequencing to determine the nucleotide sequence of either individual nucleic acid molecules (for example, in single molecule sequencing) or clonally expanded proxies for individual nucleic acid molecules in a highly parallel fashion.

[0280] Next generation sequencing methods are known in the art, and are described, for example, in Metzker, M. (2010) Nature Biotechnology Reviews 11 :31-46. Next generation sequencing collectively refers to several DNA / RNA sequencing technologies that vary according to the input material, length of read, and portion of the genome to be sequenced. Broadly, the two major next generation sequencing technologies are short-read sequencing and long-read sequencing. Short-read sequencing generally refers to reads that are shorter than 300 bp, whereas long-read sequencing refers to reads that are longer than 2.5 Kb. Short-read sequencing is a relatively inexpensive option (low costs per Gb) that has a high level of accuracy and is used more frequently in clinical practice for the detection of specific mutation hotspots. Moreover, based on the initial input material, different sequencing approaches can be used (for example, genomic DNA [DNA-seq], messenger or noncoding RNA [RNA-seq], or any nucleic or ribonucleic material obtained following the use of certain procedures).

[0281] Current next generation sequencing approaches also differ based on the extent of target enrichment and sequencing involved, with the 3 major types being whole genome sequencing (WGS), whole-exome sequencing (WES), and targeted gene panels. WGS refers to sequencing the entire genome, including coding and noncoding regions. It allows detection of several types of genetic aberrations, including single nucleotide variants and / or such structural alterations as insertions or deletions (also called indels), copy number variations involving duplications or deletions of long stretches of a chromosomal region, and rearrangements involving gross alterations in chromosomes or large chromosomal regions. WES involves sequencing only the coding regions of the genome and is limited in its ability to detect rearrangements between genes with breakpoints that frequently occur in intronic PATENT

[0282] ATTORNEY DOCKET NO.: 51432-056WO2 regions. RNA-based whole-transcriptome approaches can be another strategy to identify gene rearrangements.

[0283] Another next generation sequencing strategy, which is currently the most commonly used approach to cancer genotyping in clinical use, is targeted gene panels that interrogate a discrete number of genes. This approach has the advantage of being able to focus on clinically relevant targets with deeper sequencer and focused analyses. Targeted gene panels can be performed with either ampliconbased or hybrid-capture enrichment strategies and can range from small, hotspot-only panels focusing on less than fifty genes to larger, more comprehensive panels that include hundreds to greater than a thousand genes with selected intronic tiling coverage. In addition to lower costs, the advantages of targeted gene panels include greater analytic sensitivity because of the greater depth of coverage, less complex data analysis and interpretation than would be necessary for WES and WGS, and greater flexibility that allows for tailoring the testing to genomic regions relevant to cancer. Any of the known Next generation sequencing approaches can be practiced for the methods described herein and a skilled person will be able to select the best sequencing strategy to practice the methods described herein.

[0284] In certain embodiments, determining the genotype of one or more biomarker genes comprises measuring the expression level of one or more biomarker genes. The expression level can be measured in a number of ways, including, but not limited to measuring the mRNA encoded by the biomarker genes; measuring the amount of protein encoded by the biomarker genes; and measuring the activity of the protein encoded by the biomarker genes. In certain embodiments, a genotype of a biomarker gene is determined by measuring RNA, cDNA, protein or any combination thereof. When a genotype is determined by measuring RNA, the RNA can be reverse transcribed to produce cDNA (such as by RT- PCR), and the produced cDNA expression level can be detected. The expression level of a biomarker gene can be detected by forming a complex between a nucleic acid corresponding to a biomarker gene and a labeled probe or primer. When the nucleic acid is RNA or cDNA, the RNA or cDNA can be detected by forming a complex between the RNA or cDNA and a labeled nucleic acid probe or primer. The complex between the RNA or cDNA and the labeled nucleic acid probe or primer can be a hybridization complex.

[0285] Another method of determining the genotype of a biomarker gene at the nucleic acid level is the use of an amplification method such as, for example, RT-PCR or quantitative RT« PCR (qRT- PCR). Methods for determining the level of mRNA in a sample can involve the process of nucleic acid amplification, for example, by RT-PCR, ligase chain reaction or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. Numerous different PCR or qRT-PCR protocols are known in the art and can be directly applied or adapted for use using the presently described compositions for the detection and / or quantification of expression of biomarker genes in a sample.

[0286] Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, PCR analyses and probe arrays. One method for the detection of mRNA levels involves contacting the isolated mRNA or synthesized cDNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a cDNA, or a portion thereof, such as an oligonucleotide of at least about 7, about 15, PATENT

[0287] ATTORNEY DOCKET NO.: 51432-056WO2 about 30, about 50, about 100, about 250, or about 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to the non-natural cDNA or mRNA.

[0288] As described herein, biomarker gene expression can be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed molecule or protein. Non-limiting examples of such methods include immunological methods for detection of secreted, cell-surface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.

[0289] In certain embodiments, activity of a particular biomarker gene is characterized by a measure of gene transcript (for example mRNA), by a measure of the quantity of translated protein, or by a measure of gene product activity. Biomarker gene expression can be monitored in a variety of ways, including by detecting mRNA levels, protein levels, or protein activity, any of which can be measured using standard techniques. Detection can involve quantification of the level of gene expression (for example, genomic DNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can be a qualitative assessment of the level of gene expression, in particular in comparison with a control level. The type of level being detected will be clear to the skilled person from the context.

[0290] In certain embodiments, detecting or determining expression levels of a biomarker gene and functionally similar homologs thereof, including a fragment or genetic alteration thereof (for example, in regulatory or promoter regions thereof) comprises detecting or determining RNA levels for the biomarker marker gene. In certain embodiments, one or more cells from the subject to be tested are obtained and RNA is isolated from the cells.

[0291] General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al, ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999. Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker (Lab Invest. 56:A67, 1987) and De Andres et al. (Biotechniques 18:42-44, 1995). In particular, RNA isolation can be performed using a purification kit, a buffer set and protease from commercial manufacturers according to the manufacturers' instructions. RNA prepared from a tumor can be isolated, for example, by cesium chloride density gradient centrifugation.

