Combination of a KRAS inhibitor and an EGFR inhibitor for the treatment of colorectal cancer

Abstract

The disclosure addresses the challenge of treating KRAS-mutant colorectal cancer (CRC), a significant subset of metastatic CRC with limited treatment options due to inherent resistance mechanisms. The disclosure involves a pharmaceutical composition combining a KRAS inhibitor, such as MRTX1133, with an epidermal growth factor receptor (EGFR) inhibitor, like Osimertinib. This combination aims to enhance the efficacy of KRAS inhibitors and overcome resistance in CRC cells. The method includes administering these inhibitors concurrently or sequentially to patients with KRAS mutations, potentially improving treatment outcomes by targeting multiple pathways involved in tumor growth and resistance. The disclosure provides a novel approach to managing KRAS-mutant CRC, offering a treatment option with the potential for more durable responses.

Description

C-1572 (EIC0016PCT)COMBINATION OF A KRAS INHIBITOR AND AN EGFR INHIBITOR FOR THE TREATMENT OF COLORECTAL CANCERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority from U.S. Provisional Application No.63 / 746,435, filed on January 17, 2025. the entire contents of which are incorporated herein by reference for all purposes.FEDERAL RESEARCH STATEMENT

[0002] This invention was made with government support under grant number P30CA013330, R00GM138758 and R35GM155249 awarded by the by the National Institute of Health. The government has certain rights in the invention.BACKGROUND

[0003] Colorectal cancer (CRC) is the third deadliest cancer globally. Death due to CRC is almost exclusively caused by the progression of metastatic CRC (mCRC). The standard treatment options for mCRC are combination cytotoxic chemotherapies and biologic agents such as EGFR- and VEGF-inhibiting antibodies. However, the vast majority of mCRC patients progress through all approved treatments and eventually die from their disease. Thus, novel therapies for mCRC are desperately needed. KRAS-mutant CRC represents approximately 50% of the mCRC population. RAS oncogenes drive the activation of the mitogen- activated protein kinase (MAPK) pathway and are a principal cancer driver pathway in KRAS-mutant CRCs. These oncogenes also represent a viable treatment target, as demonstrated by numerous recent advances in KRAS-directed treatments. The KRASG12Dmutation is the most common KRAS mutation found in CRC patients, making this particular allele a high-priority therapeutic target.

[0004] Previously, the KRAS protein was considered undruggable until the FDA approved Sotorasib, which targets KRASG12Cmutant tumors, for the treatment of non-small cell lung cancer. This was a landmark event but also highlighted drawbacks to KRAS allele-specific inhibitors. KRAS-mutant CRCs have many adaptive and long-term resistance mechanisms, making single-agent treatment with KRAS inhibitors ineffective for long-term disease control. Another drawback with G12C specific inhibitors is the patients in the clinical studiesC-1572 (EIC0016PCT)experienced toxicity and acquired resistance to the point specific inhibitors. The CodeBreaK100 (NCT03600883) trial assessed Sotorasib in advanced KRASG12C-mutant colorectal cancer (CRC), involving 62 patients. The study showed a 9.7% objective response rate, all partial responses. Despite not meeting the benchmark response rate, once daily Sotorasib demonstrated modest anti-tumor activity and manageable safety. Many patients eventually develop progressive disease (PD) due to mechanisms of resistance that remain largely unknown. The only small molecule KRAS inhibitor currently approved for CRC patients is Adagrasib, which covalently binds in the inactive form of KRASG12Cin the switch II pocket, like Sotorasib. In June 2024, Adagrasib, combined with the EGFR-directed antibody, Cetuximab, demonstrated promising clinical activity, resulting in accelerated FDA approval for progressive KRASG12Cmutant tumors. Moreover, KRASG12Cmutations are relatively infrequent in colorectal cancers, representing only 11% of KRAS mutations in these malignancies, and are only present in 3% of colorectal cancers overall. In contrast, KRASG12Dmutation occurs in approximately 20% of CRC patients.

[0005] The KRAS G12D mutation is the most common KRAS mutation in CRC, leading to the constitutive activation of KRAS, which drives abnormal cell growth and survival through persistent activation of downstream signaling pathways such as the RAS-RAF-MEK-ERK and PI3K-AKT pathways. Despite its high prevalence, KRAS-mutant CRC has proven to be particularly challenging to treat due to its inherent resistance mechanisms. KRAS-mutant tumors develop various adaptive and long-term resistance mechanisms, rendering traditional therapies, including chemotherapy and single-agent targeted therapies, largely ineffective in achieving sustained disease control.

[0006] More recently, the KRASG12Dinhibitor MRTX1133 was reported (Fig. 1 A). This compound selectively binds to the G12D mutant form of KRAS, inhibiting its activity and thereby disrupting the downstream signaling pathways that drive tumor growth and survival. Recent preclinical studies have demonstrated the potent efficacy of MRTX1133 in reducing tumor proliferation in models of KRASG12Dmutant CRC. However, the therapeutic efficacy in preclinical data showed distinct patterns in cancer xenograft models, showing lower therapeutic effect in colorectal cancer compared to pancreatic cancer. The early clinical experience with KRAS inhibitors has also revealed several challenges. There is a diversity of on-target and off-target mechanisms that can confer resistance to KRAS inhibitors and support the need for theC-1572 (EIC0016PCT)development of additional KRAS-targeting therapeutic strategies. Many such mechanisms, including alternative KRAS mutations and mutations in other MAPK proteins, were detected in response to KRASG12Ctreatment in patients. Similarly, in preclinical models of pancreatic cancer, MRTX1133 treatment led to diverse resistance mechanisms including epithelial-mesenchymal transition, PI3K-AKT-mTOR signaling, and amplification of Kras, Yapl, Myc, Cdk6, and Abcbla / b. Combination therapy is a promising approach to overcome these limitations and induce deeper, more durable responses in patients.SUMMARY

[0007] Herein, through agnostic screening of over 2,600 kinase inhibitors against both parental and resistant colorectal cancer cells, novel drug combinations that potentiate the efficacy of MRTX1133 and overcome acquired drug resistance were identified.

[0008] This disclosure provides a pharmaceutical composition that potentiates the efficacy of KRAS inhibitors that can overcome the resistant colorectal cancer. The pharmaceutical composition comprises a combination of a KRAS inhibitor and an epidermal growth factor receptor (EGFR) inhibitor. The KRAS inhibitor comprises MRTX1133 (CAS Reg. No. 2621928-55-8), RMC-6236 (CAS Reg. No. 2765081-21-6), RMC-7977 (CAS Reg. No. 2765082-12-8), RMC-9805 (CAS Reg. No. 2922732-54-3), or RMC-6291 (CAS Reg. No.2641998-63-0). The EGFR inhibitor comprises Osimertinib (CAS Reg. No. 1421373-65-0), AZ-5104 (CAS Reg. No. 1421373-98-9), Limertinib (CAS Reg. No. 1934259-00-3), Afatinib (CAS Reg. No. 850140-72-6), Dacomitinib (CAS Reg. No. 1110813-31-4), Erlotinib (CAS Reg. No.183319-69-9), Gefitinib (CAS Reg. No. 184475-35-2), Lazertinib (CAS Reg. No. 1903008-80-9), Neratinib (CAS Reg. No. 698387-09-6), Vandetanib (CAS Reg. No. 443913-73-3), or mutant EGFR inhibitor (CAS Reg. No. 1421373-62-7).

[0009] The disclosure also provides a method of inhibiting the proliferation or growth of cancer cells comprising contacting cancer cells, concurrently or sequentially, with a KRAS inhibitor and an EGFR inhibitor. The KRAS inhibitor comprises MRTX1133, RMC-6236, RMC-7977, RMC-9805, or RMC-6291. The EGFR inhibitor comprises Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor. The cancer cells are colorectal cancer (CRC) cells, metastatic colorectal cancer (mCRC) cells, colon cancer cells, or rectal cancer cells.C-1572 (EIC0016PCT)

[0010] The disclosure further provides a method of treating cancer in a subject in need thereof, and the method comprises determining whether the subject has a cancer with a KRAS mutation; and if the subject has a cancer with a KRAS mutation, and administering to the subject concurrently or sequentially, a therapeutically effective amount of a KRAS inhibitor and an EGFR inhibitor. The KRAS inhibitor comprises MRTX1133, RMC-6236, RMC-7977, RMC-9805. or RMC-6291. The EGFR inhibitor comprises Osimertinib. AZ-5104, Limertinib. or mutant EGFR inhibitor. The KRAS mutation includes but is not limited to KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS G12S, KRAS Q61H, KRAS Q61L or a KRAS G13D. One or both of the EGFR inhibitor and the KRAS inhibitor are administered orally, rectally, sublingually, in the buccal cavity, intravenously, intratumorally, percutaneously, intramuscularly, transdermally, cutaneously. subcutaneously, intrathecally, nasally, or vaginally.

[0011] The above described and other features are exemplified by the following figures and detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] A better understanding of features and advantages of the present disclosure will be obtained by reference to the following detailed description, which sets forth illustrative embodiments of the disclosure, and the accompanying drawings. The following figures are exemplified embodiments.

[0013] Figs. 1A-E show KRAS G12D inhibitor MRTX1133 and generation of resistant cell lines. Fig. 1A; Chemical structure of the KRAS G12D Inhibitor MRTX1133. Fig. 1B:MRTX1133 selectively inhibits the LS-174T cells (KRAS G12D mutation) compared to HCT116 CRC cells (KRAS G13D mutation) or RKO cells (WT KRAS). Fig. 1C: Generation of MRTX1133- resistant LS-174T cell lines. Concentration-dependent inhibition curves of cell viability against parental (circle) or resistant (square) cells are shown. Fig. ID: Immunoblotting confirmed the increased expression levels of key proteins involved in the MAPK signaling pathway in the resistant cells compared to their parental counterparts. Fig. IE: Genomic mutational profiling identified the emergence of a new KRAS mutation in the resistant cell line.

[0014] Figs.2A-D show schematics of the screening assay and screening triage. Fig. 2A: Overview of Assay set up for high throughput screening in LS-174T Parental (P) and LS-174T Resistant (R) cells. Fig. 2B: Screening triage. Detailed triage, hit cutoff, hit numbers, and theirC-1572 (EIC0016PCT)structures are shown in supporting information. Fig. 2C: Known targets of hit molecules shown in the schematics of cancer signaling pathways. Fig. 2D: Identified “hit” molecules sorted into 16 distinct categories.

[0015] Figs.3A-C show the validation of High-throughput screening (HTS) drug combinations for overcoming MRTX1133 resistance. Fig. 3A: Seven compounds were chosen to validate using multi-dose synergy testing based on manual inspection for pan-assay interference and un-druglike properties. Fig. 3B and Fig. 3C: NT-1 significantly enhances the synergistic effect in the parental and resistant cell lines, showing the strong synergistic effect at low doses in panels.

[0016] Figs.4A-B. Irreversible EGFR inhibitors synergizer comparison in vitro. Fig. 4A and 4B: Dose-response of NT-1, and the FDA approved wildtype EGFR treatment, Cetuximab, and the superiority of NT-1.

[0017] Figs. 5A-C. Combination therapy blocks downstream MAPK pathway in mrtx1133 resistant cells. Fig. 5 A: Western blot showing the monotherapy and combined effect of NT-land MRTX1133 in the resistant cells. Figs 5B and 5C: Western blot showing the cells cotreated with NT-1 failed to activate EGFR, indicating effective suppression of the feedback loop.

[0018] Figs.6A-D. Irreversible EGFR inhibitors synergize and overcome MRTX1133 resistance in PDO models. Figs. 6A and B: the drug combination in vitro to determine if the heterogeneity of CRC PDO models could be overcome using the top synergistic compound. Fig.6C: A histological view of the treated organoid after 72 hours, shows apoptosis in both monotherapy and combination therapy. Fig. 6D: the combination treatment immunoblot reveals similar suppression of p-EGFR, p-ERK and p-S6. which is not observed in monotherapy alone.

[0019] Fig. 7. Structure of four additional KRAS inhibitors: RMC-6236, RMC-7977, RMC-9805, and RMC-6291.

[0020] Fig. 8. Structure of 4 EGFR inhibitors: Fig. 8A, Osimertinib; Fig. 8B, AZ-5104; Fig. 8C, Limertinib; and Fig. 8D, mutant EGFR inhibitor (Mutant EGFRi, or NT-1).

[0021] Fig. 9A. Dose-response curves of NT-1 (left) and Osimertinib (right) treated alone and in combination with MRTX1133 for 72 hours in LS-174T parental. Fig.9B. Doseresponse curves of NT-1 (left) and Cetuximab (right) treated alone and in combination with MRTX1133 for 72 hours in LS-174T / MRTX1133 resistant cell line. Fig. 9C. Immunoblot dataC-1572 (EIC0016PCT)of LS-174T-R cell line treated with MRTX alone (left) and in combination with NT-1 at 60nM (right), focusing on various downstream MARK pathways.

[0022] Fig. 10 Table of synergy scores from top screening hit compounds treated in 8x8 synergy matrix with MRTX1133. Bolded numbers over 10 considered synergistic. Concentration starting at 1000nM. 8x8 dose-response inhibition matrices were also determined for top 6 hit compounds in combination with MRTX 1133 for 72 hours. The cell line is a combination in LS-174T-MRTX1133 Resistant cell line (data not shown)

[0023] Fig. 11A. Table of synergy scores of multiple EGFRi treated in 8x8 synergy matrix with MRTX1133. Bolded numbers over 10 are considered synergistic. Concentrations starting at 100nM were evaluated. Fig. 11B. Proliferation assay over 3 days in three different KRASG12DCRC cell lines treated with DMSO, MRTX1133 (100nM), Gefitinib (100nM), Osimertinib (100nM), Limertinib, (100nM), NT-1 (100nM), Afatinib (100nM) and Erlotinib (100nM) or combination. Graph reflects the relative change in cell number (log2 fold scale to best visualize loss of cells) compared with day 0.

