Combination therapy with dual farnesyltransferase and geranylgeranyltransferase-1 inhibitors and KRAS inhibitors

Abstract

A method of treating cancer in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of a dual farnesyltransferase and geranylgernayltransferase-1 inhibitor (FGTI) or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of a KRAS inhibitor or a pharmaceutically acceptable salt thereof. Dosage compositions and kits including the FGTI and the KRAS inhibitor or pharmaceutically acceptable salts thereof are also provided.

Description

COMBINATION THERAPY WITH DUAL FARNESYLTRANSFERASE AND GERANYLGERANYLTRANSFERASE-1 INHIBITORS AND KRAS INHIBITORS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of United States provisional patent application 63 / 743,006, filed January 8, 2025, the contents of which are incorporated herein by reference.STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENTThis invention was made with government support under grant number R35 CA197731 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.FIELD OF THE INVENTIONThe invention is generally related to a combination of a dual famesyltransferase (FT) and geranylgernayltransferase-1 (GGT-1) inhibitor, and a KRAS inhibitor for the treatment of cancer.BACKGROUND OF THE INVENTIONPancreatic cancer is a significant health concern in the United States, contributing substantially to cancer-related morbidity and mortality, with an estimated 64,050 new cases and 50,550 deaths in 2023, reflecting its high mortality rate and poor prognosis1. Despite advances in diagnosis and treatment, the overall prognosis remains poor, emphasizing the urgent need for novel therapeutic strategies to improve patient outcomes for this major deadly cancel'2’3. Mutations in Kirsten rat sarcoma viral oncogene homolog (KRAS) are significant contributors to tumorigenesis in pancreatic cancer. The RAS GTPase family members — HRAS, KRAS, and NRAS — act as molecular switches, cycling between GDP (inactive) and GTP (active) states4, regulated by GTPase- activating proteins (GAPs) leading to the inactive RAS-GDP state, and guanine-nucleotide-exchange factors (GEFs) leading to the active RAS-GTP state5. RAS proteins transduce signals from external stimuli to pathways like Raf / Mek / Erk, PI3K / Akt / mTOR, and RalGDS / Ral, affecting gene expression, cell division, differentiation, and survival6,7. Mutations in RAS reduce its affinity for GAP, resulting in persistently active GTP-bound RAS, which drives oncogenic events such as uncontrolledproliferation, apoptosis resistance, angiogenesis, immune evasion, invasion, metastasis, and drug resistance7 10. KRAS mutations occur in 90% of pancreatic ductal adenocarcinoma, 40% of colorectal cancer, and 30% of non-small cell lung cancer (NSCLC)8,9, with the KRAS G12D mutation present in 40-50% of pancreatic, 10-15% of colon, and 2-5% of lung cancer cases7 10. This prevalence across these deadly cancers highlights the need for targeted therapies to mitigate KRAS oncogenic impact.Recent breakthroughs have led to the FDA approval of KRAS G12C inhibitors sotorasib and adagrasib for patients with advanced KRAS G12C NSCLC11 17. However, while these drugs show promise, the overall response rate (ORR) (37-43%) and progression-free survival (PFS) (6.8-6.9 months) remain modest14 18due to the emergence of clinically-relevant resistance mechanisms involving adaptive reactivation of ERK, overexpression of receptor tyrosine kinases (RTKs, e.g., MET, EGFR, HER2), RTK mutations (e.g., EGFR-A289V, RET-M918T), acquired secondary KRAS oncogenic mutations, and secondary mutations within the sotorasib switch II binding site in KRAS12 17,19-22.KRAS G12C is present in a relatively small proportion of human cancers, approximately 13%. In contrast, the KRAS G12D mutation is found in about 27% of cancers, double the prevalence of KRAS G12C8,9. Despite this higher prevalence, there are currently no FDA-approved therapies specifically targeting the KRAS G12D isoform. Consequently, drugs targeting KRAS G12D are highly sought after, and several inhibitors (e.g., MRTX1133,, RMC-6236, RMC-7977, RMC-9805, ASP3082, QLC1101, PF-4040, BL2582, ) have been identified and some entered clinical trials including the highly potent and KRAS G12D-selective inhibitor MRTX113323’26. Although the discovery of MRTX1133 is a breakthrough for targeting KRAS G12D-harboring cancers such as pancreatic and colon cancers, just as described above for sotorasib and adagrasib, challenges remain due to several MRTX1133 resistance mechanisms, including amplification of RTKs, KRAS, YAP1, Myc, and CDK6, activation of the PI3K / AKT / mTOR pathway, secondary KRAS mutations, and adaptive feedback WT Ras-dependent ERK reactivation25’33. Similar resistance mechanisms with other KRAS inhibitors such as RMC-6236 and RMC-7977 have also been observed.Therapies that can enhance the efficacy of KRAS inhibitors are needed.SUMMARYAs described herein, dual farnesyltransferase (FT) and geranylgernayltransferase- 1(GGT-1) inhibitors (FGTIs) overcome resistance and enhance KRAS inhibitor anti -tumor activity by inhibiting ERK reactivation providing a synergistic anti-cancer therapy.An aspect of the disclosure provides a method of treating cancer in a subject in need thereof, wherein the cancer is mediated by a KRAS mutation, comprising administering to the subject i) a therapeutically effective amount of a FGTI or a pharmaceutically acceptable salt thereof; and ii) a therapeutically effective amount of a KRAS inhibitor or a pharmaceutically acceptable salt thereof, wherein administration of the FGTI and the KRAS inhibitor provides a synergistic enhancement of anti-tumor activity compared to administration of either agent alone. In some embodiments, the