[0292] The population of RNA can optionally be enriched, and further be amplified. For example, where RNA is mRNA, an amplification process such as RT-PCR can be utilized to amplify the mRNA, such that a signal is detectable or detection is enhanced. Such an amplification process is beneficial particularly when the biological, tissue, or tumor sample is of a small size or volume. Various amplification and detection methods can be used. For example, it is within the scope of the present disclosure to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps, or reverse transcribe mRNA into cDNA followed by symmetric gap ligase chain reaction (RT-AGLCR).

[0293] Many techniques are known in the state of the art for determining absolute and relative levels of gene expression, commonly used techniques suitable for use in the present disclosure include Northern analysis, RNase protection assays (RPA), microarrays and PCR* based techniques, such as quantitative PCR and differential display PCR. For example, Northern blotting involves running a preparation of RNA PATENT

[0294] ATTORNEY DOCKET NO.: 51432-056WO2 on a denaturing agarose gel, and transferring it to a suitable support, such as activated cellulose, nitrocellulose or glass or nylon membranes. Radiolabeled cDNA or RNA is then hybridized to the preparation, washed, and analyzed by autoradiography.

[0295] In situ hybridization visualization can also be employed, wherein a radioactively labeled antisense RNA probe is hybridized with a thin section of a biopsy sample, washed, cleaved with RNase and exposed to a sensitive emulsion for autoradiography. The samples can be stained with hematoxylin to demonstrate the histological composition of the sample, and dark field imaging with a suitable light filter shows the developed emulsion. Non-radioactive labels such as digoxigenin can also be used.

[0296] Alternatively, mRNA expression can be detected on a DNA array, chip or a microarray. Labeled nucleic acids of a test sample obtained from a subject can be hybridized to a solid surface comprising biomarker DNA Positive hybridization signal is obtained with the sample containing biomarker transcripts. In one embodiment, gene expression can be detected by microarray analysis. Differential gene expression can also be identified or confirmed using a microarray technique. The expression levels of one or more biomarker genes can be measured in either fresh or fixed tissue, using microarray technology. In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are plated, or arrayed, on a microchip substrate. The arrayed sequences are then hybridized with specific DNA probes from cells or tissues of interest. Fluorescently labeled cDNA probes can be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the microarray chip is scanned by a device such as, confocal laser microscopy or by another detection method. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance. Microarray analysis can be performed by commercially available equipment, following manufacturer's protocols.

[0297] Types of probes that can be used in the methods described herein include cDNA, riboprobes, synthetic oligonucleotides and genomic probes. The type of probe used will generally be dictated by the particular situation, such as riboprobes for in situ hybridization, and cDNA for Northern blotting, for example. In one embodiment, the probe is directed to nucleotide regions unique to the RNA The probes can be as short as is required to differentially recognize marker mRNA transcripts, and can be as short as, for example, about 15 bases; however, probes of at least 17, 18, 19 or 20 or more bases can be used. In one embodiment, the primers and probes hybridize specifically under stringent conditions to a DNA fragment having the nucleotide sequence corresponding to the marker. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least about 95% identity in nucleotide sequences. In another embodiment, hybridization under "stringent conditions" occurs when there is at least 97% identity between the sequences.

[0298] The activity, level or presence of a protein encoded by a biomarker gene can be detected and / or quantified by detecting or quantifying the expressed polypeptide. The polypeptide can be detected and quantified by any of a number of means well known to those of skill in the art. Any method known in the art for detecting polypeptides can be used. Such methods include, but are not limited to, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, binder-ligand assays, immunohistochemical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer PATENT

[0299] ATTORNEY DOCKET NO.: 51432-056WO2 chromatography (TLC), hyperdiffusion chromatography, and the like (for example, Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn, pp 217-262, 1991 which is incorporated by reference).

[0300] ELISA and RIA procedures can be conducted such that a desired protein standard is labeled (with a radioisotope such as 1251 or 35S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and, together with the unlabelled sample, brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay). Alternatively, the protein in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled antibody is allowed to react with the system, and radioactivity or the enzyme assayed (ELISA-sandwich assay). Other conventional methods can also be employed as suitable.

[0301] Enzymatic and radiolabeling of a protein encoded by a biomarker gene and / or the antibodies can be effected by conventional means. It is possible to immobilize the enzyme itself on a support, but if solidphase enzyme is required, then this is generally best achieved by binding to antibody and affixing the antibody to a support, models, and systems for which are well-known in the art.

[0302] Other techniques can be used to detect protein corresponding to a biomarker gene according to a practitioner's preference based upon the present disclosure. One such technique is Western blotting (Towbin et al., Proc. Nat. Acad. Sci. 76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter. Antibodies specific for the protein (unlabeled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-immunoglobulin (suitable labels including 1251 , horseradish peroxidase and alkaline phosphatase). Chromatographic detection can also be used.

[0303] Immunohistochemistry can be used to detect expression of a protein corresponding to a biomarker gene, for example, in a biopsy sample. A suitable antibody is brought into contact with, for example, a thin layer of cells, washed, and then contacted with a second, labeled antibody. Labeling can be by fluorescent markers, enzymes, such as peroxidase, avidin, or radiolabelling. The assay is scored visually, using microscopy. Any other art-known method can be used to detect a protein corresponding to a biomarker gene.

[0304] In various embodiments, the disclosure provides a method of treating cancer in a subject comprising administering to the subject a RAS(ON) inhibitor or combination of compounds described herein, wherein the subject has one or more tumors that are resistant or unresponsive to treatment. In various embodiments, the subject has one or more tumors that are resistant or unresponsive to one or more treatments selected from the group consisting of surgery, radiation, chemotherapy, biologic agents, small molecules, cell-based therapy, hormone therapy, and immunotherapy. In various embodiments, treatment is a standard of care therapy, first-line therapy, second-line therapy, or third* line therapy. In various embodiments, the subject has one or more tumors that have progressed during one or more treatments, wherein the treatments are standard of care therapy, first-line therapy, second-line therapy, or third-line therapy.