[0024] Fig. 12A. Dose-response data with NT-1 (starting at 300nM) in combination with MRTX1133 (starting at 3µM) in the G12D-PDO-resistant organoid. Fig. 12 B. presents the dose response curve along with respective CI values for each concentration. Fig. 12C. Proliferation assay over 3 days in three different KRASG12DPDO-models treated MRTX1133 (3000nM), NT-1 (300nM), or combination. Graph reflects the relative change in cell number (log2 fold scale to best visualize loss of cells) compared with day 0.

[0025] Fig. 13A. Dose-response curves for RMC-7977 and NT-1, administered as single agents or in combination for 72 hours, in KRAS G12D colorectal cancer cell lines (LS-174T and LS-513), KRAS G13D CRC Cell line (HCT116) and in LS-174T / MRTX1133-resistant cells. Fig.13B. Dose-response curves of RMC-7977 treated with NT-1 alone and in combination in 4 different CRC cell lines over 72 hours.

[0026] Fig. 14. Dose-response curves for RMC-6236 and NT-1, administered as single agents or in combination for 72 hours, in KRAS colorectal cancer cell lines (LS-174T and LS-513) and a KRAS colorectal cancer cell line (HCT116). Dose-response curves for RMC-6236 and NT-1, administered as single agents or in combination for 72 hours, in two KRAS -resistant colorectal cancer cell lines (LS-174T / MRTX1133 and LS-174T / RMC-6236).C-1572 (EIC0016PCT)

[0027] Fig. 15. Table of synergy scores from KRAS inhibitors treated in 8x8 synergy matrix with NT-1. Bolded numbers over 10 considered synergistic. Concentration starting at 1000nM. 8x8 dose-response inhibition matrix were also obtained for NT-1 and RMC-6236 treated for 72 hours in four different cell lines, (data not shown)

[0028] Fig. 16A. Biochemical data of NT-1 reveals kinase targets, showing NT-l’s highest potency potent towards EGFRWildtype’ JAK3, and EGFRT790M. Fig. 16B. Healthy NOD / SCID mice were treated daily via oral gavage at different doses of NT-1 to watch for adverse reactions. All mice remained active and healthy, no apparent hair loss or weight loss observed.

[0029] Fig. 17 illustrates dose-response curves for monotherapy treatment with four KRAS inhibitors (MRTX1133, RMC-9805, RMC-7977, and RMC-6236) across colorectal cancer (CRC) cell lines (RKO, DLD1, HCT116, LS-513, and LS-174T) following 72-hour exposure. KRAS cell lines are highlighted in green, KRAS cell lines are highlighted in red, and RKO is included as a KRAS wild-type control.

[0030] Fig. 18 illustrates dose-response curves for RMC-9808 and NT-1, administered as single agents or in combination for 72 hours, in two parental colorectal cancer cell lines (LS-174T and LS-513) and LS-174T / MRTX1133-resistant cells (right). Combination treatments are highlighted in pink to illustrate enhanced inhibitory effects relative to monotherapy. Fig. 19 illustrates dose-response curves for RMC-6236 and Osimertinib, administered as single agents or in combination for 72 hours, in KRAS colorectal cancer cell lines (LS-174T and LS-513) and in the RMC-6236-resistant derivative LS-174T / RMC-6236.DETAILED DESCRIPTION

[0031] Metastatic colorectal cancer (mCRC) remains a significant clinical challenge, with a 5-year survival rate of 10%. Over half of all CRC cases harbor mutations in the KRAS gene, leading to poor response to standard therapy. This underscores the crucial need for novel therapeutics targeting KRAS and overcoming the growing barrier of resistance. To address these critical challenges, a high-throughput screen was conducted to identify small molecules that synergize with KRASG12D inhibitor MRTX1133 against CRC. Through the screening of a 2,652 kinase inhibitor library in four distinct conditions followed by rigorous validations, it was discovered that Osimertinib and its analogs strongly synergize with MRTX1133 against bothC-1572 (EIC0016PCT)parental and MRTX1133-resistant cells. The novel drug combinations were provided with the potential to overcome the limitations of MRTX1133 with direct translational applications, ultimately improving patient outcomes.

[0032] Acquired resistance limits the efficacy of KRAS inhibition in CRC patients. Using unbiased high- throughput screening of novel resistant preclinical models, new genetic and nongenetic resistance mechanisms were provided herein. This research guides combination therapy approaches to enhance the efficacy and durability of promising treatments for these patients.

[0033] In various aspects, through agnostic screening of over 2,500 kinase inhibitors against both parental and resistant colorectal cancer cells, it has been surprisingly discovered that the combination of a KRAS inhibitor and an epidermal growth factor receptor (EGFR) inhibitor, demonstrates synergistic efficacy in inhibiting the proliferation or growth of colorectal cancer cells and can overcome the KRAS inhibitor-resistant colorectal cancer cells.

[0034] In one aspect, disclosed herein is a pharmaceutical composition for effectively treating diseases associated with cells expressing KRAS mutants, including but not limited to KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS G12S, KRAS Q61H, KRAS Q61L, and KRAS G13D. The diseases include cancer diseases such as colorectal cancer, pancreatic ductal madenocarcinoma (PDAC), pancreatic cancer, colorectal cancer (CRC), metastatic colorectal cancer (mCRC), colon cancer, or rectal cancer. Particularly preferred cancer diseases are colorectal cancer and the metastases thereof. The pharmaceutical composition comprises a therapeutically effective amount of at least one first active agent selected from an EGFR inhibitor and a second active agent selected from a KRAS inhibitor.

[0035] In aspects, the pharmaceutical composition comprises a combination of at least one EGFR inhibitor selected from Osimertinib (CAS Reg. No. 1421373-65-0), AZ-5104 (CAS Reg. No. 1421373-98-9), Limertinib (CAS Reg. No. 1934259-00-3), or mutant EGFR inhibitor (Mutant EGFRi; CAS Reg. No. 1421373-62-7), or a pharmaceutically acceptable salt of any of the foregoing; and at least one KRAS inhibitor selected from MRTX1133 (CAS Reg. No.2621928-55-8), RMC-6236 (CAS Reg. No. 2765081-21-6), RMC-7977 (CAS Reg. No.2765082-12-8), RMC-9805 (CAS Reg. No. 2922732-54-3), or RMC-6291 (CAS Reg. No.2641998-63-0), or a pharmaceutically acceptable salt of any of the foregoing.C-1572 (EIC0016PCT)

[0036] The combination results in a synergistic treatment effect. In other words, the combination of a KRAS inhibitor and an EGFR inhibitor results in improved efficacy as compared to either compound alone. Preferably, the combination of a KRAS inhibitor and an EGFR inhibitor allows a treatment effect to be achieved at a lower dose of the KRAS inhibitor than in the absence of the EGFR inhibitor.

[0037] In one embodiment, the pharmaceutical composition is a combination of MRTX1133 and an EGFR inhibitor, for example Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor.

[0038] In one embodiment, the pharmaceutical composition is a combination of RMC-6236 and an EGFR inhibitor, for example Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor.

[0039] In one embodiment, the pharmaceutical composition is a combination of RMC-7977 and an EGFR inhibitor, for example Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor.

[0040] In one embodiment, the pharmaceutical composition is a combination of RMC-6291 and an EGFR inhibitor, for example Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor.

[0041] In one embodiment, the pharmaceutical composition is a combination of RMC-9805 and an EGFR inhibitor, for example Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor.METHODS OF TREATMENTS

[0042] This disclosure includes a method of inhibiting the proliferation or growth of cancer cells, expressing KRAS mutants, including but not limited to KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS G12S, KRAS Q61H, KRAS Q61L, and KRAS G13D. The method comprises contacting the cancer cells with, concurrently or sequentially, a combination of a therapeutically effective amount of at least one first active agent selected from an EGFR inhibitor and a second active agent selected from a KRAS inhibitor.

[0043] In a further aspect, disclosed herein is a method treating a subject having cancer comprising steps of a) determining the subject has a cancer with at least one KRAS mutation; and b) administering to the subject in need thereof, concurrently or sequentially, a combination of a therapeutically effective amount of at least one first active agent selected from an EGFRC-1572 (EIC0016PCT)inhibitor and a second active agent selected from a KRAS inhibitor. The step of determining the presence of a KRAS mutation can be performed by any assay or method for detecting a mutant DNA or RNA sequence such as genotyping, genome sequencing, next generation sequencing, gene expression analysis of a sample obtained from the subject, thereby identifying the presence of a KRAS mutation in the sample. The method can include determining the type of KRAS mutation in the sample. In various embodiments, the KRAS mutants include but are not limited to KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS G12S, KRAS Q61H, KRAS Q61L, and KRAS G13D.

[0044] In some embodiments, the subject has cancer that was determined to have one or more cells having a KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS G12S, KRAS Q61H, KRAS Q61L, and / or KRAS G13D mutation. In some embodiments, an assay, and / or a method are used to determine whether the subject has wild type KRAS or a KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS G12S, KRAS Q61H, KRAS Q61L and / or KRAS G13D mutation, using a biological sample from a subject suspected of having wild type KRAS or a KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS G12S, KRAS Q61H, KRAS Q61L and / or KRAS G13D mutation. The assays or methods include, but are not limited to, genotyping, genome sequencing, next generation sequencing, gene expression analysis, immunohistochemistry, Southern blotting, Western blotting, and polymerase chain reaction (PCR)-based amplification, reverse transcription polymerase chain reaction, real-time reverse transcription polymerase chain reaction (RT-PCR), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). mutant allele-specific PCR amplification (MASA) assays, oligonucleotide ligation assays, hybridization assays, TaqMan assays, and microarray analyses.

[0045] In a further aspect, disclosed herein is a method treating a subject having cancer comprising steps of selecting the subject, wherein the step of selecting comprises obtaining a sample containing a nucleic acid from the subject and determining whether the subject has a cancer with a KRAS mutation and the mutation types by performing assays and analysis, which include, but are not limited to, genotyping, genome sequencing, next generation sequencing, gene expression analysis; and administering to the subject in need thereof, concurrently or sequentially, a combination of a therapeutically effective amount of at least one first active agentC-1572 (EIC0016PCT)selected from Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor, or a pharmaceutically acceptable salt of any of the foregoing; and at least one second active agent selected from MRTX1133, RMC-6236, RMC-7977, RMC-9805, or RMC-6291, or a pharmaceutically acceptable salt of any of the foregoing. In various embodiments, the KRAS mutants include but are not limited to KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS G12S, KRAS Q61H, KRAS Q61L, and KRAS G13D.

[0046] The first active agent, selected from Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor, may be administered by any method of pharmaceutical administration, including oral, topical, parenteral, intravenous, subcutaneous injection, intramuscular injection, inhalation or spray, sublingual, transdermal, intravenous, intrathecal, buccal, and rectal administration. In certain embodiments, administration of the first active agent is oral.

[0047] The second active agent, selected from MRTX1133, RMC-6236, RMC-7977, RMC-9805, or RMC-6291, may be administered by any method of pharmaceutical administration, including oral, topical, parenteral, intravenous, subcutaneous injection, intramuscular injection, inhalation or spray, sublingual, transdermal, intravenous, intrathecal, buccal, and rectal administration. In certain embodiments, administration of the second active agent is oral.

[0048] In some embodiments, the subject administered at least one first active agent (e.g., Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor) and at least one second active agent (e.g., MRTX1133, RMC-6236, RMC-7977, RMC-9805, or RMC-6291) described herein have been previously treated with at least one anti-cancer therapy.

[0049] In some embodiments, the prior cancer therapy is a therapy with a KRAS G12A inhibitor, a KRAS G12C inhibitor, a KRAS G12D inhibitor, a KRAS G12R inhibitor, a KRAS G12V inhibitor, a KRAS G12S inhibitor, a KRAS G13D inhibitor, a KRAS Q61H inhibitor, or a KRAS Q61L inhibitor. In certain embodiments, the subject exhibits reduced sensitivity to a therapy with a KRAS G12A inhibitor, a KRAS G12C inhibitor, a KRAS G12D inhibitor, a KRAS G12R inhibitor, a KRAS G12V inhibitor, a KRAS G12S inhibitor, a KRAS G13D inhibitor, a KRAS Q61H inhibitor, and / or a KRAS Q61L inhibitor. In some embodiments, the patient is resistant to a therapy with a KRAS G12A inhibitor, a KRAS G12C inhibitor, a KRAS G12D inhibitor, a KRAS G12R inhibitor, a KRAS G12V inhibitor, a KRAS G12S inhibitor, a KRAS G13D inhibitor, a KRAS Q61H inhibitor, and / or a KRAS Q61L inhibitor.C-1572 (EIC0016PCT)

[0050] Methods of treatment include providing certain dosage amounts of the first active agent to a patient. Dosage levels of Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor range from about 0.01 mg to about 140 mg per kilogram of body weight per day. In certain embodiments, 0.1 mg to 5000 mg, 1 mg to 2000 mg, 1 mg to 1000 mg, 1 mg to 500 mg, 1 mg to 200 mg, 1 mg to 100 mg, 1 mg to 50 mg, 10 mg to 5000 mg, 10 mg to 2000 mg, 10 mg to lOOOmg, 10 mg to 500 mg 10 mg to 300 mg, 10 mg to 200 mg, 10 mg to 100 mg, 50 mg to 5000mg, 50 mg to 2000 mg, 50 mg to 1000 mg, 50 mg to 500 mg, 50 mg to 200 mg, of Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor are provided daily to a patient. In certain embodiments, 0.1 mg to 5000 mg, 1 mg to 2000 mg, 1 mg to 1000 mg, 1 mg to 500 mg, 1 mg to 200 mg, 1 mg to 100 mg, 1 mg to 50 mg, 10 mg to 5000 mg, 10 mg to 2000 mg, 10 mg to lOOOmg, 10 mg to 500 mg 10 mg to 300 mg, 10 mg to 200 mg, 10 mg to 100 mg, 50 mg to 5000mg, 50 mg to 2000 mg, 50 mg to 1000 mg, 50 mg to 500 mg, 50 mg to 200 mg per dose of Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor are provided to the patient.