cancer is selected from the group consisting of pancreatic cancer, lung cancer, colon cancer, and breast cancer. In some embodiments, the KRAS mutation is selected from the group consisting of G12D, G12C, G13D, and G12V mutations. In some embodiments, the FGTI is FGTI-2734. In some embodiments, the KRAS inhibitor is a KRAS G12D inhibitor, such as MRTX1133. In some embodiments, the KRAS inhibitor is a pan RAS inhibitor, such as RMC-6236 or RMC-7977. In some embodiments, the cancer is resistant to treatment with the KRAS inhibitor alone. In some embodiments, the synergistic anti-tumor activity comprises increased Annexin V positivity relative to single-agent treatment, enhanced activation of caspases relative to single-agent treatment, and / or increased PARP cleavage relative to single-agent treatment. In some embodiments, the FGTI or pharmaceutically acceptable salt thereof and the KRAS inhibitor or pharmaceutically acceptable salt thereof are administered simultaneously. In some embodiments, the FGTI or pharmaceutically acceptable salt thereof is administered prior to the KRAS inhibitor or pharmaceutically acceptable salt thereof.Another aspect of the disclosure provides a method of inhibiting tumor cell viability of or inducing apoptosis in KRAS-mutant cancer cells, comprising exposing the KRAS-mutant cancer cells to i) a FGTI or a pharmaceutically acceptable salt thereof; and ii) a KRAS inhibitor or a pharmaceutically acceptable salt thereof. In some embodiments, the cancer cells are pancreatic cancer cells, lung cancer cells, colon cancer cells, or breast cancer cells. In some embodiments, the cancer cells have a KRAS mutation selected from the group consisting of G12D, G12C, G13D, and G12V mutations.Another aspect of the disclosure provides a dosage composition comprising a FGTI or a pharmaceutically acceptable salt thereof, and a KRAS inhibitor or a pharmaceutically acceptable salt thereof, wherein the FGTI and KRAS inhibitor or pharmaceutically acceptablesalts thereof are present together in a single dosage form. In some embodiments, the single dosage form is selected from the group consisting of a tablet, dragee, liquid, drop, capsule, caplet, and gelcap.Another aspect of the disclosure provides a kit, comprising a first dosage composition comprising a FGTI or a pharmaceutically acceptable salt thereof; and a second dosage composition comprising a KRAS inhibitor or a pharmaceutically acceptable salt thereof.BRIEF DESCRIPTION OF THE DRAWINGSFigures 1A-P. FGTI-2734 and MRTX1133 are synergistic at inhibiting the viability of human KRAS G12D pancreatic and colon cancer cells. A, E, I, M: Pancreatic (Panc0403, Panc00203, HP AC) and colon (LS174T) cancer cells were treated with various concentrations of either MRTX1133 or FGTI-2734 for 72 hours and processed the cells for viability by CellTiter-Glo® (CTG) assay to determine IC50 values as described in Methods.B, F, J, N: Synergy studies using the combination index method; the cells were treated with FGTI-2734 and MRTX1133 alone or in combination in a constant ratio to one another, and CalcuSyn software was used to generate Combination Index-Fraction affected (CI-Fa) plots as described in Methods. For each cell line, the data are representative of three independent experiments. C, G, K, O: Synergy studies using the SynergyFinder method; the cells were treated in a matrix format with various concentrations each of FGTI-2734 and MRTX1133 alone or in combination, and the tool SynergyFinder was used to calculate synergy scores and to plot the corresponding 3D response surface plots as described in Methods. D, H, L, P:Loewe and HSA scores were calculated using the SynergyFinder tool. For each cell line, the data are representative of at least 3 independent experiments. SynergyFinder p values ranged from p = 5.97e-12 to p = 1.28e-04.Figures 2A-D. FGTI-2734 enhances the ability of MRTX1133 to induce apoptosis in KRAS G12D pancreatic cancer cells. (A, B) Panc0403 and Panc0203 cells were treated for 24 hours with MRTX1133 (30 nM) and FGTI-2734 (20 pM) individually or in combination, and processed for Annexin V-FITC staining as a read out for apoptosis and DAPI to stain cell nuclei as described in Methods. (C, D) Panc0403 and Panc0203 cells were treated for 48 hours with MRTX1133 (30 nM) and FGTI-2734 (20 pM) individually or in combination, and processed for flow cytometry analysis after staining with annexin V and propidium iodide as described in the Methods. The data are representative of 2 independentexperiments.Figures 3A-E. FGTI-2734 blocks MRTX1133-induced ERK adaptive reactivation in KRAS G12D pancreatic and colon cancer cells in vitro and in xenografts in vivo. KRAS G12D (A) pancreatic cancer Panc0403, (B) colon cancer LS174T, (C) pancreatic cancer Panc0203 and (D) pancreatic cancer HPAC cells were treated with vehicle, FTI-2734, MRTX1133 alone or in combination for 2, 24 and / or 48 hours and processed for Western blotting as described in Methods. (E) Panc0203 tumor xenografts from the anti -tumor studies of Fig. 4E& F were collected 2 and 24 hours after drug treatment of mice on the last day of treatment, lysed, and the lysates were subjected to Western blotting as described in Methods.Figures 4A-F. FGTI-2734 enhances the anti-tumor activity of MRTX1133 in an orthotopic PDX from a KRAS G12D pancreatic cancer patient and in Panc0403 and panc0203 xenografts. (A & B) The fresh biopsies derived from a KRAS G12D pancreatic cancer patient were processed and orthotopically implanted in NSG mice, and after the tumors were 100-150 mm3, mice were treated daily with vehicle, MRTX1133 (3 mpk, BID, IP), FGTI-2734 (100 mpk, once daily, oral), or the FGTI-2734 and MRTX1133 combination up to 28 days as described in Methods. (C & D) Panc0403 and (E & F) Panc0203 cells were subcutaneously implanted in nude mice, and when the average tumor volumes were 170-185 mm3, mice were treated daily with vehicle, MRTX1133 (BID, IP), FGTI-2734 (once daily / orally), or the combination of FGTI-2734 and MRTX1133 as describedin Methods. For Panc0403 xenograft mice, the MRTX1133 and FGTI-2734 doses over time were as follows. For MRTX1133: Day 0 to 16 (2 mpk) and day 17 to 21 (1.5 mpk). For FGTI-2734: day 0 to 7 (100 mpk), day 8 to 16 (150 mpk) and day 17 to 21 (200 mpk) FGTI-2734. For Panc0203 xenograft mice, the MRTX1133 and FGTI-2734 doses over time were as follows. For MRTX1133: Day 0 to 11 (1.5 mpk), day 12 to 26 (2 mpk) and day 27 to 39 (3 mpk); for FGTI-2734: Day 0 to 18 (100 mpk) and day 19 to 39 (150 mpk).Figures 5A-B. FGTI-2734 and MRTX1133 combination but not single-agent treatment causes tumor regression in an orthotopic PDX from a patient with KRAS G12D pancreatic cancer. Tumor biopsies from a KRAS G12D pancreatic cancer patient were prepared and implanted orthotopically into NSG mice, and the mice treated with either vehicle, MRTX1133 (3mpk, BID, IP), FGTI-2734 (100 mpk, once daily, oral), or the FGTI-2734 and MRTX1133 combination for 28 days as described in the Figure 4 legend. Tumors were monitored by ultrasound and tumor measurements continued until day 114 after implantation.Yellow encircled area designates tumor (T). K designates left kidney. Fig. 5A shows representative ultrasound images of larger tumors at the start of treatment (day 0) (171 mm3to 283 mm3), and Fig. 5B shows representative ultrasound images of smaller tumors at day 0 (86 mm3to 121 mm3). Regardless of the starting tumor size, the FGTI-2734 and MRTX1133 combination but not single-agent treatment causes tumor regression in this orthotopic PDX from a patient with KRAS G12D pancreatic cancer.Figures 6A-X. FGTI-2734 synergizes with the pan-RAS inhibitor RMC-6236 to inhibit viability of KRAS-mutant cancer cells. (A-F) Dose-response curves showing the effects of RMC-6236 alone and with increasing concentrations of FGTI-2734 on the viability of KRAS G12C lung (LU99, H358, H2122), KRAS G12D pancreatic (Panc0203, Panc0403), and KRAS G13D colon (HCT116) cancer cell lines following 72-hour treatment, and viability analyzed by CellTiter-Glo®. (G-L) Bar graph comparisons of selected single-agent doses of FGTI-2734 and RMC6236 versus the combination treatment in each cell line. Data are presented as mean ± SD with corresponding p-values. (M-R) SynergyFinder 3D synergy surface plots generated from matrix-format treatments (FGTI-2734: 0.03-100 pM; RMC-6236: 0.03-3000 nM). A synergy score >0 denotes synergy and a synergy score <0 denotes antagonism. Strong synergistic interactions were observed in all six cell lines. (S-X) Calcusyn Combination Index (CI) versus Fraction affected (Fa) plots for constant-ratio combinations of FGTI-2734 and RMC-6236. CI values <1 indicate synergy. All six KRAS-mutant lines displayed synergistic interactions across multiple Fa levels.Figures 7A-P. FGTI-2734 synergizes with the RMC-6236 analogue RMC-7977 in KRAS-mutant cancer cell lines. (A-D) Dose-response curves showing the effects of RMC-7977 alone and with increasing concentrations of FGTI-2734 on the viability of KRAS G12C lung (LU99, H2122) and KRAS G12D pancreatic (Panc0203, and Panc0403) cells following 72-hour treatment (cell viability analyzed by CellTiter-Glo®). (E-H) Bar graph comparisons of selected single-agent doses of FGTI-2734 and RMC6236 versus the combination treatment in each cell line. Data are presented as mean ± SD with corresponding p-values. (I-L) SynergyFinder 3D synergy surface plots derived from FGTI-2734 / RMC-7977 matrix treatments. Positive synergy scores indicate synergistic drug interactions, which were consistently observed across all four KRAS-mutant cell lines. (M-P) Calcusyn CI-Fa plots confirming robust synergy between FGTI-2734 and RMC-7977. CI values <1 were obtained in all four lines, indicating strong cooperative activity between the two agents.Figures 8A-I. FGTI-2734 enhances RMC-6236-induced apoptosis in KRAS-mutant pancreatic, lung, and colon cancer cell lines. Representative fluorescence microscopy images of LU99 (A), HCT116 (D), and Panc0403 (G) cells treated for 24 hours with FGTI-2734 (LU99: 10 pM; Panc0403 and HCT116: 20 pM) and RMC-6236 (LU99 and HCT116: 5 nM; Panc0403: 3 nM), alone or in combination. Cells were stained with Annexin V-FITC to detect apoptotic cells and DAPI to visualize nuclei. Flow cytometry analysis of LU99 (B), HCT116 (E), and Panc0403 (H) cells treated for 48 hours with FGTI-2734 and RMC-6236 as single agents or in combination. Cells were stained with Annexin V-FITC and propidium iodide (PI) as described in Example 2. Quadrants indicate the distribution of live, early apoptotic, late apoptotic, and necrotic cells. (C, F, I) Quantification of apoptotic cells (% early + late apoptosis) corresponding to the flow cytometry data.Figures 9A-D. Chemical structures of (A) FGTI-2734, (B) MRTX1133, (C) RMC-6236, and (D) RMC-7977.DETAILED DESCRIPTIONEmbodiments of the disclosure provide compositions and methods for a synergistic anticancer treatment comprising a combination of a dual farnesyltransferase and geranylgemayltransferase-1 inhibitor (FGTI) or a pharmaceutically acceptable salt thereof and a KRAS inhibitor or a pharmaceutically acceptable salt thereof.FGTIs are anticancer agents based on an ethylenediamine scaffold. Suitable FGTIs include FGTI-2734 or N-[2-[(4-cyano-2-fluorophenyl)[(1-methyl-1H-imidazol-5-yl)methyl]amino]ethyl]-N-(cyclohexylmethyl)-2-pyridinesulfonamide. FGTI inhibits membrane localization of WT and mutant KRAS, HRAS, and NRAS proteins regardless of mutation type. Alternative FGTIs that