[0305] First-line therapy is defined as a treatment that is administered to a subject suffering from cancer who has not received any prior treatment. Second-line therapy is defined as treatment that is PATENT

[0306] ATTORNEY DOCKET NO.: 51432-056WO2 administered to a subject suffering from cancer who has received prior first-line therapy but experienced disease progression during first-line treatment. Third-line therapy is defined as treatment that is administered to a subject suffering from cancer who has received prior first and second-line treatment but has experienced disease progression during second-line treatment. Each particular type of cancer has a first-line, second-line, and third-line therapy. The first-, second-, and third-line therapies for types of cancer are known in the art. In addition, FDA approved drug labels will indicate if a particular drug is approved as a first-, second-, or third* line therapy.

[0307] In various embodiments, the disclosure provides a method of treating cancer in a subject comprising administering to the subject a RAS(ON) inhibitor or combination of compounds described herein, wherein the subject cannot tolerate standard of care therapy, first-line therapy, second-line therapy, or third-line therapy. In various embodiments, the disclosure provides a method of treating cancer in a subject comprising administering to the subject a RAS(ON) inhibitor or combination of compounds described herein, wherein the subject has experienced tumor recurrence after surgical resection of the primary tumor. In various embodiments, the disclosure provides a method of treating cancer in a subject comprising administering to the subject a RAS(ON) inhibitor or combination of compounds described herein, wherein the subject has a tumor that cannot be surgically removed. In various embodiments, the disclosure provides a method of treating cancer in a subject comprising administering to the subject a RAS(ON) inhibitor or combination of compounds described herein, wherein the subject has no treatment options available.

[0308] The methods of the disclosure may include a therapy disclosed herein in combination with one or more additional therapies (e.g., non-drug treatments or therapeutic agents). The dosages of one or more of the additional therapies (e.g., non-drug treatments or therapeutic agents) may be reduced from standard dosages when administered alone. For example, doses may be determined empirically from drug combinations and permutations or may be deduced by isobolographic analysis (e.g., Black et al., Neurology 65:S3-S6 (2005)).

[0309] A composition of the disclosure may be administered before, after, or concurrently with one or more of such additional therapies. When combined, dosages of a compound of the disclosure and dosages of the one or more additional therapies (e.g., non-drug treatment or therapeutic agent) provide a therapeutic effect (e.g., synergistic or additive therapeutic effect). A composition of the disclosure and an additional therapy, such as an anti-cancer agent, may be administered together, such as in a unitary pharmaceutical composition, or separately and, when administered separately, this may occur simultaneously or sequentially. Such sequential administration may be close or remote in time.

[0310] In some embodiments, the additional therapy is the administration of side-effect limiting agents (e.g., agents intended to lessen the occurrence or severity of side effects of treatment. For example, in some embodiments, the compounds of the present disclosure can also be used in combination with a therapeutic agent that treats nausea. Examples of agents that can be used to treat nausea include: dronabinol, granisetron, metoclopramide, ondansetron, and prochlorperazine, or pharmaceutically acceptable salts thereof.

[0311] In some embodiments, the one or more additional therapies includes a non-drug treatment (e.g., surgery or radiation therapy). In some embodiments, the one or more additional therapies includes a therapeutic agent (e.g., a compound or biologic that is an anti-angiogenic agent, signal transduction PATENT

[0312] ATTORNEY DOCKET NO.: 51432-056WO2 inhibitor, antiproliferative agent, glycolysis inhibitor, or autophagy inhibitor). In some embodiments, the one or more additional therapies includes a non-drug treatment (e.g., surgery or radiation therapy) and a therapeutic agent (e.g., a compound or biologic that is an anti-angiogenic agent, signal transduction inhibitor, antiproliferative agent, glycolysis inhibitor, or autophagy inhibitor). In other embodiments, the one or more additional therapies includes two therapeutic agents. In still other embodiments, the one or more additional therapies includes three therapeutic agents. In some embodiments, the one or more additional therapies includes four or more therapeutic agents.

[0313] The compositions described herein can be used in combination with the agents disclosed herein or other suitable agents, depending on the condition being treated. Hence, in some embodiments the one or more compounds of the disclosure will be co-administered with other therapies as described herein. When used in combination therapy, the compounds described herein may be administered with the second agent simultaneously or separately. This administration in combination can include simultaneous administration of the two agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. That is, a composition described herein and any of the agents described herein can be formulated together in the same dosage form and administered simultaneously. Alternatively, a composition of the disclosure and any of the therapies described herein can be simultaneously administered, wherein both the agents are present in separate formulations. In another alternative, a composition of the disclosure can be administered and followed by any of the therapies described herein, or vice versa. In some embodiments of the separate administration protocol, a compound of the disclosure and any of the therapies described herein are administered a few minutes apart, or a few hours apart, or a few days apart.

[0314] In some embodiments of any of the methods described herein, the first therapy (e.g., a compound of the disclosure) and one or more additional therapies are administered simultaneously or sequentially, in either order. The first therapeutic agent may be administered immediately, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to, 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, 14 hours, up to hours 16, up to 17 hours, up 18 hours, up to 19 hours up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, up to 24 hours, or up to 1-7, 1-14, 1-21 or 1 -30 days before or after the one or more additional therapies.

[0315] The disclosure also features kits including (a) a pharmaceutical composition including an agent (e.g., a compound of the disclosure) described herein, and (b) a package insert with instructions to perform any of the methods described herein. In some embodiments, the kit includes (a) a pharmaceutical composition including an agent (e.g., a compound of the disclosure) described herein, (b) one or more additional therapies (e.g., non-drug treatment or therapeutic agent), and (c) a package insert with instructions to perform any of the methods described herein.