[0051] Methods of treatment include providing certain dosage amounts of the second active agent to a patient. Dosage levels of MRTX1133, RMC-6236, RMC-7977, RMC-9805, or RMC-6291 range from about 0.01 mg to about 140 mg per kilogram of body weight per day. In certain embodiments, 0.1 mg to 5000 mg, 1 mg to 2000 mg, 1 mg to 1000 mg, 1 mg to 500 mg, 1 mg to 200 mg, 1 mg to 100 mg, 1 mg to 50 mg, 10 mg to 5000 mg, 10 mg to 2000 mg, 10 mg to lOOOmg, 10 mg to 500 mg 10 mg to 300 mg, 10 mg to 200 mg, 10 mg to 100 mg, 50 mg to 5000mg, 50 mg to 2000 mg, 50 mg to 1000 mg, 50 mg to 500 mg, 50 mg to 200 mg, of MRTX1133, RMC-6236, RMC-7977, RMC-9805, or RMC-6291 are provided daily to a patient. In certain embodiments. 0.1 mg to 5000 mg, 1 mg to 2000 mg, 1 mg to 1000 mg, 1 mg to 500 mg, 1 mg to 200 mg, 1 mg to 100 mg, 1 mg to 50 mg, 10 mg to 5000 mg, 10 mg to 2000 mg, 10 mg to lOOOmg, 10 mg to 500 mg 10 mg to 300 mg, 10 mg to 200 mg, 10 mg to 100 mg, 50 mg to 5000mg, 50 mg to 2000 mg, 50 mg to 1000 mg, 50 mg to 500 mg, 50 mg to 200 mg per dose of MRTX1133, RMC-6236, RMC-7977, RMC-9805, or RMC-6291 are provided to the patient.

[0052] Frequency of dosage may also vary, depending on the particular cancer or disease treated. Treatment regimens may also include administering the first active agent (Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor) and the second active agent (MRTX1133, RMC-6236, RMC-7977, RMC-9805, or RMC-6291) to the patient for a number of consecutive days, for example for at least 5, 7, 10, 15, 20, 25, 30, 40, 50, or 60 consecutive days. In certainC-1572 (EIC0016PCT)embodiments the first active agent is administered for a period of 1 to 10 weeks and the amount and frequency of dosage is such that concentration of the compound in the patient’s plasma in never less than 50% of the patient’s plasma Cmax. In certain embodiments the second active agent is administered for a period of 1 to 10 weeks and the amount and frequency of dosage is such that concentration of the compound in the patient’s plasma in never less than 50% of the patient’s plasma Cmax.

[0053] Treatment regimens may also include administering the first active agent (Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor) and the second active agent (MRTX1133, RMC-6236, RMC-7977, RMC-9805, or RMC-6291) to the patient for a number of days prior to cancer surgery. For example, the first active agent and the second active agent may be administered to the patient for a number of consecutive days at 1 to 4 months prior to surgery. Treatment regimens may also include administering the first active agent and the second active agent to the patient in conjunction with radiation therapy, e.g., before, during, or after radiation therapy.

[0054] The methods of treatment disclosed herein regarding administration of the at least one first active agent (e.g., Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor) and at least one second active agent (e.g., MRTX1133, RMC-6236, RMC-7977, RMC-9805, or RMC-6291) to a subject, concurrently or sequentially, include concurrent or concomitant administration of the therapeutics (e.g., within 5 minutes, within 10 minutes, within 15 minutes, within 30 minutes, within 45 minutes, or within 1 hour of each other), and sequential administration (e.g., administration separated by at least 1 hour, or at least two hours, or at least four hours, or at least six hours, or at least eight hours, or at least ten hours, or at least twelve hours, or at least 24 hours, or at least 2 days, or at least 3 days).

[0055] Response rates or results for subjects administered at least one first active agent (e.g., Osimertinib, AZ-5104, Limertinib. or mutant EGFR inhibitor) and at least one second active agent (e.g., MRTX1133, RMC-6236, RMC-7977, RMC-9805, or RMC-6291) disclosed herein can be measured after a suitable length of time, which includes but is not limited to at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 15 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months.C-1572 (EIC0016PCT)

[0056] Response can be evaluated through several factors, including reduction in tumor size, inhibition or slowing of tumor growth, shrinkage or reduction in tumor lesions, delayed disease progression, absence of new tumors or lesions, decreased formation of new tumors, improved overall survival, or extended progression-free survival (PFS), and the lack of metastasis, hi certain embodiments, the progression of a patient’s disease can be monitored by measuring tumor size, tumor lesions, or new tumor formation. This assessment can be conducted using various imaging techniques, such as computerized tomography (CT) scans, positron emission tomography (PET) scans, magnetic resonance imaging (MRI), X-rays, ultrasounds, or a combination thereof.

[0057] The concentration and route of administration to the subject will vary based on the specific type of cancer being treated. The pharmaceutical compositions, including their pharmaceutically acceptable salts, can be administered in combination with other anti-neoplastic compounds, such as chemotherapeutic drugs. Additionally, these pharmaceutical compositions may be used alongside other treatment modalities, such as radiation therapy or surgical interventions. In some cases, the treatment may serve as an adjuvant therapy, either administered prior to surgery to reduce tumor size or post-operatively to prevent recurrence.

[0058] It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disorder for the patient undergoing therapy. A person skilled in the art will understand that human clinical trials, including dose-ranging and efficacy trials in both healthy subjects and subjects with the relevant disorder, may be conducted and completed following well-established methodologies widely recognized in clinical and medical research.

[0059] The pharmaceutical composition is administered to a subject, and in particular, a subject having cancer. The subject is a mammalian subject. The mammalian subject can be, for example, a human, a rodent, a monkey, a cat, a dog, a bovine animal (cow, steer, bull), a sheep, a monkey, or a primate. In aspects, the mammalian subject is a human.

[0060] Disclosed herein is a method of treating cancer in a subject comprising administering to the subject a pharmaceutical composition comprising a combination of a KRAS inhibitor, selected from MRTX1133, RMC-6236, RMC-7977, RMC-9805, RMC-6291, and anC-1572 (EIC0016PCT)EGFR inhibitor, selected from Osimertinib, AZ-5104, Limertinib, mutant EGFR inhibitor, in an amount effective to treat the cancer in the subject. The cancer can be acidophil carcinoma, acinar cell carcinoma, acral lentiginous melanomas, acute granulocytic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, acute myeloid leukemia, adenocarcinoma, adenoid cystic carcinoma, adenomatoid tumor, adenosquamous carcinoma, adrenal carcinoma, adrenal cortex carcinoma, adrenal cortical carcinoma, alveolar (bronchiolar) carcinoma, alveolar rhabdomyosarcoma, amelanotic melanoma, ameloblastic fibrosarcoma, ameloblastic odonto sarcoma, ameloblastoma, ampullary carcinoma, androblastoma, angioma, angiosarcoma, apocrine adenocarcinoma, astroblastoma, astrocytoma, basal cell carcinoma, basophil carcinoma, basophilic leukemia, B-cell lymphoma, benign chondroma, bladder cancer, bladder carcinoma, botryoid sarcoma, embryonal rhabdomyosarcoma, brain or spinal cord cancer, branchiolo-alveolar adenocarcinoma, breast cancer, breast carcinoma, brenner tumor, bronchial adenoma, bronchogenic carcinoma, carcinoid tumors, carcinosarcoma, central nervous system cancer, cerebellar sarcoma, ceruminous adenocarcinoma, cervical carcinoma, cervical hyperplasia, cholangiocarcinoma, osteogenic sarcoma, osteosarcoma, chondroblastoma, chondromatous hamartoma, chondromyxofibroma, chondrosarcoma, chordoma, choriocarcinoma, chromophobe carcinoma, chronic granulocytic leukemia, chronic lymphocytic leukemia, chronic myeloblastic leukemia, chronic myelogenous leukemia, clear cell adenocarcinoma, colon cancer, colon carcinoma, colorectal cancer, congenital tumors, cystadenocarcinoma, dermatofibroma, dysgerminoma, embryonal carcinoma, endocrine cancer, endometrial carcinoma, endometroid carcinoma, eosinophilic leukemia, ependymoma, epithelioid cell melanoma, erythroleukemia, esophageal carcinoma, essential thrombocytosis, ewing's sarcoma, extra-mammary paraganglioma, eye cancer, fibrillary astrocytoma, fibroadenoma, fibroma, fibrosarcoma, fibrous histiocytoma, follicular adenocarcinoma, ganglioneuroblastoma, gastrinoma, genitourinary cancer, genitourinary carcinoma, germinoma, pinealoma, giant and spindle cell carcinoma, giant cell tumor of bone, glioblastoma, glioma, glomangiosarcoma, glucagonoma, granular cell carcinoma, granular cell tumor, granuloma, granulosa cell tumor, granulosa-thecal cell tumor, hairy cell leukemia, hamartoma, head-neck cancer, hemangioendothelioma, hemangioma, gall bladder carcinoma, hemangiopericytoma, hemangiosarcoma, hematopoietic cancer, hepatoblastoma, hepatocellular adenoma, hepatocellular carcinoma, infiltrating duct carcinoma, inflammatory carcinoma, insulinoma,C-1572 (EIC0016PCT)interstitial cell carcinoma, intraepithelial carcinoma, juxtacortical osteosarcoma, Kaposi's sarcoma, keloids, kidney cancer, leiomyoma, leiomyosarcoma, melanoma, leukemia, leydig cell tumor, lipid cell tumor, lipoma, hepatoma, liposarcoma, liver cancer, lobular carcinoma, lung carcinoma, lung cancer, lymphangiosarcoma, lymphoepithelial carcinoma, lymphoid leukemia, lymphoma, lymphosarcoma cell leukemia, malignant carcinoid carcinoma, malignant fibrous histiocytoma, malignant giant cell tumor chordoma, malignant histiocytosis, malignant hypercalcemia, malignant lymphoma, malignant melanoma, malignant pancreatic insulinoma, malignant teratoma, malignant tissue, mantle cell lymphoma, mast cell leukemia, mast cell sarcoma, medullary carcinoma, medulloblastoma, megakaryoblastic leukemia, meningioma, meningiosarcoma, mesenchymal chondrosarcoma, mesenchymoma, mesonephroma, mesothelioma, metastatic colorectal cancer, mucinous adenocarcinoma, mucinous cystadenocarcinoma, mucoepidermoid carcinoma, mullerian mixed tumor, multiple myeloma, mycosis fimgoides, mycosis fungoides, myeloid leukemia, myeloid sarcoma, myeloma, myxoma, myxosarcoma, nephroblastoma, neuroblastoma, neurofibroma, neurofibrosarcoma, nodular melanomas, nonencapsulating sclerosing carcinoma, non-Hodgkin's lymphoma, nonsmall cell lung cancer (NSCLC), odontogenic tumor, olfactory neurogenic tumor, oligodendroblastoma, oligodendroglioma, osteitis deformans, osteochronfroma, osteocartilaginous exostoses, non-hodgkin's lymphomas, ovarian cancer, ovarian carcinoma, ovarian stromal tumor, oxyphilic adenocarcinoma, ductal adenocarcinoma, pancreatic cancer, pancreatic carcinoma, papillary adenocarcinoma, papillary and follicular adenocarcinoma, papillary carcinoma, papillary cystadenocarcinoma, papillary serous cystadenocarcinoma, papillary transitional cell carcinoma, paragranuloma, pheochromocytoma, phyllodes tumor, pilomatrix carcinoma, placental cancer, plasma cell leukemia, polycythemia vera, pre-tumor cervical dysplasia, primary brain carcinoma, primary macroglobulinemia, prostate cancer, prostatic carcinoma, protoplasmic astrocytoma, neuroblastoma, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, sarcoma, schwannoma, sebaceous adenocarcinoma, sertoli cell carcinoma, Sertoli-Leydig cell tumors, signet ring cell carcinoma, skin appendage carcinoma, skin cancer, small cell carcinoma, small cell lung cancer (SCLC), small lymphocytic (SL) NHL, soft tissue cancer, soft-tissue sarcoma, solid carcinoma, spinal cord neurofibroma, squamous cell carcinoma, stomach carcinoma, stomach cancer, stromal sarcoma, superficial spreading melanoma, synovial sarcoma, teratocarcinoma, teratoma,C-1572 (EIC0016PCT)testicular carcinoma, the cancer is colon cancer, thecoma, thymoma, thyroid carcinoma, trabecular adenocarcinoma, transitional cell carcinoma, tubular adenoma, villous adenoma, Wilm's tumor, xanthoma or a combination thereof.

[0061] In one embodiment, the cancer is selected from the group consisting of colorectal cancer, pancreatic ductal madenocarcinoma (PDAC), pancreatic cancer, colorectal cancer, metastatic colorectal cancer (mCRC), colon cancer, or rectal cancer. In one embodiment, the cancer is colorectal cancer. In one embodiment, the cancer is metastatic colorectal cancer.EGFR INHIBITORS

[0062] An epidermal growth factor receptor (EGFR) inhibitor is a type of substance, agent, chemical, or compound capable of inhibiting or negatively modulating all or a portion of the enzymatic activity of EGFR, a transmembrane receptor tyrosine kinase that is critical in regulating cell growth, proliferation, and survival. EGFR is often overexpressed or mutated in various cancers, leading to uncontrolled cell signaling and tumor progression. EGFR inhibitors can function by preventing the binding of natural ligands to the receptor or by inhibiting the receptor's kinase activity, thereby disrupting downstream signaling pathways like the RAS-RAF-MEK-ERK and PI3K-AKT pathways.

[0063] EGFR inhibitors suitable for use in the pharmaceutical combinations, compositions, and methods of treatment of this disclosure include Osimertinib (CAS Reg. No.1421373-65-0, 2-Propenamide, A-[2-[[2-(dimethylamino)ethyl]methylamino]-4-methoxy-5-[[4-( 1-methyl- lH-indol-3-yl)-2-pyrimidinyl]amino]phenyl]-), AZD 9291, and the pharmaceutically acceptable salts of any of the foregoing.

[0064] EGFR inhibitors suitable for use in the pharmaceutical combinations, compositions, and methods of treatment of this disclosure include AZ-5104 (CAS Reg. No. 1421373-98-9. 2-Propenamide. A-[2-[[2-(dimethylamino)ethyl]methylamino]-5-[[4-(lH-indol-3-yl)-2-pyrimidinyl]amino]-4-methoxyphenyl] discussed in WO2013 / 014448), and the pharmaceutically acceptable salts of any of the foregoing.