may be used in the context of the disclosure, including the synthetic routes, are described in US Patent Publication 2013 / 0190355 incorporated herein by reference.KRAS inhibitors block the activity of mutated KRAS proteins which drive cell growth in various cancers. KRAS inhibitors include pan RAS inhibitors which target multiple KRAS mutations and inhibitors that target specific mutations such as G12D, G12C, G13D, and G12V mutations. Suitable pan RAS inhibitors include, but are not limited to, RMC-6236, RMC-7977, Divarasib (GDC-6036, Roche / Genentech), JDQ443 (Novartis), LY3537982 (Eli Lilly), RMC-9805 / RMC-6291, MK-1084, ADT-007, MCB-294, and BI-2493. Other suitable inhibitors include KRAS G12D inhibitors such as MRTX1133. In some embodiments, the KRASinhibitor is not a KRAS G12C inhibitor, such as sotorasib, adagrasib, or garsorasib.Chemical structures of exemplary inhibitors as described herein are provided in Figures 9A-D.Embodiments provide methods for treating cancer by the administration of a combination as described herein. As used herein “treating” or “treatment” means any manner of managing the cancer by medicinal or other therapies, such that the cancer no longer increases in size, metastasizes, or otherwise progresses in severity on a diagnosis scale, such as Duke's classification or any other classification system known. In some embodiments, the treatment ameliorates the disease through a reduction in size or otherwise beneficially improves the severity on a diagnosis scale.As used herein, the term “cancer” refers to a neoplasm, cancer, or precancerous lesion. The neoplasm or cancer may be benign or malignant. This includes cells or tissues that have characteristics relating to changes that may lead to malignancy or cancer, such as mutations controlling cell growth and proliferation. Examples of cancers, e.g. solid tumors, to be treated include but are not limited to: lung cancer, including non-small cell lung cancer, colon cancer, colorectal cancer, pancreatic cancer, including pancreatic ductal adenocarcinoma, gall bladder cancer, thyroid cancer, bile duct cancer, breast cancer, urothelial cancer, head and neck cancer, esophagus cancer, thyroid cancer, oral cancer, cervical cancer, ovarian cancer, and liver cancer (e.g., hepatocellular carcinoma). Further examples include hematological malignancies (e.g., cancers that affect blood, bone marrow and / or lymph nodes). Such malignancies include, but are not limited to leukemias and lymphomas. For example, the presently disclosed compounds can be used for treatment of diseases such as Acute lymphoblastic leukemia (ALL), Acute myelogenous leukemia (AML), Chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Chronic myelogenous leukemia (CML), Acute monocytic leukemia (AMoL) and / or other leukemias. In other embodiments, the compounds are useful for treatment of lymphomas such as all subtypes of Hodgkins lymphoma or non-Hodgkins lymphoma. In various embodiments, the compounds are useful for treatment of plasma cell malignancies such as multiple myeloma, mantle cell lymphoma, and Waldenstrom's macroglubunemia.In some embodiments, the cancer is mediated or characterized by a KRAS, HRAS, or NRAS mutation, such as a G12D, G12C, G13D, Q61L, or G12V KRAS, HRAS or NRAS mutation. In some embodiments the disclosure provides a method of treating cancer, whereinthe method comprises determining if the subject has a KRAS, HR AS or NRAS mutation and if the subject is determined to have the KRAS, HRAS or NRAS mutation, then administering to the subject a therapeutically effective dose of a combination therapy as disclosed herein.Determining whether a tumor or cancer comprises a KRAS, HRAS or NRAS mutation can be undertaken by assessing the nucleotide sequence encoding the KRAS, HRAS or NRAS protein, by assessing the amino acid sequence of the KRAS, HRAS or NRAS protein, or by assessing the characteristics of a putative KRAS, HRAS or NRAS mutant protein. The sequence of wild-type human KRAS, HRAS or NRAS is known in the art, (e.g. Accession No. NP203524).Methods for detecting a mutation in a KRAS, HRAS or NRAS nucleotide sequence are known by those of skill in the art. These methods include, but are not limited to, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) assays, real-time PCR assays, PCR sequencing, mutant allele-specific PCR amplification (MASA) assays, direct sequencing, primer extension reactions, electrophoresis, oligonucleotide ligation assays, hybridization assays, TaqMan assays, SNP genotyping assays, high resolution melting assays and microarray analyses. In some embodiments, samples are evaluated for KRAS, HRAS or NRAS mutations by real-time PCR. In real-time PCR, fluorescent probes specific for the KRAS, HRAS or NRAS mutation are used. When a mutation is present, the probe binds and fluorescence is detected. In some embodiments, the KRAS, HRAS or NRAS mutation is identified using a direct sequencing method of specific regions (e.g., exon 2 and / or exon 3) in the KRAS, HRAS or NRAS gene. This technique will identify all possible mutations in the region sequenced.Methods for detecting a mutation in a KRAS, HRAS or NRAS protein are known by those of skill in the art. These methods include, but are not limited to, detection of a KRAS, HRAS or NRAS mutant using a binding agent (e.g., an antibody) specific for the mutant protein, protein electrophoresis and Western blotting, and direct peptide sequencing.Methods for determining whether a tumor or cancer comprises a KRAS, HRAS or NRAS mutation can use a variety of samples. In some embodiments, the sample is taken from a subject having a tumor or cancer. In some embodiments, the sample is a fresh tumor / cancer sample. In some embodiments, the sample is