[0316] As one aspect of the present disclosure contemplates the treatment of the disease or symptoms associated therewith with a combination of pharmaceutically active compounds that may be administered separately, the disclosure further relates to combining separate pharmaceutical compositions in kit form. The kit may comprise two separate pharmaceutical compositions: a compound of the present disclosure, and one or more additional therapies. The kit may comprise a container for containing the separate compositions such as a divided bottle or a divided foil packet. Additional examples of containers include syringes, boxes, and bags. In some embodiments, the kit may comprise directions for the use of the PATENT

[0317] ATTORNEY DOCKET NO.: 51432-056WO2 separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing health care professional.

[0318] Examples

[0319] The disclosure is further illustrated by the following examples and synthesis examples, which are not to be construed as limiting this disclosure in scope or spirit to the specific procedures herein described. It is to be understood that the examples are provided to illustrate certain embodiments and that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure or scope of the appended claims.

[0320] Example 1. Mutational analysis of response to a RAS(ON) inhibitor therapy

[0321] RAS mutations, particularly in the KRASG12Xcodons, are frequently observed across a range of solid tumors and represent a major oncogenic driver. Despite their prevalence, therapeutic targeting of RAS has historically been challenging due to its high affinity for GTP / GDP and the absence of suitable binding pockets. The development of small molecule inhibitors capable of selectively engaging RAS in its active, GTP-bound conformation ("RAS(ON)") has enabled new therapeutic opportunities. However, much remains unknown regarding the mechanisms of resistance to RAS(ON) inhibitors. Moreover, there are no validated biomarkers guiding selection of subjects that are likely to benefit from a RAS(ON) inhibitor therapy or for those subjects who are not likely to respond.

[0322] The present example provides evidence supporting the broad antitumor efficacy of daraxonrasib, (RMC-6236) as well as the identification of biomarkers that predict differential response to RAS(ON) inhibitor compounds, including CDKN2A loss and KEAP1 mutation.

[0323] As shown in FIG. 1A-1 C, once-daily oral administration of daraxonrasib (RMC-6236) (25 mg / kg) led to significant tumor volume reductions in xenograft models of NSCLC, PDAC, and CRC bearing KRASG12Xmutations. Tumor response was assessed via waterfall plots representing mean percent tumor volume change from baseline at response call date. Among 29 NSCLC models (FIG. 1A), 22 PDAC models (FIG. 1 B), and 23 CRC models (FIG. 1 C), multiple models in each tumor type showed partial or complete regression, indicating a broad spectrum of activity.

[0324] Importantly, no significant correlation was observed between tumor response and the presence or absence of common co-mutations or gene expression profiles (see oncoplots beneath each waterfall plot), including those in TP53, STK11 , CDKN2A, KEAP1 , or SMARCA4. These findings suggest that no significant association was found between RAS(ON) multi-selective inhibitor, RMC-6236, tumor volume response and the presence of functional alterations in genes reflecting major co-mutation classes and associated with disease etiology in each indication.

[0325] Next, the impact of occurrence of oncogenic mutations in KEAP1 and SMARCA4, as well as loss of expression of CDKN2A was examined on PFS in KRASG12XNSCLC models treated with RAS(ON) multi-selective inhibitor RMC-6236 (FIG. 2). To determine whether specific genomic features impact PATENT

[0326] ATTORNEY DOCKET NO.: 51432-056WO2 treatment durability, progression-free survival (PFS) in KRASG12X NSCLC xenograft models stratified by CDKN2A expression status was evaluated in FIG. 2. Among 29 models, 10 exhibited CDKN2A loss and 19 retained expression. In response to daraxonrasib, CDKN2A-deficient models showed significantly reduced PFS compared to CDKN2A-intact models. Interestingly, KRASG12XNSCLC models with loss of expression of CDKN2A (CDKN2A loss) exhibited a significantly shorter PFS on RMC-6236 treatment as compared to those with intact CDKN2A expression. It is possible that the loss of p16, the gene product of CDKN2A, removes the negative regulation of CDK4 / CyclinD and reduces the impact of RAS inhibition on this axis.

[0327] Co-mutation of KEAP1 (KEAPMUT) was also associated with reduced durability of response to RMC-6236 (FIG. 4), while SMARCA4 co-mutation (SMARCA4MUT) was not (FIG. 5). Notably, no significant prognostic effect was observed for any of the above-mentioned genomic aberrations on the PFS of KRASG12XNSCLC models in control groups. In contrast to CDKN2A and KEAP1 , SMARCA4 mutation status did not significantly impact PFS outcomes in daraxonrasib-treated KRASG12XNSCLC models (FIG. 5). Both SMARCA4MUTand SMARCA4WTmodels exhibited similar progression-free survival distributions. These results indicate that SMARCA4, despite being a frequent co-altered gene in NSCLC, may not influence sensitivity or resistance to RAS(ON) inhibition.

[0328] To assess whether CDKN2A loss also modulates response to other RAS(ON) inhibitors, the above analysis was performed using the RAS(ON) G12C-selective inhibitor elironrasib (RMC-6291) in KRASG12CNSCLC models (FIG. 3). Consistent with the findings for daraxonrasib, CDKN2A-deficient models displayed reduced PFS, indicating that CDKN2A status may represent a biomarker for predicting broadly response durability to RAS(ON) inhibition, including RAS(ON) multi-selective and RAS(ON) mutation-selective inhibitors.

[0329] Embodiments

[0330] Embodiment 1 : A method comprising: (a) determining a genotype of one or more biomarker genes comprising CDKN2A and RAS in a biological sample obtained from a subject afflicted with cancer; and (b) classifying the subject as likely or unlikely to have a durable response to a therapy comprising a RAS(ON) inhibitor based on the genotype of each of the one or more biomarker genes in the biological sample. Embodiment 2: The method of embodiment 1 , further comprising obtaining a biological sample from the subject.

[0331] Embodiment 3: The method of embodiment 1 or 2, wherein the biological sample is a tumor sample. Embodiment 4: The method of any one of embodiments 1 to 3, wherein the tumor has a genotype comprising a mutation in the one or more biomarker genes.