[0065] EGFR inhibitors suitable for use in the pharmaceutical combinations, compositions, and methods of treatment of this disclosure include Limertinib (CAS Reg. No. 1934259-00-3, 2-Propenamide, N- [ 5- [ [5-chl oro-4- (2-naphthalenylamino)-2-pyrimidinyl] amino] -C-1572 (EIC0016PCT)2-[[2-(dimethylamino)ethyl]methylamino]-4-methoxyphenyl]- discussed in WO2015 / 082713), and the pharmaceutically acceptable salts of any of the foregoing.

[0066] EGFR inhibitors suitable for use in the pharmaceutical combinations, compositions, and methods of treatment of this disclosure include a compound called “mutant EGFR inhibitor” (CAS Reg. No. 1421373-62-7, 2-Propenamide, A-[5-[[5-chloro-4-(lH-indol-3-yl)-2-pyrimidinyl]amino]-2-[[2-(dimethylamino)ethyl]methylamino]-4-methoxyphenyl]- also discussed in WO2013 / 014448), and the pharmaceutically acceptable salts of any of the foregoing.

[0067] Additional EGFR inhibitors suitable for use in the pharmaceutical combinations, compositions, and methods of treatment of this disclosure include BMS-690514 (CAS Reg. No.859853-30-8, (3R,4R)-4-amino-l-[[4-[(3-methoxyphenyl)amino]pyrrolo[2,l-f][l,2,4]triazin-5-yl] methyl] piperidin- 3 -ol), Neratinib (CAS Reg. No. 698387-09-6, (2E)-N-[4-[[3-chloro-4-(2-pyridinylmethoxy)phenyl]amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide)), Naquotinib (mesylate) (CAS Reg. No. 1448237-05-5, 6-ethyl-3-[4-[4-(4-methylpiperazin-l-yl)piperidin-l-yl]anilino]-5-[(3R)-l-prop-2-enoylpyrrolidin-3-yl]oxypyrazine-2-carboxamide;methanesulfonic acid), PF-6274484 (CAS Reg. No. 1035638-91-5, N-[4-[(3-Chloro-4-tluorophenyl)amino]-7-methoxy-6-quinazolinyl]-2-propenamide), Pelitinib (CAS Reg. No. 257933-82-7, N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2E-butenamide), Mobocertinib (CAS Reg. No. 1847461-43-1, 2-[[4-[[2-(dimethylamino)ethyl]methylamino]-2-methoxy-5-[(l-oxo-2-propen-l-yl)amino]phenyl]amino]-4-(l-methyl-lH -indol-3-yl)-5-pyrimidinecarboxylic acid, 1-methylethyl ester). Tarlox-TKI (CAS Reg. No. 2135696-72-7), EGFR-IN-11 (CAS Reg. No. 2463200-44-2, (R)-9-(l-(cyclopropylsulfonyl)pyrrolidin-3-yl)-N2-(4-(4-methylpiperazin-l-yl)phenyl)-N8-phenyl-9H-purine-2,8-diamine)), or Rezivertinib (CAS Reg. No. 1835667-12-3, 2-Propenamide, N-(2-(2-(dimethylamino)ethoxy)-4-methoxy-5-((4-(l-methyl-lH-indol-3-yl)-2-pyrimidinyl)amino)phenyl)), and the pharmaceutically acceptable salts of any of the foregoing.

[0068] Additional examples of EGFR inhibitors include Mutant EGFR Inhibitor, AZ-5104, Limertinib, BMS-690514, Neratinib, Naquotinib (Mesylate), PF-6274484, Pelitinib, Mobocertinib, Tarlox-TKI, EGFR-IN-11, Rezivertinib, Tesevatinib, Dacomitinib, Astragaloside VI, Varlitinib, TAK-285, Theliatinib, NSC 228155. Selatinib, Zipalertinib, Oritinib, EAI045, ZD-4190, FIIN-3, BMS-599626 (Hydrochloride), AEE788, Tucatinib, Mubritinib, EMI56,C-1572 (EIC0016PCT)Almonertinib, Tuxobertinib, JCN037, Osimertinib, HKI-357, Zorifertinib, Tyrphostin 23, CNX-2006, Epitinib (Succinate), Gefitinib (Hydrochloride), EGFR-IN-9, Mavelertinib, JND3229, ARRY-380 (Analog), Mobocertinib (Succinate), WHI-P180, Tucatinib (Hemiethanolate).Lirafugratinib (Hydrochloride), Avitinib (Maleate), (E / Z)-AG490, Tyrphostin AG 879, PD-161570, AV-412, EGFR-IN-17, Canertinib (Dihydrochloride), PP 3, SU5204, Icotinib (Hydrochloride). CL-387785. Lifirafenib, Gefitinib Impurity 2, Tyrphostin AG30. Epertinib (Hydrochloride), RTC-5, O-Desmethyl Gefitinib, EGFR-IN-69, TAS0728, Rociletinib Hydrobromide, Befotertinib, Methyl 2,5-Dihydroxycinnamate, EAI001, TX1-85-1, Lazertinib, WZ-3146, JBJ-09-063 (TFA), PD153035 (Hydrochloride), BAY 2476568, Allitinib Tosylate, PKI-166, WHI-P154, Canertinib, Osimertinib (Mesylate), B-Hydroxyisovalerylshikonin, (Rac)-JBJ-04- 125-02, RG13022, CUDC-101, PD 174265, EGFR Protein Tyrosine Kinase Substrate, Osimertinib, MS9427, EGFR-IN-12, Lirafugratinib, MTX-211, Nazartinib, Olmutinib, EMI48, Almonertinib (Mesylate), PD158780, AG 555, Sulforaphene, PD153035, Delphinidin 3-Glucoside (Chloride), AG-825. Sapitinib, AV-412 (Free Base), AG1557, BI-4142, NRC-2694, AG-1478, HER2 / Neu (654-662) GP2, Icotinib, DBPR112, Tarloxotinib (Bromide), JBJ-09-063 (Hydrochloride), CZC-8004, EGFR- IN- 1 (TFA), (E)-AG 99, LY456236, (E)-AG 556, PF-06459988, Almonertinib (Hydrochloride), (+)-Tyrphostin B44, Olafertinib, Mutated EGFR-IN-1, Falnidamol, (3S,4S)-PF-06459988, RG14620, WZ4002, Tyrphostin AG 528, CP-724714, Simotinib, WZ8040, Cyasterone, EGFR / Erbb-2 / Erbb-4 Inhibitor-2, Chrysophanol, Rociletinib, and BLU-945.

[0069] In further embodiments, the concentration of the EGFR inhibitor (e.g.Osimertinib, Limertinib, AZ-5104, or mutant EGFR inhibitor) in the colon, at the tumor, at the cancer cells, or in the patient’s blood or plasma is about 1 nM to about 1000 nM. In additional embodiments, the concentration of the EGFR inhibitor is about 0.01 nM to about 1 nM, 1 nM to about 100 nM, 10 nM to about 100 nM, about 20 nM to about 150 nM, about 30 nM to about 200 nM, about 40 nM to about 250 nM, about 50 nM to about 300 nM, about 100 nM to about 400 nM, about 150 nM to about 500 nM, about 200 nM to about 750 nM, about 250 nM to about 1000 nM, about 500 nM to about 1000 nM, or about 1000 nM to about 3000 nM.KRAS INHIBITORS

[0070] A KRAS inhibitor is a type of substance, agent, chemical, or compound capable of specifically inhibiting or negatively modulating all or a portion of the enzymatic activity ofC-1572 (EIC0016PCT)KRAS protein or KRAS mutants. KRAS mutations are frequently observed in various cancers, leading to uncontrolled signaling through the MAPK and PI3K pathways. KRAS inhibitors bind to the mutant KRAS protein, blocking its GTPase function or inhibiting downstream signaling, thereby preventing aberrant cell growth and tumor progression. These inhibitors are primarily developed to target specific mutations, such as KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS G12S, KRAS Q61H, KRAS Q61L, and KRAS G13D.

[0071] KRAS inhibitors suitable for use in this disclosure include MRTX1133 (CAS Reg. No. 2621928-55-8, 2-Naphthalenol, 4-[4-(3,8-diazabicyclo[3.2.1]oct-3-yl)-8-fluoro-2-[[(27?,7aS)-2-fluorotetrahydro-lH-pyrrolizin-7a(5H)-yl]methoxy]pyrido[4,3-d]pyrimidin-7-yl]-5-ethynyl-6-fluoro-, discussed in W02021 / 041671), RMC-6236 (CAS Reg. No. 2765081-21-6, Cyclopropanecarboxamide, -[(27?,145,185)-l-ethyl-18,19,20,21-tetrahydro-2-[2-[(15)-l-methoxyethyl]-5-(4-methyl-l-piperazinyl)-3-pyridinyl]-25,25-dimethyl-15,22-dioxo-17 / / -5,3-([4,2]-e / 76 / o-thiazolopropano[ l,3]-en< / o-pyridazinomethanoxypropano)-l -indol-14-yl]-2-methyl-, (15.25)-, discussed in W02022 / 060583), RMC-7977 (CAS Reg. No. 2765082-12-8. 3-Oxabicyclo[3.1.0]hexane-6-carboxamide, N-[(27?,145,185)-2-[5-(4-cyclopropyl-l-piperazinyl)-2-[(15)-l-methoxyethyl]-3-pyridinyl]-l-ethyl-18,19,20,21-tetrahydro-25,25-dimethyl-15,22-dioxo-17H-5,3-([4,2]-ew / o-thiazolopropano[ l,3]-enrfo-pyridazinomethanoxypropano)-177-indol-14-yl]-, (la, 5a, 6a)- discussed in W02022 / 060386), or RMC-6291 (CAS Reg. No. 2641998-63-0, 3-Pyridazinecarboxylic acid, N1-[N-[[l-[4-(dimethylamino)-4-methyl-l-oxo-2-pentyn-l-yl]-4-fluoro-4-piperidinyl]carbonyl]-N-methyl-L-valyl-3-[4-[(27?)-l-ethyl-3-(3-hydroxy-2,2-dimethylpropyl)-2-[2-[(15)-l-methoxyethyl]-3-pyridinyl]-lH-indol-5-yl]-2-morpholinyl]-L-alanyl]hexahydro-, (3→2)-lactone discussed in US2021-0130303), or a pharmaceutically acceptable salt of any of the foregoing.

[0072] The therapeutic efficacies of RMC-6236, RMC-7977, RMC-9805, and RMC-6291 have been reported in the following references. RMC-6236 is a potent RAS(ON)MULTI inhibitor and is reported in Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers Cancer Discovery. 2024 Jun 3;14(6):994-1017.). RMC-7977 is a reversible, tri-complex RAS inhibitor with broad spectrum activity for both mutant and wild-type (WT) KRAS, NRAS, and HRAS variants. RMC-7977 is reported in Concurrent inhibition of oncogenic and wildtype RAS-GTP for cancer therapy (Nature. 2024 May;629(8013):919-926.). RMC-6291 is an orally active and covalent inhibitor ofC-1572 (EIC0016PCT)KRASG12C(ON). RMC-6291 is reported in Chemical remodeling of a cellular chaperone to target the active state of mutant KRAS (Science. 2023 Aug 18; 381(6659): 794-799.). RMC-9805 has been reported to treat KRASG12D-mutated solid tumors. RMC-9805 was reported in Abstract 3475: RMC-9805, a first-in-class, mutant-selective, covalent and orally bioavailable KRASG12D(ON) inhibitor, promotes cancer-associated neoantigen recognition and synergizes with immunotherapy in preclinical models (Cancer Res (2023) 83 (7_Supplement): 3475.).

[0073] In various other embodiments, the concentration of the KRAS inhibitor, e.g., MRTX1133, is in the colon, at the tumor, at the cancer cells or in the patient’s plasma or blood is about 0.01 nM to about 20 nM. In additional embodiments, the concentration of the KRAS inhibitor is about 0.01 nM to about 5 nM, about 1 nM to about 10 nM, about 2 nM to about 15 nM, about 3 nM to about 20 nM, about 4 nM to about 25 nM, about 5 nM to about 30 nM, about 10 nM to about 40 nM, about 15 nM to about 50 nM, about 20 nM to about 75 nM, about 25 nM to about 100 nM, about 50 nM to about 100 nM, about 100 nM to about 500 nM, about 500 nM to about 1000 nM, or about 1000 nM to about 3000 nM. In additional embodiments, the concentration of the KRAS inhibitor is about 3.9 nM to about 1,000 nM. In additional embodiments, the concentration of the KRAS inhibitor is about 1.0 nM to about 50 nM, about 10 nM to about 100 nM, about 20 nM to about 150 nM, about 30 nM to about 200 nM, about 40 nM to about 250 nM, about 50 nM to about 300 nM, about 100 nM to about 400 nM, about 150 nM to about 500 nM, about 200 nM to about 750 nM, about 250 nM to about 1000 nM, about 500 nM to about 1000 nM, or about 1000 nM to about 3000 nM.TERMINOLOGY

[0074] Unless otherwise indicated, the disclosure is not limited to specific procedures, starting materials, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless clearly contraindicated by the context, each compound name includes the free acid or free base form of the compound as well as hydrates and pharmaceutically acceptable salts of the compound.

[0075] The “first active agent” means a compound selected from any of Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor., and pharmaceutically acceptable salts and hydrates of any of the foregoing.C-1572 (EIC0016PCT)

[0076] The “second active agent” means a compound selected from any of MRTX1133, RMC-6236, RMC-7977, RMC-9805, or RMC-6291, and pharmaceutically acceptable salts and hydrates of any of the foregoing.

[0077] “KRAS G12A” is a mutant form of a KRAS protein that contains an amino acid substitution of glycine with alanine at amino acid position 12. A “KRAS G12A inhibitor” refers to substances, agents, chemicals or compounds capable of inhibiting or negatively modulating all or a portion of the enzymatic activity of KRAS G12A.

[0078] “KRAS G12C” is a mutant form of a KRAS protein that contains an amino acid substitution of glycine with cysteine at amino acid position 12. A “KRAS G12C inhibitor” refers to substances, agents, chemicals or compounds capable of inhibiting or negatively modulating all or a portion of the enzymatic activity of KRAS G12C.