a frozen tumor / cancer sample. In some embodiments, the sample is a formalin-fixed paraffin-embedded sample. In someembodiments, the sample is a circulating tumor cell (CTC) sample. In some embodiments, the sample is processed to a cell lysate. In some embodiments, the sample is processed to DNA or RNA.In some embodiments, the cancer is resistant to treatment with a KRAS inhibitor or FGTI treatment alone. In some embodiments, the subject has previously been administered a KRAS inhibitor or FGTI. In some embodiments, the subject has not previously been administered a KRAS inhibitor or FGTI.As described in the Examples, an FGTI synergistically enhances the anti-tumor activity of KRAS inhibitors. In some embodiments, the synergistic anti-tumor activity comprises increased Annexin V positivity, enhanced activation of caspases, and / or increased PARP cleavage relative to single-agent treatment.In some embodiments, the FGTI or pharmaceutically acceptable salt thereof and the KRAS inhibitor or pharmaceutically acceptable salt thereof are administered simultaneously. In some embodiments, the FGTI or pharmaceutically acceptable salt thereof is administered prior to the KRAS inhibitor or pharmaceutically acceptable salt thereof.The anti-cancer agents described herein may be administered in vivo by any suitable route (e.g. parenterally or enterally) including but not limited to: inoculation or injection (e.g. intravenous, intraperitoneal, intramuscular, subcutaneous, intra-aural, intraarticular, intramammary, and the like), topical application, and by absorption through epithelial or mucocutaneous linings (e.g., nasal, oral, vaginal, rectal, gastrointestinal mucosa, and the like). Other suitable means include but are not limited to: inhalation (e.g. as a mist or spray), orally (e.g. as a pill, capsule, liquid, etc.), intravaginally, intranasally, rectally, by ingestion of a food or probiotic product containing the compound, as eye drops, etc. In preferred embodiments, the mode of administration is oral or by injection. The anti-cancer agents described herein may be administered simultaneously or sequentially. The present disclosure also provides a method of treatment comprising administering to a subject a formulation as described herein, with or without an additional biological active agent or anti-cancer agent, e.g. an immunotherapy agent, chemotherapeutic agent, anti-angiogenesis agent, signal transduction inhibitor, antiproliferative agent, glycolysis inhibitor, or autophagy inhibitor. In some embodiments, the additional therapeutic agent is selected from an anti-PD-1 antibody, a chemotherapeutic agent, a MEK inhibitor, an EGFR inhibitor, a TOR inhibitor, a SHP2 inhibitor, PI3K inhibitor, and an AKT inhibitor. In some embodiments, the treatmentdescribed herein is administered with or without radiation therapy.A patient or subject to be treated by any of the compositions or methods of the present disclosure can mean either a human or a non-human animal including, but not limited to mammals, dogs, horses, cats, rabbits, gerbils, hamsters, rodents, birds, aquatic mammals, cattle, pigs, camelids, and other zoological animals.In some embodiments, the formulation or active agent is administered to the subject in a therapeutically effective amount. By a "therapeutically effective amount" or an “effective amount” is meant a sufficient amount to treat the disease or disorder at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder: activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific active agent employed; and like factors well known in the medical arts. In the case of cancer, the effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and / or recurrence of tumor; and / or (vii) relieve to some extent one or more of the symptoms associated with the cancer. It is well within the skill of the art to start doses of the compound at levels or frequencies lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage or frequency until the desired effect is achieved. However, the daily dosage of the active agent may be varied over a wide range from 1 to 3000 mg per adult per day. In particular, the compositions contain at least or up to 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, or 3000 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 3000 mg of the active ingredient, in particular from 100 mg to about 3000 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level at least or up to 1 mg / kg to 100mg / kg of body weight per day, e.g. about 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg, or 100 mg / kg of body weight per day. Such doses may be administered in a single dose or it may be divided into multiple doses.Further embodiments provide a method of inhibiting tumor cell viability of or inducing apoptosis in KRAS-mutant cancer cells in vitro or in vivo comprising the step of exposing the cancer cells to a combination therapy as described herein.Embodiments of the disclosure also provide compositions comprising the anti-cancer agents described herein. For example, the FGTI and KRAS inhibitor, or the pharmaceutically acceptable salts thereof, may be present together in a single dosage form. In some embodiments, the single dosage form is selected from the group consisting of a tablet, dragee, liquid, drop, capsule, caplet and gelcap.Embodiments of the disclosure further provide combining separate pharmaceutical compositions in kit form. The kit comprises two separate pharmaceutical compositions: FGTI and KRAS inhibitor. The kit comprises a container for containing the separate compositions such as a divided bottle or a divided foil packet. Additional examples of containers include syringes, boxes, and bags. In some embodiments, the kit comprises directions for