[0332] Embodiment 5: The method of embodiment 4, wherein the mutation inactivates the one or more biomarker genes.

[0333] Embodiment 6: The method of any one of embodiments 1 to 5, further comprising comparing the genotype with a reference genotype.

[0334] Embodiment 7: The method of any one of embodiments 1 to 6, wherein the genotype is reported as a score.

[0335] Embodiment 8: The method of any one of embodiments 1 to 7, wherein determining the genotype comprises genomic profiling. PATENT

[0336] ATTORNEY DOCKET NO.: 51432-056WO2

[0337] Embodiment 9: The method of any one of embodiments 1 to 8, wherein determining the genotype comprises measuring gene expression.

[0338] Embodiment 10: The method of embodiment 9, wherein measuring gene expression comprises detection of ribonucleic acids (RNAs) or polypeptides.

[0339] Embodiment 11 : The method of any one of embodiments 1 to 10, wherein the subject is classified as likely to have a durable response to a RAS(ON) inhibitor treatment.

[0340] Embodiment 12: The method of any one of embodiments 1 to 11 , wherein the subject is classified as unlikely to have a durable response to a RAS(ON) inhibitor treatment.

[0341] Embodiment 13: The method of any one of embodiments 1 to 12, wherein the cancer is selected from the group consisting of skin cancer, lung cancer, pancreatic cancer, breast cancer, and colorectal cancer. Embodiment 14: The method of embodiment 13, wherein the cancer is lung cancer.

[0342] Embodiment 15: The method of embodiment 14, wherein the lung cancer is non-small cell lung cancer (NSCLC).

[0343] Embodiment 16: The method of embodiment 15, wherein the NSCLC is lung adenocarcinoma.

[0344] Embodiment 17: The method of any one of embodiments 1 to 16, wherein the durable response is progression free survival.

[0345] Embodiment 18: A method of predicting duration of tumor growth inhibition in response to a therapy comprising a RAS(ON) inhibitor, the method comprising: (a) detecting in a biological sample previously obtained from a subject afflicted with cancer a genotype of one or more biomarker genes; and (b) predicting duration of tumor growth inhibition in response to the therapy comprising a RAS(ON) inhibitor by detecting a RAS mutation, and a CDKN2A mutation.

[0346] Embodiment 19: A method of predicting reduced duration of tumor growth inhibition to a therapy comprising a RAS(ON) inhibitor, the method comprising: (a) detecting in a biological sample previously obtained from a subject afflicted with cancer a genotype of one or more biomarker genes; (b) analyzing the genotype of the one or more biomarker genes in the tumor sample; and (c) predicting reduced duration of tumor cell growth to inhibition by the therapy comprising a RAS(ON) inhibitor, if the tumor sample comprises (i) a RAS mutation, and (ii) an inactivating CDKN2A mutation, a decreased copy number of CDKN2A, or a decreased expression of CDKN2A mRNA or protein.

[0347] Embodiment 20: A method of predicting increased duration of tumor growth to inhibition by a therapy comprising a RAS(ON) inhibitor, the method comprising: (a) detecting in a biological sample previously obtained from a subject afflicted with cancer a genotype of one or more biomarker genes; (b) analyzing the genotype of the one or more biomarker genes in the tumor sample; and (c) predicting increased duration of tumor cell growth to inhibition by the therapy comprising a RAS(ON) inhibitor, if the tumor sample comprises (i) a RAS mutation, and (ii) wild-type CDKN2A or normal expression of wild-type CDKN2A.

[0348] Embodiment 21 : A method of predicting resistance of tumor growth to inhibition by a therapy comprising a RAS(ON) inhibitor, the method comprising: (a) determining in a biological sample previously obtained from a subject afflicted with cancer a genotype of one or more biomarker genes comprising CDKN2A and RAS; (b) analyzing the genotype of the one or more biomarker genes in the tumor sample; and (c) predicting the resistance of tumor cell growth to inhibition by the therapy comprising a RAS(ON) inhibitor, PATENT

[0349] ATTORNEY DOCKET NO.: 51432-056WO2 if the tumor sample comprises (i) a RAS mutation, and (ii) an inactivating CDKN2A mutation, a decreased copy number of CDKN2A, or a decreased expression of CDKN2A mRNA or protein.

[0350] Embodiment 22: A method of selecting a subject for treatment with a combination therapy comprising a RAS(ON) inhibitor and a cell cycle inhibitor, the method comprising: (a) determining in a biological sample previously obtained from a subject afflicted with cancer a genotype of one or more biomarker genes comprising CDKN2A and RAS; (b) selecting the subject for the combination therapy, if the tumor sample comprises (i) a RAS mutation, and (ii) an inactivating CDKN2A mutation, a decreased copy number of CDKN2A, or a decreased expression of CDKN2A mRNA or protein.

[0351] Embodiment 23: A method of selecting a subject for treatment with a combination therapy comprising a RAS(ON) inhibitor and a DDR inhibitor, the method comprising: (a) determining in a biological sample previously obtained from a subject afflicted with cancer a genotype of one or more biomarker genes comprising CDKN2A and RAS; (b) selecting the subject for the combination therapy, if the tumor sample comprises (i) a RAS mutation, and (ii) an inactivating CDKN2A mutation, a decreased copy number of CDKN2A, or a decreased expression of CDKN2A mRNA or protein.

[0352] Embodiment 24: A method of enriching a prospective patient population for subjects likely to respond to a RAS(ON) inhibitor therapy, the method comprising: performing the method of any one of embodiments 1 to 17 on two or more individual subjects within the prospective patient population.

[0353] Embodiment 25: A method of treating a tumor in a subject, wherein the tumor comprises a RAS mutation and an inactivating CDKN2A mutation, a decreased copy number of CDKN2A, or a decreased expression of CDKN2A mRNA or protein, the method comprising: administering to the subject an amount of a RAS(ON) inhibitor and an amount of a cell cycle inhibitor effective to treat the tumor.