[0079] “KRAS G12D” is a mutant form of a KRAS protein that contains an amino acid substitution of glycine with aspartic acid at amino acid position 12. A “KRAS G12D inhibitor” refers substances, agents, chemicals or compounds capable of inhibiting or negatively modulating all or a portion of the enzymatic activity of KRAS G12D.

[0080] “KRAS G12R” is a mutant form of a KRAS protein that contains an amino acid substitution of glycine with arginine at amino acid position 12. A “KRAS G12R inhibitor” refers to substances, agents, chemicals or compounds capable of inhibiting or negatively modulating all or a portion of the enzymatic activity of KRAS G12R.

[0081] “KRAS G12V” is a mutant form of a KRAS protein that contains an amino acid substitution of glycine with valine at amino acid position 12. A “KRAS G12V inhibitor” refers to substances, agents, chemicals or compounds capable of inhibiting or negatively modulating all or a portion of the enzymatic activity of KRAS G12V.

[0082] “KRAS G12S” is a mutant form of a KRAS protein that contains an amino acid substitution of glycine with serine at amino acid position 12. A “KRAS G12S inhibitor” refers to substances, agents, chemicals or compounds capable of inhibiting or negatively modulating all or a portion of the enzymatic activity of KRAS G12S.

[0083] “KRAS G13D” is a mutant form of a KRAS protein that contains an amino acid substitution of glycine with aspartic acid at amino acid position 13. A “KRAS G13D inhibitor” refers to substances, agents, chemicals or compounds capable of inhibiting or negatively modulating all or a portion of the enzymatic activity of KRAS G13D.C-1572 (EIC0016PCT)

[0084] “KRAS Q61H” is a mutant form of a KRAS protein that contains an amino acid substitution of glutamine with histidine at amino acid position 61. A “KRAS Q61H inhibitor” refers to substances, agents, chemicals or compounds capable of inhibiting or negatively modulating all or a portion of the enzymatic activity of KRAS Q61H.

[0085] KRAS Q61L is a mutant form of a KRAS protein that contains an amino acid substitution of glutamine with leucine at amino acid position 61. A “KRAS Q61L inhibitor” refers to substances, agents, chemicals or compounds capable of inhibiting or negatively modulating all or a portion of the enzymatic activity of KRAS Q61L.

[0086] The terms “first,” “second,” and the like, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another.

[0087] The terms “a” and “an” do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. The term “or” means “and / or”. The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”). The open-ended term “comprising” encompasses the terms “consisting of’ and “consisting essentially of.”

[0088] Recitation of ranges of values is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.

[0089] The phrases “for example,” “for instance,” “such as,” or “including” are meant to introduce examples that further clarify more general subject matter. These examples are provided only as an aid for understanding the disclosure and are not meant to be limiting in any fashion.

[0090] Reference throughout the specification to “some embodiments”, “an embodiment”, and so forth, means that a particular element described in connection with the embodiment is included in at least one embodiment described herein, and may or may not beC-1572 (EIC0016PCT)present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments. A “combination thereof’ is open and includes any combination comprising at least one of the listed components or properties optionally together with a like or equivalent component or property not listed

[0091] “Pharmaceutical compositions” are compositions comprising at least one active agent, and at least one other substance, such as a carrier, excipient, or diluent. Pharmaceutical compositions meet the U. S. FDA’s GMP (good manufacturing practice) standards for human or non-human drugs.

[0092] “Pharmaceutically acceptable salts” include derivatives of the disclosed compounds in which the parent compound is modified by making inorganic and organic, nontoxic, acid or base addition salts thereof. The salts of the present compounds can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca. Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred, where practicable. Salts of the present compounds further include solvates of the compounds and of the compound salts.

[0093] A “therapeutically effective amount” means an amount effective, when administered to a patient, to provide a therapeutic benefit such as an amelioration of at least a symptom of the disorder, decrease the frequency or severity of symptoms, or effect a change in a clinical marker for a disease or disorder, slowing the progression of a disease or disorder, halting the progression of a disease or disorder, or reversing the course of a disorder. In the context of triple negative breast cancer, a “therapeutically effective amount” also includes an amount sufficient for any of slowing the rate of tumor growth and formation, slowing the metastasis of the cancer, halting tumor growth, halting the formation of new tumors, halting the metastasis of the cancer, reducing tumor size, reducing the number of tumors, causing a remission so cancer is no longer observable in the patient, or reducing any marker of triple negative breast cancer in the patient.C-1572 (EIC0016PCT)

[0094] The term “cancer disease” or “cancer” includes a disease characterized by aberrantly regulated cellular growth, proliferation, differentiation, adhesion, and / or migration. “Cancer cell” means an abnormal cell that grows by rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Preferably, a “cancer disease” is characterized by cells expressing KRAS mutant(s), and a cancer cell expresses KRAS mutant(s).

[0095] " Metastasis" refers to the process by which cancer cells spread from their original site to other parts of the body. This complex phenomenon involves several stages, including the detachment of malignant cells from the primary tumor, invasion into the extracellular matrix, penetration through the endothelial basement membranes to access blood vessels and body cavities, and eventual transport through the bloodstream to infiltrate target organs. The establishment of new tumors at these sites relies on angiogenesis, the formation of new blood vessels. Notably, metastasis can occur even after the primary tumor has been removed, as residual cancer cells may persist and acquire metastatic capabilities. In the context of this invention, "metastasis" specifically refers to "distant metastasis," which indicates the spread of cancer cells far from the primary tumor and regional lymph nodes. Additionally, lymph node metastasis is included in this definition. A specific type of metastasis that can be addressed by the proposed therapy originates from colorectal cancer. Preferred embodiments include metastasis to lymph nodes, the lungs, and / or the liver, particularly from pancreatic cancer.

[0096] The term “treating” or “treatment of a disease” includes curing, shortening the duration, ameliorating, preventing, slowing down or inhibiting progression or worsening, or preventing or delaying the onset of a disease or the symptoms thereof.

[0097] The term “subject”, “patient” or “individual” used interchangeably, refers to any animal, including mammals such as cows, mice, pigs, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, primates, and humans. In some embodiments, the patient is a human. In some embodiments, the subject has experienced and / or exhibited at least one symptom of the disease or disorder to be treated. In some embodiments, the subject has been diagnosed as having a disease having wild type KRAS or a KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS G12S, KRAS Q61H, KRAS Q61L and / or KRAS G13D mutation. The subject can be a subject with a tumor(s) that is positive for wild type KRAS or a KRAS G12A,C-1572 (EIC0016PCT)KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS G12S, KRAS Q61H, KRAS Q61L and / or KRAS G13D mutation.

[0098] The term "contacting" refers to the act of making contact or bringing into immediate or close proximity, whether at the cellular or molecular level. This can involve initiating a physiological reaction, chemical reaction, or physical change, and may occur in various environments, such as in a solution, reaction mixture, or in vitro and in vivo conditions. For example, “contacting” wild type KRAS, or a KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS G12S, KRAS Q61H, KRAS Q61L and / or KRAS G13D with a pharmaceutical composition provided herein includes introducing an effective amount of the composition provided herein into a sample containing a cellular or preparation containing wild type KRAS, or a KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS G12S, KRAS Q61H, KRAS Q61L or KRAS G13D mutation, to negatively modulate the activity of one or more of wild type KRAS, or a KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS G12S, KRAS Q61H, KRAS Q61L or KRAS G13D mutation.

[0099] The term “sensitivity” refers to the way a cancer reacts to a drug, a compound, or a pharmaceutical composition. In various aspects, “sensitivity” means “responsive to treatment” and the concepts of “sensitivity” and “responsiveness” are positively associated in that a cancer or tumor that is responsive to a drug treatment is said to be sensitive to that drug. As used herein, “sensitivity” is defined as the ability of a subject, an individual, a cell or a tissue, relative to the abilities of others, to respond in a qualitatively normal fashion to a particular drug dose. The smaller the dose required producing an effect, the more sensitive is the responding system. In exemplary aspects, “sensitivity” is opposite to “resistance” and the concept of “resistance” is negatively associated with “sensitivity”. For example, a cancer that is resistant to a drug treatment is either not sensitive nor responsive to that drug or was initially sensitive to the drug and is no longer sensitive upon acquiring resistance; that drug is not or no longer an effective treatment for that tumor or cancer cell.

[0100] The term “concurrently” means simultaneously in time, or at different times during the course of a common treatment schedule or the same treatment scheme.

[0101] The term “sequentially” refers to the administration of one active agent used in the method followed by administration of another active agent. After administration of one active agent, the next active agent can be administered substantially immediately after the first, or theC-1572 (EIC0016PCT)next active agent can be administered after an effective period after the first active agent. The effective time period is the amount of time given for realization of maximum benefit from the administration of the first active agent.

[0102] An “effective amount” of an active ingredient, or a pharmaceutical composition or combination including the active ingredient, is an amount effective, when administered to a subject, to provide a therapeutic benefit.

[0103] Throughout this disclosure and in the claims, the open-ended transitional phrase “comprising” includes the intermediate transitional phrase “consisting essentially of’ and the closed transitional phrases “consists” or “consisting of.” Claims using “comprising” can be amended with the intermediate and closed transitional phrases to designate particular embodiments.

[0104] The compositions, methods, and articles can alternatively comprise, consist of, or consist essentially of, any appropriate materials, steps, or components herein disclosed. The compositions, methods, and articles can additionally, or alternatively, be formulated so as to be devoid, or substantially free, of any materials (or species), steps, or components, that are otherwise not necessary to the achievement of the function or objectives of the compositions, methods, and articles.

[0105] All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other (e.g., ranges of “up to 25 wt.%, or, more specifically, 5 wt.% to 20 wt.%”, is inclusive of the endpoints and all intermediate values of the ranges of “5 wt.% to 25 wt.%,” etc.). “Combinations” is inclusive of blends, mixtures, alloys, reaction products, and the like.

[0106] Unless specified to the contrary herein, all test standards are the most recent standard in effect as of the filing date of this application, or, if priority is claimed, the filing date of the earliest priority application in which the test standard appears.

[0107] Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this application belongs. All cited patents, patent applications, and other references are incorporated herein by reference in their entirety. However, if a term in the present application contradicts or conflicts with a term in the incorporated reference, the term from the present application takes precedence over the conflicting term from the incorporated reference.C-1572 (EIC0016PCT)

[0108] While particular embodiments have been described, alternatives, modifications, variations, improvements, and substantial equivalents that are or may be presently unforeseen may arise to applicants or others skilled in the art. Accordingly, the appended claims as filed and as they may be amended are intended to embrace all such alternatives, modifications variations, improvements, and substantial equivalents.

[0109] This invention will be better understood from the experimental details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.PHARMACEUTICAL PREPARATIONS

[0110] Osimertinib, AZ-5104, Limertinib, mutant EGFR inhibitor, MRTX1133, RMC-6236, RMC-7977, RMC-9805, and RMC-6291, and the pharmaceutically acceptable salts and hydrates of any of the foregoing can be administered as neat chemicals but are preferably administered as a pharmaceutical composition.

[0111] Accordingly, the disclosure provides pharmaceutical compositions comprising a first active agent, selected from any of Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor and a second active agent selected from any of MRTX1133, RMC-6236, RMC-7977, RMC-9805, or RMC-6291 and the pharmaceutically acceptable salts and hydrates, together with at least one pharmaceutically acceptable carrier. The pharmaceutical composition may contain a compound or salt of Osimertinib, AZ-5104, Limertinib, mutant EGFR inhibitor, MRTX1133, RMC-6236, RMC-7977, RMC-9805, and RMC-6291 as the only active agent, or may contain one or more additional active agents.

[0112] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,C-1572 (EIC0016PCT)maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH2)n-COOH where n is 0-4, and the like.

[0113] The first active agent or the second active agent may be administered orally, topically, parenterally, by inhalation or spray, sublingually, transdermally, intravenously, intrathecally. via buccal administration, or rectally, or by other means, in dosage unit formulations containing conventional pharmaceutically acceptable carriers. In certain embodiments the first active agent or the second active agent is administered orally. In certain embodiments the first active agent or the second active agent is administered subcutaneously or intravenously. The pharmaceutical composition may be formulated as any pharmaceutically useful form, e.g., as an aerosol, a cream, a gel, a pill, a capsule, a tablet, a syrup, a transdermal patch, or an ophthalmic solution. Some dosage forms, such as tablets and capsules, are subdivided into suitably sized unit doses containing appropriate quantities of the active components, e.g., an effective amount to achieve the desired purpose.

[0114] Carriers include excipients and diluents and must be of sufficiently high purity and sufficiently low toxicity to render them suitable for administration to the patient being treated. The carrier can be inert, or it can possess pharmaceutical benefits of its own. The amount of carriers employed in conjunction with the compound is sufficient to provide a practical quantity of material for administration per unit dose of the compound.

[0115] Classes of carriers include, but are not limited to binders, buffering agents, coloring agents, diluents, disintegrants, emulsifiers, flavorants, glidents, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. Some carriers may be listed in more than one class, for example vegetable oil may be used as a lubricant in some formulations and a diluent in others. Exemplary pharmaceutically acceptable carriers include sugars, starches, celluloses, powdered tragacanth, malt, gelatin; talc, and vegetable oils. Optional active agents may be included in a pharmaceutical composition, which do not substantially interfere with the activity of the compound of the present invention.

[0116] The pharmaceutical compositions can be formulated for oral administration.These compositions contain between 0.1 and 99 weight % (wt.%) of a compound of and usually at least about 5 wt.% of the first active agent or the second active agent. Some embodimentsC-1572 (EIC0016PCT)contain from about 25 wt. % to about 50 wt. % or from about 5 wt.% to about 75 wt.% of the first active agent or the second active agent.

[0117] In some embodiments, the disclosed EGFR inhibitors and KRAS inhibitors are delivered from a sustained-release composition. As used herein, the term “sustained-release composition” encompasses sustained-release, prolonged-release, extended-release, delayed-release. slow-release and controlled-release compositions, systems and devices. Advantages of a sustained-release composition include without limitation a more uniform blood level of the drug (e.g., avoidance of wide peak-to-trough fluctuations), delivery of a therapeutically effective amount of the drug over a prolonged time period, reduced frequency of administration, and reduced side effects (e.g., avoidance of a drug overdose). In certain embodiments, the sustained-release composition delivers the EGFR inhibitors and KRAS inhibitors over a period of at least about 1 day, 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 1 month or longer.