the use of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing health care professional.An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process recesses are formed in the plastic foil. The recesses have the size and shape of the tablets or capsules to be packed. Next, the tablets or capsules are placed in the recesses and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil which is opposite from the direction in which the recesses were formed. As a result, the tablets or capsules are sealed in the recesses between the plastic foil and the sheet. Preferably the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formedin the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.The pharmaceutical compositions can be formulated according to known methods for preparing pharmaceutically useful compositions. The active ingredients may be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients. Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, e.g. a human, as appropriate. As used herein, the phrase “pharmaceutically acceptable carrier’’ means any of the standard pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier can include diluents, adjuvants, and vehicles, as well as implant carriers, and inert, non-toxic solid or liquid fillers, diluents, or encapsulating material that does not react with the active ingredients of the invention. Examples include, but are not limited to, phosphate buffered saline, physiological saline, water, and emulsions, such as oil / water emulsions. The carrier can be a solvent or dispersing medium containing, for example, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Formulations are described in a number of sources that are well known and readily available to those skilled in the art. For example, Remington’s Pharmaceutical Sciences (Martin E W

[1995] Easton Pa., Mack Publishing Company, 19thed.) describes formulations which can be used in connection with the subject invention. The final amount of the compounds in the formulations may vary. However, in general, the amount in the formulations will be from about 0.01-99%, weight / volume.Compositions as described herein may be prepared either as liquid solutions or suspensions, or as solid forms such as tablets, pills, granules, capsules, powders, ampoules, and the like. The liquid may be an aqueous liquid. Solid forms suitable for solution in, or suspension in, liquids prior to administration may also be prepared.Formulations suitable for parenteral administration include, for example, aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, priorto use. Extemporaneous injection solutions and suspensions may be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of the subject invention can include other agents conventional in the art having regard to the type of formulation in question. The pharmaceutical composition can be adapted for various forms of administration. Administration can be continuous or at distinct intervals as can be determined by a person skilled in the art.The compositions of the present disclosure may also contain other components such as, but not limited to, additives, adjuvants, buffers, tonicity agents, bioadhesive polymers, and preservatives. In any of the compositions of this disclosure, the mixtures are preferably formulated at about pH 5 to about pH 8. This pH range may be achieved by the addition of buffers to the composition. It should be appreciated that the compositions of the present disclosure may be buffered by any common buffer system such as phosphate, borate, acetate, citrate, carbonate and borate-polyol complexes, with the pH and osmolality adjusted in accordance with well-known techniques to proper physiological values.An additive such as a sugar, a glycerol, and other sugar alcohols, can be included in the compositions of the present disclosure. Pharmaceutical additives can be added to increase the efficacy or potency of other ingredients in the composition. For example, a pharmaceutical additive can be added to a composition of the present disclosure to improve the stability of the bioactive agent, to adjust the osmolality of the composition, to adjust the viscosity of the composition, or for another reason, such as effecting drug delivery. Non-limiting examples of pharmaceutical additives of the present disclosure include sugars, such as, trehalose, mannose, D-galactose, and lactose.In an embodiment, if a preservative is desired, the compositions may optionally be preserved with any well-known system such as benzyl alcohol with / without EDTA, benzalkonium chloride, chlorhexidine, Cosmocil® CQ, or Dowicil 200.“Salts” or “pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds of the present disclosure. These salts can be prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Exemplary acid addition salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate (e.g. FGTI-2734 mesylate as used in Example 1), glucoheptonate, lactiobionate, sulfamates, malonates, salicylates, propionates, methylene-bis-.beta.-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates and laurylsulfonate salts, and the like. See, for example S. M. Berge, et al., " Pharmaceutical Salts," J. Pharm. Sci., 66, 1-19 (1977) which is incorporated herein by reference. Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. The sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like. Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use such as ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N, N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, and dicyclohexylamine, and the like.The compounds of the present disclosure may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water (hydrate), ethanol, and the like. A solvate is the result of solvation which is an interaction of a solute (i.e. compound of the disclosure) with a solvent. Solvation leads to stabilization of the solute species in the solution. A solvate refers to the solvated state, whereby an ion in a solution is surrounded or complexed by solvent molecules. Exemplary solvents include, but are not limited to,propylene glycol; polypropylene glycol; polyethylene glycol (for example, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 900, polyethylene glycol 540 (all available from Union Carbide) and the like); pharmaceutically acceptable alcohols (for example, ethanol or 2-(2-ethoxyethoxy)ethanol (Transcutol®, Gattefosse, Westwood, N. J. 07675) and the like); polyoxyethylene castor oil derivatives (for example, polyoxyethyleneglycerol triricinoleate or polyoxyl 35 castor oil (Cremophor®EL, BASF Corp.), polyoxyethyleneglycerol oxystearate (Cremophor®RH 40 (polyethyleneglycol 40 hydrogenated castor oil) or Cremophor®RH 60 (polyethyleneglycol 60 hydrogenated castor oil), BASF Corp.) and the like); fractionated coconut oil (for example, mixed triglycerides with caprylic acid and capric acid (Miglyol®812, available from Huis AG, Witten, Germany) and the like); Tween®80; isopropyl palmitate; isopropyl myristate; pharmaceutically acceptable silicon fluids; and the like.Before exemplary embodiments of the present invention are described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, 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, since the scope of the present invention will be limited only by the appended claims.Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individuallyindicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended, nor should they be interpreted to, limit the scope of the invention.EXAMPLE 1. FGTI-2734 Enhances MRTX1133 Anti-tumor Activity by Preventing ERK Adaptive Reactivation in KRAS G12D Pancreatic CancerThe KRAS G12D inhibitor MRTX1133 has shown promise as an anti-cancer drug; however, resistance mechanisms, particularly the adaptive reactivation of ERK requiring wild-type (WT) RAS membrane localization, pose a substantial challenge. In this study, we report that the dual farnesyltransferase (FT) and geranylgeranyltransferase-1 (GGT-1) inhibitor FGTI-2734, which inhibits WT RAS membrane localization, suppresses MRTX1133-induced ERK feedback reactivation, thereby overcoming adaptive resistance to MRTX1133. The combination of FGTI-2734 and MRTX1133 demonstrates synergistic effects in inhibiting viability and inducing apoptosis in KRAS G12D pancreatic and coloncancer cell lines. In vivo, FGTI-2734 enhances the anti-tumor activity of MRTX1133, leading to significant tumor regression in orthotopic xenografts from a KRAS G12D pancreatic cancer patient who relapsed after radiation and chemotherapy, as well as KRAS G12D human pancreatic cancer xenografts. Notably, treatment of mice with FGTI-2734 inhibited MRTX1133-induced ERK reactivation in KRAS G12D xenografts. The comprehensive and robust synergy between FGTI-2734 and MRTX1133 suggests that this combination provides a treatment option for both early-stage and advanced metastatic pancreatic cancers, including those resistant to therapy.METHODSCells lines, cell culture, and reagentsKRAS G12D human pancreatic cancer cell lines Panc0203, Panc0403, HPAC and the KRAS G12D human colon cancer cell line CT174T were obtained from American Type Culture Collection (ATCC). Except HPAC, all other cell lines were cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, USA) and HPAC cells were cultured in DMEM media (Gibco, Thermo Fisher Scientific, USA). Both culture media were supplemented with 10% heat -inactivated fetal bovine serum (FBS) (R& D Systems, USA), 1% penicillin¬ streptomycin solution (Sigma, USA). All cell lines were mycoplasma-free, monitored regularly using InvivoGen (cat# rep-pt1) HEK-blue2 cells Mycoplasma Detection Kit. The authentication of cell lines was done by the University of Arizona Genetics Core facility. FGTI-2734 mesylate was synthesized by WuXi as described previously37,48. MRTX1133 (Cat# CT-MRTX1133) was purchased from ChemieTek, Indianapolis, IN, USA.Cell viability assay and synergy analysisCell viability was measured using CellTiter-Glo® luminescent cell viability assay (Promega, Madison, WI, USA) as per manufacturer’s protocol. Cells were seeded at a density of 2,000 cells / well in 96-well plates and treated the next day in a matrix format with several concentrations of FGTI-2734 and MRTX1133. After 72 hours of incubation, the cell viability was determined by CellTiter-Glo® and IC50 values were determined using GraphPad Prism® software as described by us previously42. The online SynergyFinder tool was used to evaluate drug combination synergy scores and to plot corresponding 3D surface plots42. Moreover, we also used CalcuSyn software (Biosoft; Cambridge, UK) to determine synergy using combination index method as described by us43. Several concentrations of FGTI-2734 and MRTX1133 alone and in combination at a constant ratio were used to determine their effectson cell viability, which was entered into CalcuSyn to determine a CI value for each fraction affected as described by us previously43. The resulting values were used to construct a plot of CI values over a range of fractions affected (Fa-CI plot) as described by us43. Additive effects (CI=1), synergy (CI<1). and antagonism (CI>1).Western blot analysis for cell lines and tumor xenograftsAfter drug treatment, human KRAS G12D pancreatic and colon cancer cells were washed twice with cold phosphate -buffered saline (PBS), and lysed in mammalian protein extraction reagent (product no. 78501, Thermo Fisher Scientific, Rockford, IL, USA) supplemented with protease inhibitors (Product no. A32953, Thermo