[0354] Embodiment 26: A method of treating a tumor in a subject, wherein the tumor comprises a RAS mutation and an inactivating CDKN2A mutation, a decreased copy number of CDKN2A, or a decreased expression of CDKN2A mRNA or protein, the method comprising: administering to the subject an amount of a RAS(ON) inhibitor and an amount of a DDR inhibitor effective to treat the tumor.

[0355] Embodiment 27: The method of any one of embodiments 18-26, wherein the cancer is lung cancer or the tumor is a lung cancer tumor.

[0356] Embodiment 28: The method of embodiment 27, wherein the lung cancer is non-small cell lung cancer (NSCLC).

[0357] Embodiment 29: The method of embodiment 28, wherein the NSCLC is lung adenocarcinoma. Embodiment 30: The method of embodiment 25 or 26, wherein the inhibitors are administered simultaneously or sequentially.

[0358] Embodiment 31 : The method of any one of embodiments 25 to 30, wherein the inhibitors are administered as a single formulation or in separate formulations.

[0359] Embodiment 32: The method of any one of embodiments 1 to 31 , wherein the RAS(ON) inhibitor is a RAS(ON) G12C-selective inhibitor.

[0360] Embodiment 33: The method of any one of embodiments 1 to 31 , wherein the RAS(ON) inhibitor is a RAS(ON) multi-selective inhibitor.

[0361] Embodiment 34: A method of treating a subject afflicted with a tumor resistant to a RAS(ON) inhibitor therapy comprising administering to the subject an amount of a RAS(ON) inhibitor and an amount of a cell cycle inhibitor, effective to treat the tumor. PATENT

[0362] ATTORNEY DOCKET NO.: 51432-056WO2

[0363] Embodiment 35: A method of treating a subject afflicted with a tumor resistant to a RAS(ON) inhibitor therapy comprising administering to the subject an amount of a RAS(ON) inhibitor and an amount of a DNA damage response inhibitor, effective to treat the tumor.

[0364] Embodiment 36: The method of claim 34 or 35, wherein the tumor has a RAS mutation and an inactivating CDKN2A mutation, a decreased copy number of CDKN2A, or a decreased expression of CDKN2A mRNA or protein.

[0365] Embodiment 37. The method of embodiment 36, wherein the tumor has a CDKN2A copy number or expression lower than a predetermined reference value.

[0366] Embodiment 38: The method of embodiment 36 or 37, wherein the RAS mutation is a mutation of KRAS, NRAS or HRAS.

[0367] Embodiment 39: The method of any one of embodiments 34 to 38, wherein the RAS mutation is a G12C mutation.

[0368] Embodiment 40: The method of any one of embodiments 34 to 38, wherein the RAS mutation is a G12D mutation.

[0369] Embodiment 41 : The method of any one of embodiments 34 to 38, wherein the RAS mutation is a G12V mutation.

[0370] Embodiment 42: The method of any one of embodiments 34 to 38, wherein the RAS mutation is a G13C mutation.

[0371] Embodiment 43: The method of any one of embodiments 34 to 38, wherein the RAS mutation is a Q61 H mutation.

[0372] Embodiment 44: The method of any one of embodiments 34 to 43, wherein the tumor is a lung cancer tumor, pancreatic cancer tumor, or colorectal cancer tumor.

[0373] Embodiment 45: The method of embodiment 44, wherein the lung cancer is a non-small cell lung cancer. Embodiment 46: The method of embodiment 44, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

[0374] Embodiment 47: The method of embodiment 39, wherein the RAS(ON) inhibitor is a RAS(ON) G12C- selective inhibitor.

[0375] Embodiment 48: The method of embodiment 47, wherein the RAS(ON) G12C-selective inhibitor is elironrasib.

[0376] Embodiment 49: The method of embodiment 40, wherein the RAS(ON) inhibitor is a RAS(ON) G12D- selective inhibitor.

[0377] Embodiment 50: The method of embodiment 49, wherein the RAS(ON) G12D-selective inhibitor is zoldonrasib.

[0378] Embodiment 51 : The method of embodiment 41 , wherein the RAS(ON) is a RAS(ON) G12V-selective inhibitor.

[0379] Embodiment 52: The method of embodiment 51 , wherein the RAS(ON) G12V-selective inhibitor is RMC- 5127.

[0380] Embodiment 53: The method of embodiment 42, wherein the RAS(ON) inhibitor is a RAS(ON) G1 SC- selective inhibitor.

[0381] Embodiment 54: The method of embodiment 53, wherein RAS(ON) G13C-selective inhibitor is RMC- 8839. PATENT

[0382] ATTORNEY DOCKET NO.: 51432-056WO2

[0383] Embodiment 55: The method of embodiment 43, wherein the RAS(ON) inhibitor is a RAS(ON) Q61 H- selective inhibitor.

[0384] Embodiment 56: The method of embodiment 55, wherein the RAS(ON) Q61 H-selective inhibitor is RMC- 0708.

[0385] Embodiment 57: The method of any one of embodiments 34 to 46, wherein the RAS(ON) inhibitor is a RAS(ON) multi-selective inhibitor.

[0386] Embodiment 58: The method of embodiment 57, wherein the RAS(ON) multi-selective inhibitor is daraxonrasib.

[0387] Embodiment 59: The method of any one of embodiments 34 or 36 to 58, wherein the cell cycle inhibitor is a CDK inhibitor.

[0388] Embodiment 60: The method of embodiment 59, wherein the CDK inhibitor is a CDK2, CDK4 / 6, CDK7 or CDK9 inhibitor.

[0389] Embodiment 61 : The method of embodiment 58, wherein the CDK inhibitor is palbociclib, ribociclib, abemaciclib, trilaciclib, tagtociclib, seliciclib, voruciclib (P1446A-05), BLU-222, dinaciclib, AT-7519, RGB286638, or AZD4573.

[0390] Embodiment 62: The method of any one of embodiments 34 or 36 to 58, wherein the cell cycle inhibitor is a Wee1 inhibitor.