[0118] In some embodiments, the sustained-release composition is a drug-encapsulation system, such as nanoparticles, microparticles or a capsule made of, e.g., a biodegradable polymer or / and a hydrogel. In certain embodiments, the sustained-release composition comprises a hydrogel. Non-limiting examples of polymers of which a hydrogel can be composed include polyvinyl alcohol, acrylate polymers (e.g., sodium polyacrylate), and other homopolymers and copolymers having a relatively large number of hydrophilic groups (e.g., hydroxyl or / and carboxylate groups). In other embodiments, the sustained-release drug-encapsulation system comprises a membrane-enclosed reservoir, wherein the reservoir contains a drug and the membrane is permeable to the drug. Such a drug-delivery system can be in the form of, e.g., a transdermal patch.

[0119] In some embodiments, the sustained-release composition is formulated as polymeric nanoparticles or microparticles, wherein the polymeric particles can be delivered, e.g., by injection or from an implant. In some embodiments, the polymeric implant or polymeric nanoparticles or microparticles are composed of a biodegradable polymer. In certain embodiments, the biodegradable polymer comprises lactic acid or / and glycolic acid [e.g., an L-lactic acid-based copolymer, such as poly(L-lactide-co-glycolide) or poly(L-lactic acid-co-D, L-2-hydroxyoctanoic acid)]. For example, biodegradable polymeric microspheres composed of polylactic acid or / and polyglycolic acid can serve as sustained-release pulmonary drug-delivery systems. The biodegradable polymer of the polymeric implant or polymeric nanoparticles orC-1572 (EIC0016PCT)microparticles can be selected so that the polymer substantially completely degrades around the time the period of treatment is expected to end, and so that the byproducts of the polymer’ s degradation, like the polymer, are biocompatible.

[0120] In further embodiments, a sustained-release composition comprises a dendrimer. In certain embodiments, the dendrimer is a water-soluble dendrimer, such as a poly(amidoamine) (PAMAM) dendrimer. In some embodiments, a dendrimer encapsulates a drug through the formation of a dendrimer-drug supramolecular assembly. In other embodiments, a sustained-release composition comprises a water-soluble polymer [e.g., poly(DL-lactide)] or a liposome encapsulating a drug complexed with a dendrimer.

[0121] In other embodiments, the sustained-release composition is an oral dosage form, such as a tablet or capsule. For example, a drug can be embedded in an insoluble porous matrix such that the dissolving drug must make its way out of the matrix before it can be absorbed through the GI tract. Alternatively, a drug can be embedded in a matrix that swells to form a gel through which the drug exits. Sustained release can also be achieved by way of a single-layer or multi-layer osmotic controlled-release oral delivery system (OROS). An OROS is a tablet with a semi-permeable outer membrane and one or more small laser-drilled holes in it. As the tablet passes through the body, water is absorbed through the semi-permeable membrane via osmosis, and the resulting osmotic pressure pushes the drug out through the hole(s) in the tablet and into the GI tract where it can be absorbed.

[0122] For a delayed or sustained release of the disclosed EGFR inhibitors and KRAS inhibitors, a composition can also be formulated as, e.g., a depot that can be implanted in or injected into a subject, e.g., intramuscularly, intracutaneously or subcutaneously. A depot formulation can be designed to deliver the EGFR inhibitors and KRAS inhibitors over an extended period of time, e.g., over a period of at least about 1 week, 2 weeks, 3 weeks, 1 month or longer. For example, the EGFR inhibitors and KRAS inhibitors can be formulated with a polymeric material (e.g., polyethylene glycol [PEG], polylactic acid [PLA] or polyglycolic acid [PGA], or a copolymer thereof [e.g., PLGA or PLA-PEG]), with a hydrophobic material (e.g., as an emulsion in an oil) and / or an ion-exchange resin, as a more lipophilic derivative (e.g., as an ester of or a salt with a fatty acid such as a C8-C20fatty acid [e.g., decanoic acid]), or as a sparingly soluble derivative (e.g., a sparingly soluble salt). As an illustrative example, theC-1572 (EIC0016PCT)disclosed EGFR inhibitors and KRAS inhibitors can be incorporated or embedded in sustained-release microparticles composed of PLGA and formulated as a monthly depot.

[0123] The disclosed EGFR inhibitors and KRAS inhibitors can also be contained or dispersed in a matrix material. The matrix material can comprise a polymer (e.g., ethylene-vinyl acetate) and controls the release of the drug by controlling dissolution and / or diffusion of the drug from, e.g., a reservoir, and can enhance the stability of the drug while contained in the reservoir. Such a release system can be designed as a sustained-release system, can be configured as, e.g., a transdermal or transmucosal patch, and can contain an excipient that can accelerate the drug’s release, such as a water-swellable material (e.g., a hydrogel) that aids in expelling the drug out of the reservoir. US Pat. Nos. 4,144,317 and 5,797,898 describe examples of such a release system.

[0124] The release system can provide a temporally modulated release profile (e.g., pulsatile release) when time variation in plasma levels is desired, or a more continuous or consistent release profile when a constant plasma level is desired. Pulsatile release can be achieved from an individual reservoir or from a plurality of reservoirs. For example, where each reservoir provides a single pulse, multiple pulses (“pulsatile” release) are achieved by temporally staggering the single pulse release from each of multiple reservoirs. Alternatively, multiple pulses can be achieved from a single reservoir by incorporating several layers of a release system and other materials into a single reservoir. Continuous release can be achieved by incorporating a release system that degrades, dissolves, or allows diffusion of a drug through it over an extended time period. In addition, continuous release can be approximated by releasing several pulses of a drug in rapid succession (“digital” release). An active release system can be used alone or in conjunction with a passive release system, as described in US Pat. 5,797,898.

[0125] In addition, pharmaceutical compositions comprising disclosed EGFR inhibitors and KRAS inhibitors can be formulated as, e.g.. liposomes, micelles (e.g., those composed of biodegradable natural or / and synthetic polymers, such as lactosomes), nanoparticles (e.g., lipid nanoparticles such as solid lipid nanoparticles), microparticles or microspheres, whether or not designed for sustained release. In some embodiments, liposomes or micelles are composed of one or more phospholipids. Phospholipids include without limitation phosphatidic acids (e.g., DEPA, DLPA, DMPA, DOPA, DPPA and DSPA), phosphatidylcholines (e.g., DDPC, DEPC, DLPC, DLOPC, DMPC, DOPC, DPPC, DSPC, MPPC, MSPC, PLPC, PMPC, POPC, PSPC,C-1572 (EIC0016PCT)SMPC, SOPC and SPPC), phosphatidylethanolamines (e.g., DEPE, DLPE, DMPE, DOPE, DPPE, DSPE and POPE), phosphatidylglycerols (e.g., DEPG, DLPG, DMPG, DOPG, DPPG, DSPG and POPG), phosphatidylserines (e.g., DLPS, DMPS. DOPS, DPPS and DSPS), and salts (e.g., sodium and ammonium salts) thereof. In certain embodiments, liposomes or micelles are composed of one or more phosphatidylcholines. Liposomes have a hydrophilic core, so liposomes are particularly suited for delivery of more hydrophilic drugs, whereas micelles have a hydrophobic core, so micelles are particularly suited for delivery of more hydrophobic drugs. Liposomes and micelles can permeate across biological membranes. Moreover, liposomes and micelles composed of a fusogenic lipid (e.g., DPPG) can fuse with the plasma membrane of cells and thereby deliver a drug into those cells. Liposomes and micelles can provide sustained release of a drug based in part on the rate of degradation of the liposomes and micelles.

[0126] The pharmaceutical compositions can be manufactured in any suitable manner known in the art, such as by means of conventional mixing, dissolving, suspending, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compressing processes, or any combination thereof.

[0127] The compositions can be presented in unit dosage form as a single dose wherein all active and inactive ingredients are combined in a suitable system, and components do not need to be mixed to form the composition to be administered. A unit dosage form generally contains a therapeutically effective dose of the drug but can contain an appropriate fraction thereof so that taking multiple unit dosage forms achieves the therapeutically effective dose. Examples of a unit dosage form include a tablet, capsule, or pill for oral uptake; a solution in a pre-filled syringe of a single-use pen or a pen with a dose counter for parenteral (e.g., intravenous, subcutaneous or intramuscular) injection; a capsule, cartridge or blister pre-loaded in or manually loaded into an inhaler; and a reservoir-type transdermal patch or a drug-inadhesive patch.

[0128] Alternatively, the compositions can be presented as a kit in which the active ingredient, excipient(s) and carrier(s) [e.g., solvent(s)] are provided in two or more separate containers (e.g., ampules, vials, tubes, bottles or syringes) and need to be combined to form the composition to be administered. The kit can contain instructions for storing, preparing and administering the composition (e.g., a solution to be injected parenterally).C-1572 (EIC0016PCT)

[0129] A kit can contain all active and inactive ingredients in unit dosage form or the active ingredient and inactive ingredients in two or more separate containers, and can contain instructions for administering or using the pharmaceutical composition to treat a medical condition. A kit can further contain a device for delivering the composition, such as a needle and a syringe, an injection pen, an inhaler or a transdermal patch.

[0130] In some embodiments, a kit contains the disclosed EGFR inhibitors and KRAS inhibitors described herein or a pharmaceutical composition comprising the same, and instructions for administering or using the disclosed EGFR inhibitors and KRAS inhibitors or the composition to treat a medical condition disclosed herein. In certain embodiments, the kit further contains a device for delivering the disclosed EGFR inhibitors and KRAS inhibitors or the composition, such as an injection pen, an inhaler or a transdermal patch.GENERAL METHODS CELL VIABILITY ASSAY

[0131] Cell viability was assessed using the CellTiterGlo Luminescent Cell Viability Assay (Promega). Cells were seeded in 96-well plates at a density of 3,000 cells per well or 384-well plates at a density of 1,000 cells per well and incubated overnight at 37 °C in a 5% CO2atmosphere to allow for adherence. Post-treatment with experimental compounds for 72 hours, 20-100 µL of CellTiter-Glo reagent was added to each well, followed by gentle mixing on an orbital shaker for 2 minutes. After a 10-minute incubation at room temperature, luminescence was measured using a relative luminescence unit (RLU) from treated wells were normalized to control wells to determine cell viability. IC50 values were calculated by GraphPad prism 9 using 3-paramter dose-response model.CELL LINES AND CELL CULTURE

[0132] LS513 (also referred to as LS-513). LS-174T, HCT116. RKO, and HT29 human colon cancer cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). CACO-2, a KRAS wild- type human colon cancer cell line, was purchased from RIKEN Cell Bank (Ibaraki, Japan). LS513 cells were cultured in RPMI1640 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum and 1% P / S. LS-174T cells were cultured in Dulbecco’s modified Eagles medium (DMEM; FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum andC-1572 (EIC0016PCT)1% P / S. All cell lines were incubated at 37°C and 5% CO2. Representative colorectal cancer cell lines include LS-513, LS-174T and GP2D, each of which harbors a KRAS G12D mutation; HCT116 and DLD1, each of which harbors a KRAS G13D mutation; and RKO, which does not exhibit a detectable KRAS mutation (KRAS wild-type).ESTABLISHMENT OF MRTX1133 RESISTANT CELLS

[0133] MRTX1133-resistant cells were established by exposure of KRASG12D induced LS-174T cells to the increasing concentrations of MRTX1133. The initial concentration of MRTX1133 was 10 nM. When the cells adapted to the drag, the concentration of MRTX1133 was gradually increased by 1.5-2 times every week to a final concentration of 1 µM.CELL SEEDING AND DRUG SCREENING

[0134] Freshly trypsinized colorectal cell lines were seeded into black CELLSTAR® uCLEAR® 384 well plates (Greiner, Monroe, NC, USA) with 3000 cells per well. After cell seeding, the cells were incubated overnight to adhere to the bottom of the plate before drugs were added. Prior to drug screening the drugs were re-suspended as 4µM stock solutions and dispensed into 384-well plates using a BRAVO liquid handler (Agilent) in a randomized plate layout to control for plate effects. The kinase library was based on purchasable drags from the vendor (MedChem Express) of n = 2, 652. All kinase inhibitors were tested at 10 nM, and, for selected hit compounds, a concentration range of 1-1000 nM was tested. Cells were incubated for 72 h with drugs in media at 37 °C, 5% CO2 before MTS assay read out.C-1572 (EIC0016PCT)SYNERGY DETERMINATION WITH THE SYNERGYFINDER METHOD

[0135] CRC Cell lines were seeded at a density of 0.05 × 106cells / well in 384-well black pclear plates (Greiner) and were further treated as described above. Synergy scoring was determined using the “inhibition readout” (calculated as “100 - Cell Viability”) on the online SynergyFinder software) and implementing the ZIP calculation method. The matrix started with 1µM in the hit compounds and 300 nM or 3000 nM in the MRTX1133 starting concentration with a 3-fold decrease in the parental and resistant, respectively. The matrices were created in a DMSO 384 format that can be re-used for multiple tests. After testing the cell viability using Cell titer gio, the results were input into synergyfinge3.0 to determine synergistic potential from these specific compounds. The synergy score used for this high throughout screen was ZIP synergy score that assumes the two drugs are non-interacting. Scores between -10 and 10 are additive and below -10 are considered antagonistic.WESTERN IMMUNOBLOT

[0136] Cell lysates were harvested using a lysis buffer supplemented with protease and phosphatase inhibitors. The total protein concentration of the cell lysates was determined using a BCA protein quantification kit according to the manufacturer’s protocol. Proteins from each sample (10–50 µg) were separated on 4-20% mini-PROTEAN TGX gels and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). After blocking in milk with 5% Trisbuffered saline with Tween (TBST) buffer, the membranes were probed with primary antibodies overnight at 4 °C. The membranes were washed three times in TBST and incubated with secondary antibodies for 1 h at room temperature. Image acquisition and band intensity quantification were performed using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, USA) and Image J software (NIH, Bethesda, USA), respectively. The following primary antibodies were obtained from Cell Signaling Technology and used at a dilution of 1:1000: anti-EGFR (#4267), anti-phospho-EGFRY1068 (#3777), anti-ERKl / 2 (#9102), anti-phosphoERKl / 2 (#4370), anti-AKT (#9272), anti-phospho-AKTS473 (#4060), and anti-PARP (#9542).C-1572 (EIC0016PCT)COLONY FORMATION ASSAY