[0391] Embodiment 63: The method of embodiment 62, wherein the Wee1 inhibitor is imp7068, adavosertib, or ZNL-02-096.

[0392] Embodiment 64: The method of any one of embodiments 34 or 36 to 58, wherein the cell cycle inhibitor is a CHK inhibitor.

[0393] Embodiment 65: The method of embodiment 64, wherein the CHK inhibitor is a CHK1 or CHK2 inhibitor.

[0394] Embodiment 66: The method of embodiment 63, wherein the CHK inhibitor is BBI-355, rabusertib, LY2606368, LY2880070, GDC-0575, MK-8776, BEBT-260, or PEP07.

[0395] Embodiment 67: The method of any one of embodiments 35 to 58, wherein the DNA damage response inhibitor is an ATM inhibitor.

[0396] Embodiment 68: The method of embodiment 67, wherein the ATM inhibitor is lartesertib, AZD1390, AZD0156, KU-60019, M4076, M3541 , WSD-0628, ZN-B-2262, SYH2051 , or VE-821 .

[0397] Embodiment 69: The method of any one of embodiments 35 to 58, wherein the DNA damage response inhibitor is an ATR inhibitor.

[0398] Embodiment 70: The method of claim 69, wherein the ATR inhibitor is berzosertib, gartisertib, camonsertib, ceralaertib, VE-821 , RP-3500, AZ20, VX-970, abd110, VX-803, or elimusertib (BAY 1895344).

[0399] Embodiment 71 : The method of any one of embodiments 35 to 58, wherein the DNA damage response inhibitor is a PARP inhibitor.

[0400] Embodiment 72: The method of embodiment 71 , wherein the PARP inhibitor is olaparib, rucaparib, niraparib, or veliparib (ABT-888).

[0401] Embodiment 73: The method of any one of embodiments 35 to 58, wherein the DNA damage response inhibitor is a DNA-PK inhibitor.

[0402] Embodiment 74: The method of embodiment 71 , wherein the DNA-PK inhibitor is NU7441 , AZD7648, VX- 984, peposertib (M3814), or CC-115. PATENT

[0403] ATTORNEY DOCKET NO.: 51432-056WO2

[0404] Embodiment 75: The method of any one of embodiments 34 to 74, wherein the subject has not received a prior cancer therapy.

[0405] Embodiment 76: The method of any one of embodiments 34 to 74, wherein the subject has received at least one prior cancer therapy to treat the cancer.

[0406] Embodiment 77: The method of any one of embodiments 34 to 76, wherein the RAS(ON) inhibitor and the cell cycle inhibitor or DNA damage response inhibitor are administered sequentially.

[0407] Embodiment 78: The method of any one of embodiments 34 to 76, wherein the RAS(ON) inhibitor and the cell cycle inhibitor or DNA damage response inhibitor are administered sequentially or concurrently. Embodiment 79: A method of identifying a subject afflicted with cancer for treatment with a RAS(ON) inhibitor and a cell cycle inhibitor, the method comprising:

[0408] (a) obtaining a biological sample from the subject;

[0409] (b) determining the RAS and CDKN2A mutation status of the subject; and

[0410] (c) identifying the subject as a candidate for treatment with the RAS(ON) inhibitor and cell cycle inhibitor combination when the cancer comprises a RAS mutation and an inactivating CDKN2A mutation, a decreased copy number of CDKN2A, or a decreased expression of CDKN2A mRNA or protein. Embodiment 80: The method of embodiment 79, wherein the subject identified in (c) is administered a RAS(ON) inhibitor and a cell cycle inhibitor.

[0411] Embodiment 81 : A method of identifying a subject afflicted with cancer for treatment with a RAS(ON) inhibitor and a DNA damage response inhibitor, the method comprising:

[0412] (a) obtaining a biological sample from the subject;

[0413] (b) determining the RAS and CDKN2A mutation status of the subject; and

[0414] (c) identifying the subject as a candidate for treatment with the RAS(ON) inhibitor and cell cycle inhibitor combination when the cancer comprises a RAS mutation and an inactivating CDKN2A mutation, a decreased copy number of CDKN2A, or a decreased expression of CDKN2A mRNA or protein. Embodiment 82: The method of embodiment 81 , wherein the subject identified in (c) is administered a RAS(ON) inhibitor and a DNA damage response inhibitor.

[0415] Embodiment 83: A pharmaceutical composition comprising a RAS(ON) inhibitor and a pharmaceutically acceptable carrier for use in any of the methods of embodiments 1 to 82.

[0416] Other Embodiments

[0417] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the disclosure that come within known or customary practice within the art to which the disclosure pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims.

Claims

PATENTATTORNEY DOCKET NO.: 51432-056WO2Claims1 . A method of treating a subject afflicted with a tumor resistant to a RAS(ON) inhibitor therapy comprising administering to the subject an amount of a RAS(ON) inhibitor and an amount of a cell cycle inhibitor or DNA damage response inhibitor that together are effective to treat the tumor.

2. The method of claim 1 , wherein the tumor has a RAS mutation and (i) an inactivating CDKN2A mutation, (ii) a decreased copy number of CDKN2A, or (iii) a decreased expression of CDKN2A mRNA or protein.

3. The method of claim 1 or 2, wherein the tumor has a CDKN2A copy number or expression lower than a predetermined reference value.

4. The method of claim 2 or 3, wherein the RAS mutation is a mutation of KRAS, NRAS or HRAS.

5. The method of any one of claims 1 to 4, wherein the RAS mutation is a G12C mutation.

6. The method of any one of claims 1 to 4, wherein the RAS mutation is a G12D mutation.

7. The method of any one of claims 1 to 4, wherein the RAS mutation is a G12V mutation.

8. The method of any one of claims 1 to 4, wherein the RAS mutation is a G13C mutation.

9. The method of any one of claims 1 to 4, wherein the RAS mutation is a Q61 H mutation.

10. The method of any one of claims 1 to 9, wherein the tumor is a lung cancer tumor, a pancreatic cancer tumor, or a colorectal cancer tumor.11 . The method of claim 10, wherein the lung cancer is a non-small cell lung cancer.

12. The method of claim 10, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

13. The method of claim 5, wherein the RAS(ON) inhibitor is a RAS(ON) G12C-selective inhibitor.

14. The method of claim 13, wherein the RAS(ON) G12C-selective inhibitor is elironrasib.

15. The method of claim 6, wherein the RAS(ON) inhibitor is a RAS(ON) G12D-selective inhibitor.

16. The method of claim 15, wherein the RAS(ON) G12D-selective inhibitor is zoldonrasib.

17. The method of claim 7, wherein the RAS(ON) is a RAS(ON) G12V-selective inhibitor.PATENTATTORNEY DOCKET NO.: 51432-056WO218. The method of claim 17, wherein the RAS(ON) G12V-selective inhibitor is RMC-5127.

19. The method of claim 8, wherein the RAS(ON) inhibitor is a RAS(ON) G13C-selective inhibitor.

20. The method of claim 19, wherein RAS(ON) G13C-selective inhibitor is RMC-8839.21 . The method of claim 9, wherein the RAS(ON) inhibitor is a RAS(ON) Q61 H-selective inhibitor.

22. The method of claim 21 , wherein the RAS(ON) Q61 H-selective inhibitor is RMC-0708.

23. The method of any one of claims 1 to 12, wherein the RAS(ON) inhibitor is a RAS(ON) multi- selective inhibitor.

24. The method of claim 23, wherein the RAS(ON) multi-selective inhibitor is daraxonrasib.

25. The method of any one of claims 1 to 24, wherein the cell cycle inhibitor is a CDK inhibitor.

26. The method of claim 25, wherein the CDK inhibitor is a CDK2, CDK4 / 6, CDK7 or CDK9 inhibitor.

27. The method of claim 25, wherein the CDK inhibitor is palbociclib, ribociclib, abemaciclib, trilaciclib, tagtociclib, seliciclib, voruciclib (P1446A-05), BLU-222, dinaciclib, AT-7519, RGB286638, or AZD4573.

28. The method of any one of claims 1 to 24, wherein the cell cycle inhibitor is a Wee1 inhibitor.

29. The method of claim 28, wherein the Wee1 inhibitor is imp7068, adavosertib, or ZNL-02-096.

30. The method of any one of claims 1 to 24, wherein the cell cycle inhibitor is a CHK inhibitor.31 . The method of claim 30, wherein the CHK inhibitor is a CHK1 or CHK2 inhibitor.

32. The method of claim 30, wherein the CHK inhibitor is BBI-355, rabusertib, LY2606368, LY2880070, GDC-0575, MK-8776, BEBT-260, or PEP07.

33. The method of any one of claims 1 to 24, wherein the DNA damage response inhibitor is an ATM inhibitor.

34. The method of claim 33, wherein the ATM inhibitor is lartesertib, AZD1390, AZD0156, KU-60019, M4076, M3541 , WSD-0628, ZN-B-2262, SYH2051 , or VE-821 .PATENTATTORNEY DOCKET NO.: 51432-056WO235. The method of any one of claims 1 to 24, wherein the DNA damage response inhibitor is an ATR inhibitor.

36. The method of claim 35, wherein the ATR inhibitor is berzosertib, gartisertib, camonsertib, ceralaertib, VE-821 , RP-3500, AZ20, VX-970, abd110, VX-803, or elimusertib (BAY 1895344).

37. The method of any one of claims 1 to 24, wherein the DNA damage response inhibitor is a PARP inhibitor.

38. The method of claim 37, wherein the PARP inhibitor is olaparib, rucaparib, niraparib, or veliparib (ABT-888).

39. The method of any one of claims 1 to 24, wherein the DNA damage response inhibitor is a DNA- PK inhibitor.

40. The method of claim 39, wherein the DNA-PK inhibitor is NU7441 , AZD7648, VX-984, peposertib (M3814), or CC-115.41 . The method of any one of claims 1 to 40, wherein the subject has not received a prior cancer therapy.

42. The method of any one of claims 1 to 40, wherein the subject has received at least one prior cancer therapy to treat the tumor.

43. The method of any one of claims 1 to 42, wherein the RAS(ON) inhibitor and the cell cycle inhibitor or DNA damage response inhibitor are administered sequentially or concurrently.

44. A method of identifying a subject afflicted with cancer for treatment with a RAS(ON) inhibitor and a cell cycle inhibitor, the method comprising:(a) obtaining a biological sample from the subject;(b) determining a RAS and CDKN2A mutation status of the subject; and(c) identifying the subject as a candidate for treatment with the RAS(ON) inhibitor and cell cycle inhibitor combination when the cancer comprises a RAS mutation and (i) an inactivating CDKN2A mutation, (ii) a decreased copy number of CDKN2A, or (iii) a decreased expression of CDKN2A mRNA or protein.

45. The method of claim 44, wherein the subject identified in (c) is administered a RAS(ON) inhibitor and a cell cycle inhibitor.

46. A method of identifying a subject afflicted with cancer for treatment with a RAS(ON) inhibitor and a DNA damage response inhibitor, the method comprising:PATENTATTORNEY DOCKET NO.: 51432-056WO2(a) obtaining a biological sample from the subject;(b) determining a RAS and CDKN2A mutation status of the subject; and(c) identifying the subject as a candidate for treatment with the RAS(ON) inhibitor and cell cycle inhibitor combination when the cancer comprises a RAS mutation and (i) an inactivating CDKN2A mutation, (ii) a decreased copy number of CDKN2A, or (iii) a decreased expression of CDKN2A mRNA or protein.

47. The method of claim 46, wherein the subject identified in (c) is administered a RAS(ON) inhibitor and a DNA damage response inhibitor.

48. A pharmaceutical composition comprising a RAS(ON) inhibitor and a pharmaceutically acceptable carrier for use in any of the methods of claims 1 to 47.

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