[0137] The cells were seeded in 12- well plates. After overnight incubation, the cells were treated with various inhibitors for 72 hours. At the end of the treatment period, the colonies were fixed and stained. Crystal violet was removed from the colonies using 10% acetic acid and the absorbance was measured at 595 nm. The relative cell viability was calculated by setting the untreated group to 100%.PATIENT-DERIVED ORGANOIDS

[0138] See supplemental methods for detailed PDO culture protocol. In brief, frozen aliquots of PDO tissue were obtained from NCI Patient-Derived Models Repository were obtained, thawed, and re-established as PDOs. Thawed tissues were embedded in Matrigel and cultured in complete PDO media which was supplemented with WRN conditional media as well as ROCK inhibitor. All PDOs used were passaged at least twice and growing with a doubling time of 3-10 days, prior to use in experiments.IMMUNOBLOT

[0139] Immunoblotting was performed as previously described [cite PMID 36313707]. Detection was performed using Clarity Western ECL Substrate (Bio-Rad) and visualized using X-ray film exposure. Densitometry quantification was performed using ImageJ (version 1.53).EXAMPLES EXAMPLE 1. GENERATION OF MRTX1133 RESISTANT CELL LINES

[0140] It was first validated that MRTX1133 (Structure shown in Fig. 1A) is selectively effective against colorectal cancer cells harboring G12D mutations. Three colorectal cancer cell lines, LS-174T cells (KRAS G12D mutation). HCT116 CRC cells (G13D). and RKO cells (WT KRAS) were treated with MRTX1133 in a concentration-dependent manner. Cell viability was measured using CellTiter-Glo after 72 hours of incubation. As expected, MRTX1133 selectively inhibits the growth of LS-174T cells that have a KRAS G12D mutation while ineffective against other two cell lines that do not have G12D mutation up to 1,000 nM (Fig. IB). This is consistent with the previously reported biochemical inhibitory potency of MRTX1133 as well as previous cellular data of MRTX1133.C-1572 (EIC0016PCT)

[0141] To screen the drug combinations to overcome resistance, LS-174T cells resistant to MRTX1133 were established, referred to “LS-174T / MRTX1133 cells.” After passaging 8 times in the presence of MRTX1133 at 1000 nM in cell culture, we obtained cells that are resistant (R) to MRTX1133, with more than 100-fold decrease of potency compared to the parent cell line (P) (Fig. 1C).

[0142] To further characterize the parental and resistant cell lines, histological analyses were conducted to elucidate the morphological differences between these cell populations. In order to understand the mechanisms of resistance, the two cell lines were analyzed using reversephase protein arrays (RPPAs). The antibody repertoire covers key oncogenic pathways such as PI3K / AKT, RAS / MAPK, Src / FAK, TGF-b / SMAD, JAK / STAT, DNA damage repair, Hippo, cell cycle, apoptosis, histone modification, and immune oncology. Analysis of the RPPA data revealed significant upregulation of genes, notably PLK1 and Src, in the resistant LS-174T cell line compared to less notable genes in the downregulation. This upregulation was corroborated by immunoblotting (Fig. ID), which confirmed increased expression levels of these key proteins involved in the MAPK signaling pathway in the resistant cells compared to their parental counterparts. The resistant cell line exhibited upregulation of EGFR expression, aligning with previous studies that confirmed the EGFR feedback mechanism upon treatment with MRTX (Cancer Res, 2023). Furthermore, genomic mutational profiling identified the emergence of a new KRAS mutation in the resistant cell line (Fig. IE), absent in the parental line. This newly acquired mutation may contribute to the observed resistance phenotype and alterations in signaling dynamics within the resistant LS-174T cells.

[0143] These results indicate that prolonged treatment with MRTX1133 can lead to diverse genomic and proteomic resistance mechanisms in CRC. The pair of parental and resistant cell lines also serve as useful tools for discovery and validation of therapies to overcome resistance, as follows.EXAMPLE 2. HIGH-THROUGHPUT DRUG SCREENING AND ANALYSIS

[0144] With the MRTX1133-resistant cells in hand, a high throughput small molecule screen was performed to identify molecules that (i) potentiate the efficacy of MRTX 1133 in parental colorectal cancer cells, and (ii) overcome MRTX1133 resistance. A kinase inhibitor library consisting of 2,652 compounds was selected for the screening campaign. The diverse kinase library encompasses compounds that target protein kinases (such as VEGFR, EGFR,C-1572 (EIC0016PCT)BTK, CDK, Akt), and lipid kinases (including PT3K, PI4K, SK), etc. and several inhibitors have received FDA approval. The library information includes the compound structures and target proteins and pathways. The cell viability assay was miniaturized to 384 well plate format.

[0145] The schematics of the screening assay are shown in Fig. 2A. Cell viability assay was miniaturized to 384 well plate format. Cells were seeded at a 0.05 M / mL density in 384 well plates and allowed to adhere to the plate overnight, and the compound library was dispensed using a robotic liquid handler Agilent Bravo with a 384ST head with the final concentration of 10 nM. The optimal concentration was determined by testing a single plate from the library at various concentrations to ensure an appropriate number of 'hits' were observed. After 72 h incubation, the cell viability was measured using Cell-Titer Gio. The combination of parental vs resistant LS-174T cells ± MRTX1133 established four distinct conditions for the screen (Table 1).

[0146] The screening assay was well-behaved, with an overall Z’ factor over 0.6 and an inhibition cut off rate of 50%. Out of the 2,652 compounds tested, a total of 186 compounds that pass the inhibition rate with a hit percentage of 6% were obtained, representing the yield from the primary synergizer hit cutoff. The screening groups consist of four groups to determine the best outcome for finding inhibitors that were resistant specific and ones that synergize with MRTX1133. The concentration of MRTX1133 was chosen based on the cellular IC50 determined in preliminary data in both parental and resistant cell lines. This concentration allowed for cell suppression without reaching the inhibition cut-off criteria and was normalized for each plate to identify synergistic compounds. Each plate was tested with or without MRTX1133 in both parental and resistant cell lines. An overview of the inhibition scores is depicted in the scatter plot.TABLE 1Condition LS-174T cell line MRTX1133[1] Parental (P) 0 nM[2] 10 nM[3] MRTX1133-resistant (R) 0 nM[4] 1000 nMC-1572 (EIC0016PCT)

[0147] Screening triage is shown in Fig. 2B. The primary screening assay had an overall Z’ factor over 0.6 and an inhibition cutoff rate of 50%. From the primary screen, potential synergizer hit molecules were selected. Next, concentration-dependent cell viability inhibition was measured in triplicate to determine the precise synergistic potency of the selected molecules. Finally, the synergistic potency of the top molecules was evaluated in a concentration-matrix format to determine the synergy score. The list of validated hit molecules and their known protein target in cancer signaling pathway is summarized in Fig. 2C. The identified “hit” molecules are divided into two main categories: 1. Vertical inhibitors of the RTK-RAS-RAF-MEK pathway, and 2. cell cycle inhibitors. Through this screening campaign, compounds were selected for further biological evaluation.

[0148] To organize these hits, these hits were sorted into 16 distinct categories (Fig. 2D) These categories were mathematically derived based on how the compounds fit within the screening parameters, allowing for a structured and comprehensive analysis of the screening data. The targeted molecules include those that were general KRAS synergizers. resistant selective KRAS synergizes, or simply targeting the cancer cell lines. Thus, the secondary synergizer hit cutoff was applied as follows. A mathematical cut off was used by sorting all the compounds that were above 50% inhibition rate in one of the four screening groups, and also showed at least a 30% difference in inhibition between the monotherapy and the MRTX1133 combination. The latter criteria allowed us to prioritize hits that are more likely to be synergizers or enhancers of MRTX1133. In an effort to avoid the nonspecific cytotoxic compounds, molecules above 80% inhibition in ALL screening groups were excluded from this study.Additionally, hit compounds were analyzed for feasibility, and pan-assay interference and undrug like compounds were removed. Following the application of the primary synergizer hit cutoff, the initial 185 hit compounds were subjected to a pathway enrichment analysis in order to assess biological or functional patterns among these kinase inhibitors. Focusing on the “hit” synergistic molecules in both the parental and resistant cell lines, the inhibition rates across various biological pathways were plotted. Each dot signifies a compound, with lighter dots representing more synergistic molecules based off the monotherapy “hit” kinase compounds. Notably, different compounds emerged as significant in the parental versus resistant cell lines. This suggests distinct adaptive mechanisms and pathway dependencies between the two cell lines. For instance, compounds targeting the MAPK / ERK and JAK / STAT pathways were moreC-1572 (EIC0016PCT)prevalent in the resistant cell lines, indicating a possible reliance on this pathway for survival. Conversely, parental cell lines showed a higher sensitivity to compounds affecting the Protein Tyrosine Kinase / RTK pathway. These findings provide insights into the differential pathway dependencies and potential targets for overcoming resistance in colorectal cancer treatment. EXAMPLE 3. VALIDATION OF HTS DRUG COMBINATIONS FOR OVERCOMING MRTX1133 RESISTANCE

[0149] An overview of the pathways that were enriched and associated with the hit molecules and their known protein targets in cancer signaling pathway (Fig. 2C). This figure presents a simplified graphical depiction of pathways that synergize with MRTX1133, demonstrating higher inhibition than monotherapy alone. It is interesting to note that the hit molecules are divided into two main categories: 1. Vertical inhibitors of the RTK-RAS-RAF-MEK pathway, and 2. cell cycle inhibitors. Following the application of the secondary synergizer hit cutoff, 27 compounds remained to be considered for validation. Based on manual inspection for pan-assay interference and un-druglike properties, seven compounds were chosen to validate using multi-dose synergy testing (Fig. 3A). Four compounds were strongly positive in the screen and are characterized as likely inhibitors of the MAPK pathway; the four compounds are Mutant EGFRi (CAS Reg. No. 1421373-62-7), Limeritinib (CAS Reg. No. 1934259-00-3), p38a inhibitor 2 (CAS Reg. No. 1095003-80-7), and ASN007 (CAS Reg. No. 2055597-12-9). The remaining three chosen compounds were also strongly positive in the screen and suppress well-described oncogene pathways (Aik kinase- 1) or are transcriptional inhibitors of special interest; the three compounds are ALK kinase inhibitor- 1 (CAS Reg. No. 1462949-64-9), CDK12-IN-E9 (CAS Reg. No. 2020052-55-3). and CDK8-IN-4 (CAS Reg. No. 1613638-82-6). A monotherapy dose response was performed in the parental and resistant cell lines to determine which combination shows the most promising results. The screening matrix initiated with 1 μM of hit compounds and MRTX1133 at starting concentrations of either 300 nM or 3000 nM. These concentrations were then subjected to a 3-fold serial dilution in parental and resistant cell lines, respectively. The ZIP synergy score, which presumes non-interaction between the two drugs, was utilized to assess synergy in this high-throughput screen. In the parental and resistant models, it was found striking synergy between the irreversible EGFR inhibitors and MRTX1133. The 3D synergy matrices identified two compounds with scores above 10 were determined to be synergistic, highlighted in red. Most notably, two compounds characterized as inhibitors ofC-1572 (EIC0016PCT)mutant oncogenic EGFR, Mutant EGFRi and Limeritinib, demonstrated the strongest synergy with MRTX1133. Mutant EGFRi (PubChem CID: 78357783), an experimental inhibitor without a formal name, will be referred to as NT-1 throughout the study to prevent confusion. NT-1 exhibited potent synergistic effects in both the parental and resistant colorectal cancer models. From the initial screen, dose-response analyses, and matrix experiments, NT-1 significantly enhanced the synergistic effect in the parental and resistant cell lines. This is further enhanced in the viability curves showing the strong synergistic effect at low doses in panels (Figs. 3B and 3C). The triangle line depicts combination data that shows increased inhibition in the sub nanomolar range, whereas the others follow a similar trend with MRTX1133 treatment. The CDK inhibitors worked well at low concentrations, but did not exhibit synergy, even when tested at these lower concentrations. Although most compounds were close to the cutoff around 10, indicating their potential utility for future treatments and combinations, they did not meet the synergy criteria for the current disclosure.[01501 Some of the validated EGFR inhibitors are described in TABLE 2.TABLE 2R (-) KRASi R (+) KRASi WT(-) KRASi WT(+) KRASi Product Name (Inhibition rate %) (Inhibition rate %) (Inhibition rate %) (Inhibition rate %)24 71 6 65 Mutant EGFR inhibitor 28 61 -32 -8 AZ-510420 60 5 14 Limertinib29 58 5 46 BMS-69051410 57 3 34 Neratinib24 54 3 65 Naquotinib (mesylate) 23 51 -6 8 PF-627448418 49 3 23 Pelitinib42 49 13 49 Mobocertinib24 48 1 47 Tarlox-TKI55 46 9 22 EGFR-IN-1131 45 -5 11 Rezivertinib EXAMPLE 4. IRREVERSIBLE EGFR INHIBITORS SYNERGIZER COMPARISON IN VITRO

[0151] Given previous literature, it is unsurprising that the top hits that emerged were small molecule EGFR inhibitors. Inhibition of mutant BRAFV600E with BRAF kinase inhibitors, and inhibition of KRASG12C or G12D with allele specific inhibitors, reliably triggers the adaptive mechanism of EGFR upregulation and subsequent increase in mitogenic signaling (cite). This has led to the expectation that co-inhibition of EGFR and KRAS would lead toC-1572 (EIC0016PCT)improved responses. However, the screen results and CRC models introduce an important alternative from this hypothetical mechanism of synergy. Unlike in non-small cell lung cancer, CRC has an incredibly low rate of EGFR mutation (<0.5%). In both the LS174T-P and -R models used in this screen, EGFR mutations were not detected. To investigate the hypothesis that co-inhibition of EGFR is the primary mechanism of synergy, the synergy between a panel of EGFR inhibitors and MRTX1133 was assessed. Upon closer examination of the best hit, NT-1, is found to be structurally and biochemically similar to Osimertinib. The synergy scores were very similar across all cell lines, though slightly lower in the resistant cell line. Osimertinib appeared in the Kinase library but did not show up as a hit, likely due to the concentration at which it was screened. By using a low concentration of library compounds (10 nM), potent molecules that synergize with MRTX1133 were identified, as highlighted in the boxed outline as the “most synergistic area” of the heat maps. Examining the biochemical similarities of the top hits compared to Osimertinib and Geftinib, which is an FDA approved first generation wildytpe EGFRi (Table 3A) revealed the potency in terms of mutant and wild-type IC50s of the different compounds. Utilizing the matrix for the small molecule inhibitors, it is clear that NT-1 is superior compared to Osimertinib, limeritinib, and Gefitnib (Table 3B) The molecules synergized with MRTX1133 but at a much higher concentration than NT-1. To further understand the difference between NT-1 and other FDA approved EGFR treatments, the doseresponse of NT-1, and the FDA approved wildtype EGFR treatment, Cetuximab was compared, and the superiority of NT-1 was quite striking (Figs. 4A and 4B). Overall, the results suggest that NT-1 is a uniquely synergistic molecule when combined with MRTX1133. The co-inhibition of EGFR does not appear to sufficiently explain the mechanism of synergy by NT-1, since no other EGFR inhibitor can achieve a similar depth of anti-tumor activity, and CRC lacks the primary known biological target of NT-1.C-1572 (EIC0016PCT)TABLE 3A I50 / Target Matrix for Small Molecule InhibitorsNT-1 Osmertinib Limeritinib Gefitinib0.2 nM 0.3nMEGFRL858 / T790MEGFR (WT)InMEGFRL858 / T790M10.2nMEGFR (WT)0.6nM 0.5nMEGFRExon19 deletion EGFR 12nML858 / T790MEGFRL858R11 nM 6nMEGFR (WT) EGFRExon 19 deletionTABLE 3B. LS-174T-PDrug Combination ZIP ScoreNT-1 + MRTX1133 24.88Osmeritinib + MRTX1133 10.34Limeritnib + MRTX1133 11.04Gefitinib + MRTX 1133 6.12EXAMPLE 5. COMBINATION THERAPY BLOCKS DOWNSTREAM MAPK PATHWAY IN MRTX1133 RESISTANT CELLS

[0152] Next, the molecular mechanism underlying the strong synergy between EGFR inhibition by NT-1 and MRTX1133 was investigated, focusing on the resistant cell line. The initial steps started with monotherapy of NT-1 to determine the effective concentration after 24 hours. Suppression of EGFR and increased apoptosis around - 60 nM were observed. The combination treatment moving forward included 60 nM of NT-1, which is 3X the IC50 found in previous cellular assays. MRTX1133 is known to downregulate the ERBB receptor feedback inhibitor 1 (ERRFI1), which is a negative regulator of EGFR, causing feedback activation. The EGFR family comprises four distinct membrane tyrosine kinase receptors: EGFR / ErbB-1, HER2 / ErbB-2, HER3 / ErbB-3, and HER4 / ErbB-4, which are activated upon ligand binding to the extracellular domain of these receptors (CITE). This induction of ERBB2 and ERBB3 expression, in turn, reactivates KRAS signaling, limiting the sensitivity to these single-agentC-1572 (EIC0016PCT)drug treatments. The monotherapy and combined effect of NT-1 and MRTX1133 in the resistant cells is shown in Fig. 5A. There is downstream suppression of p-ERK and an increase in Caspase 3, indicating that the cells are undergoing apoptosis with this combination treatment. This was further proven to be an exceptional synergistic model when compared to cetuximab and Osimertinib. Indeed, while monotherapy with MRTX1133 induced phospho-EGFR upregulation, cells co-treated with NT-1 failed to activate EGFR, indicating effective suppression of the feedback loop (Figs 5B and 5C). Cetuximab and Osimertinib combinations treatment shows decreased p-EGFR and increased PARP and Caspase 3 expression, but it is less effective compared to NT-1. Therefore, pharmacological inhibition of EGFR by NT-1 re-sensitizes the cells to the KRAS G12D inhibitor and assists in the prevention of this feedback loop, resulting in a more complete suppression of KRAS signaling. The comparison of the Osimertinib, under the same conditions with a 10-fold increase in concentration, further confirming the novel and high potency of Mutant NT-1. Whether combined NT-1 and MRTX1133 could effectively sustain inhibition of MAPK signaling after 24 to 72hrs was evaluated. Previous literature has shown the synergistic effects of EGFR and KRASi is only up to 48 hours before EGFR signaling resumes (cite). However, the current findings indicate sustained inhibition beyond this period, reaffirming the hypothesis that a driving force is enhancing synergy at an exceptionally low sub-nanomolar range. Notably, the decrease in downstream pathways such as p-ERK is statistically significant compared to Osimertinib and Cetuximab. To further validate these findings, organoid models to assess the therapeutic potential and mechanistic insights of combined NT-1 and MRTX1133 treatment were conducted.EXAMPLE 6. IRREVERSIBLE EGFR INHIBITORS SYNERGIZE AND OVERCOME MRTX1133 RESISTANCE IN PDO MODELS

[0153] To further validate the potent synergy between NT-1 and MRTX1133, the combination in patient-derived organoid models (PDO) was studied. To mimic the LS-174T parental protocol for the KRAS G12D Cell line, an NCI organoid model was treated with multiple cycles of 72-hour of MRTX1133 until the partial loss of organoid integrity was observed. Organoids that survived the 8 cycles of treatment were called NCI-10278 / MRTX1133 resistant. With the parental and resistant MRTX1133 resistant organoids ready, it was able to test the drug combination in vitro to determine if the heterogeneity of CRC PDO models could be overcome using the top synergistic compound (Figs. 6A and 6B). In the dose response curves, itC-1572 (EIC0016PCT)was observed that the combination therapy works in both models at very low concentrations. There is a strong synergistic effect as well as efficacy in monotherapy, which is notable because CRC is not driven by EGFR mutations. A histological view of the treated organoid after 72 hours, shows apoptosis in both monotherapy and combination therapy (Fig. 6C). This is further corroborated with the microscopic view of organoids on day 0 compared to seven days of treatment. Interestingly, in the combination treatment immunoblot reveals similar suppression of p-EGFR, p-ERK and p-S6, which is not observed in monotherapy alone (Fig. 6D). After just 72 hours of treatment, the organoids exhibit a rapid synergistic inhibitory effect at low concentrations. The only variable between previous cell line and organoid treatments is the duration of exposure. Organoids provide a more realistic and heterogenous model for inhibitory testing compared to cell lines. To compare NT-1 synergy with MRTX1133, Osimertinib treatment for comparison utilizing immunofluorescence staining (IF) was examined.Proliferation markers, and p-EGFR suppression were stained. These molecules synergize effectively with MRTX1133 in multiple MRTX1133-resistant models, suggesting their potential as promising combination therapies in KRAS G12D mutation profiles.

[0154] Concluding Remarks: the current disclosure provides a holistic and unbiased screening of a diverse kinase library leading the identification and discovery of Osimertinib analogs synergizing with MRTX113 in KRASG12Dmutant colorectal cancer to overcome resistance to MRTX1133 monotherapy. Patient-derived organoid models were used to confer the synergy between Mutant EGFRi and MRTX1133 and this study provides rationale for testing this drug combination in clinical trial patients with KRASG12Dmutant colorectal cancer.

Claims

1. C-1572 (EIC0016PCT)CLAIMSWhat it claimed is:

1. A pharmaceutical composition, comprisinga first active agent and a second active agent, wherein the first active agent comprises Osimertinib (CAS Reg. No. 1421373-65-0). AZ-5104 (CAS Reg. No. 1421373-98-9). Limertinib (CAS Reg. No. 1934259-00-3), or mutant EGFR inhibitor (CAS Reg. No. 1421373-62-7), or a pharmaceutically acceptable salt of any of the foregoing;wherein the second active agent comprises MRTX1133 (CAS Reg. No. 2621928-55-8), RMC-6236 (CAS Reg. No. 2765081-21-6), RMC-7977 (CAS Reg. No. 2765082-12-8), RMC-9805 (CAS Reg. No. 2922732-54-3). or RMC-6291 (CAS Reg. No. 2641998-63-0), or a pharmaceutically acceptable salt of any of the foregoing; andoptionally comprising a pharmaceutically acceptable carrier or excipient.

2. The composition of claim 1, wherein the excipient comprises a binder, a sorbent, a lubricant, a disintegrant, or a preservative.

3. The composition of claim 1, wherein the pharmaceutical composition is configured for treating a subject who has developed resistance to treatment with a KRAS inhibitor.

4. A method of inhibiting the proliferation or growth of cancer cells comprising contacting cancer cells, concurrently or sequentially, with a first active agent which is an EGFR inhibitor and a second active agent which is a KRAS inhibitor(i) wherein the first active agent comprises Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor, or a pharmaceutically acceptable salt of any of the foregoing; and(ii) wherein the second active agent comprises MRTX1133, RMC-6236, RMC-7977, RMC-9805, or RMC-6291, or a pharmaceutically acceptable salt of any of the foregoing.

5. The method of claim 4, wherein the cancer cells are colorectal cancer (CRC) cells, metastatic colorectal cancer (mCRC) cells, colon cancer cells, rectal cancer cells, or LS-174T cells resistant to MRTX1133.C-1572 (EIC0016PCT)6. The method of claim 4 or claim 5, wherein the cancer cells are contacted with a concentration of the EGFR inhibitor that is about 0.1 nM to about 333 nM and a concentration of the KRAS inhibitor that is about 0.01 nM to about 1000 nM.

7. The method of any one of claims 4 to 6 wherein the KRAS inhibitor is MRTX1133 or a salt thereof and the cancer cells are contacted with a concentration of MRTX1133 that is about 0.01 nM to about 1000 nM.

8. The method of any one of claims 4 to 7, wherein the EGFR inhibitor is Osimertinib or a salt thereof and the cancer cells are contacted with a concentration of Osimertinib that is about 0.1 nM to about 1000 nM.

9. The method of any one of claims 4 to 6, wherein the EGFR inhibitor is AZ-5104 or a salt thereof and the cancer cells are contacted with a concentration of AZ-5104 that is about 0.1 nM to about 1000 nM.

10. The method of any one of claims 4 to 7, wherein the EGFR inhibitor is Limertinib or a salt thereof and the cancer cells are contacted with a concentration of Limertinib that is about 0.1 nM to about 1000 nM.

11. The method of any one of claims 4 to 7, wherein the EGFR inhibitor is mutant EGFR inhibitor or a salt thereof and the cancer cells are contacted with a concentration of mutant EGFR inhibitor that is about 0.1 nM to about 1000 nM.

12. A method of treating a subject having cancer comprising steps ofadministering to the subject concurrently or sequentially, a therapeutically effective amount of a first active agent which is an EGFR inhibitor and a therapeutically effective amount of a second active agent which is a KRAS inhibitor, whereinthe EGFR inhibitor comprises Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor, or a pharmaceutically acceptable salt of any of the foregoing; andC-1572 (EIC0016PCT)the KRAS inhibitor comprises MRTX1133, RMC-6236, RMC-7977, RMC-9805, or RMC-6291, or a pharmaceutically acceptable salt of any of the foregoing.

13. The method of claim 12 additionally comprises determining whether the subject has a cancer with a KRAS mutation; and if the subject has a cancer with a KRAS mutation, administering the first active agent and second active agent to the subject.

14. The method of claim 12 or 13, wherein the cancer is colorectal cancer (CRC), metastatic colorectal cancer (mCRC), colon cancer, or rectal cancer.

15. The method of any one of claims 13 to 14, wherein the subject is a human and the type of KRAS mutation is a KRAS G12A, KRAS G12C, KRAS G12D, KRAS G12R, KRAS G12V, KRAS G12S, KRAS Q61H, KRAS Q61L or a KRAS G13D.

16. The method of any one of claims 12 to 15, wherein the KRAS inhibitor is MRTX1133 of a salt thereof.

17. The method of any one of claims 12 to 16, wherein the EGFR inhibitor is Osimertinib, or a salt thereof.

18. The method of any one of claims 12 to 16, wherein the EGFR inhibitor is AZ-5104, or a salt thereof.

19. The method of any one of claims 12 to 16, wherein the EGFR inhibitor is Limertinib, or a salt thereof.

20. The method of any one of claims 12 to 16, wherein the EGFR inhibitor is mutant EGFR inhibitor, or a salt thereof.

21. The method of any one of claims 12 to 20, wherein one or both of the EGFR inhibitor and the KRAS inhibitor are administered orally, rectally, sublingually, in the buccal cavity,C-1572 (EIC0016PCT)intravenously, intratumorally, percutaneously, intramuscularly, transdermaly, cutaneously, subcutaneously, intrathecally, nasally, or vaginally.

22. The method of claim 21, wherein both the EGFR inhibitor and KRAS inhibitor are administered orally.

23. The method of claim 21 or 22, wherein the EGFR inhibitor is administered about 0.01 mg to about 200 mg per kilogram of body weight per day, and the KRAS inhibitor is administered about 0.01 mg to about 200 mg per kilogram of body weight per day.

24. The method of claim 12, wherein the subject has developed resistance to treatment with a KRAS inhibitor.

25. A method of treating a subject having developed resistance to treatment with a KRAS inhibitor comprising steps ofadministering to the subject concurrently or sequentially, a therapeutically effective amount of a first active agent which is an EGFR inhibitor and a therapeutically effective amount of a second active agent which is a KRAS inhibitor, whereinthe EGFR inhibitor comprises Osimertinib, AZ-5104, Limertinib, or mutant EGFR inhibitor, or a pharmaceutically acceptable salt of any of the foregoing; andthe KRAS inhibitor comprises MRTX1133, RMC-6236, RMC-7977, RMC-9805, or RMC-6291, or a pharmaceutically acceptable salt of any of the foregoing.

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