Fisher Scientific) (consisting of 2-mM phenylmethylsulfonyl fluoride, 2-mM Na3VO4, and 6.4-mg / mL p-nitrophenylphosphate) and Phosphatase inhibitor (Product no: A32963 Thermo Fisher Scientific) as described by us4243. Cellular lysates were collected by centrifugation at 12,000g for 15 minutes, and the supernatants were collected as whole cell extracts. Protein concentration of each sample was determined using the BCA protein assay kit. Proteins were separated by S DS -PAGE and transferred to PVDF membranes, which were then blotted with antibodies specific for phospho-ERK (no. 4370S), ERK (no.8727S), cleaved-CASP-3 (no.9661S), cleaved-PARP (no. 9541 S), and Vinculin (no. 13901) from Cell Signaling (Danvers, MA, USA).Tumor tissue lysates from xenografts were generated by lysis of tumor in tissue protein extraction reagent (product no. 78510, Thermo Fisher Scientific) supplemented with protease inhibitors (product no. A32953, Thermo Fisher Scientific), consisting of 2 mM phenylmethylsulfonyl fluoride, 2 mM Na3VO4, and 6.4 mg / mL p-nitrophenylphosphate, and phosphatase inhibitor (Product no: A32963 Thermo Fisher Scientific) as described by us45. Tumor tissue homogenization was performed using automatic hand-operated OMNI-TIP homogenizer (Omni International, Inc. Kennesaw, GA, USA). Tumor homogenates were cleared by centrifugation at 12,000g for 15 minutes, and the supernatants were collected as tumor cell extracts. Proteins were separated by SDS-PAGE and transferred to PVDF membranes, which were then blotted with antibodies specific for phospho-ERK (no. 4370S), ERK (no.8727S), and Vinculin (no. 13901) from Cell Signaling (Danvers, MA, USA) as described by us45.Annexin V-FITC stainingCells (Panc0403, Panc0203) were cultured in RPMI 1640 or DMEM with 10% FBSand 100 U / mL of penicillin and 100 mg / mL of streptomycin, then seeded onto chamber slides and allowed to adhere overnight. The following day, cells were treated for 24 hours with either 0.1% DMSO (control), MRTX1133 (30 nM for Panc0203, Panc0403), FGTI-2734 (20 μM for Panc0203, Panc0403), or both drugs together. After treatment, cells were washed with PBS and stained with Annexin V-FITC [BD pharmingen cat # 556420] in 1X binding buffer (sterile 0.1M Hepes (pH 7.4), 1.4M NaCl, and 25 mM CaCl2 solution) for 20 minutes at room temperature, followed by a wash with IX binding buffer. Cells were then mounted with DAPI and apoptotic cells were identified by direct visualization of green-colored staining under a confocal microscope (LSM 880) using a 20x objective.Apoptosis by flow cytometryKRAS G12D cells (Panc0403, Panc0203) (1.4-1.7 × 106cells / mL) were treated with FGTI-2734, MRTX1133, or both, at the same concentrations used for F1TC staining described above. Cells were incubated for 48 hours at 37°C with 5% CO2. After incubation, media was collected from each sample followed by PBS wash, and collected pellet after trypsinization at low speed. Then cells were washed with PBS and resuspended in 100 pL of IX annexin binding buffer. They were then stained with 2.5-pL Annexin V-FITC and 2.5-pL propidium iodide, and incubated for 20 minutes at room temperature in the dark. After washing, cells were resuspended in 400 pL of Annexin binding buffer and analyzed in a FACS® tube. The data were analyzed and presented as percentages of total gated cells using the FlowJo® software.Anti-tumor studies of human tumor xenografts in miceFemale athymic nude mice (Crl: NU(NCr)-Foxnlnu) (6-7 weeks-old) were purchased from Charles River Laboratories, Wilmington, MA, USA) were maintained in our animal facility and treated in accordance with our Institutional Animal Care and Use Committee procedures and guidelines (IACUC Protocol Number AD10002149). Both Panc0403 and Panc0203 cells were grown, harvested, resuspended in 1: 1 Matrigel® matrix (Corning, Cat# 356237) and DPBS (Dulbecco’s PBS: Gibco) (Thermo Fisher Scientific, USA) at 5 million cells (Panc0203, and Panc0403) per 100 pL, and implanted subcutaneously on the right flank of each mouse. Tumor volumes were estimated using the formula: volume (v) = (L2W) / 2, where L = length (smallest measurement) and W = width (largest measurement) as described by us previously37. The animals were randomized to the following treatment groups once the tumors reached approximately 170-185 mm3: (1) vehicle control, (2) FGTI-2734, (3)MRTX1133, and (4) FGTI-2734 and MRTX1133 combination. The vehicle group received 30% PEG400 (Sigma-Aldrich (Millipore), USA, Cat#P3265) + 10% Kolliphor® EL (Sigma-Aldrich (Millipore), USA, Cat# C5135) + 60% MilliQ water. The above vehicle was used to dissolve FGTI-2734. MRTX1133 was dissolved in 10% Captisol® (w / v) (β-Cyclodextrin Sulfobutyl Ethers, Sodium Salts, CYDEX Pharmaceuticals, USA) in 50mM QB Citrate Buffer (pH5.0) (Cat # Q2443, Teknova, USA). Mice were treated with FGTI-2734 (once / day / orally) and MRTX1133 (BID / i.p. [intraperitoneal]) with the doses as described herein. Animal body weights were measured 2-3 per week to determine gross toxicity of drugs. All four different groups of mice did not show any evidence of gross toxicity (weight loss, decreased activity, decreased food intake). Per our IACUC Protocol (# AD 10002149) criteria, once mouse tumor reached 2000 mm3, the mice were euthanized.Anti-tumor efficacy study of orthotopically implanted PDX from a pancreatic cancer patientWith written consent we obtained fresh tumor biopsies from a 63-year-old male patient with pancreatic cancer (G160) who underwent a laparoscopic pancreaticoduodenectomy (VCU, IRB protocol HM20021072). The pathology report showed a 1.5 cm, poorly differentiated, ductal adenocarcinoma with lymphovascular and perineural invasion, 3 out of 24 involved lymph nodes, and negative surgical margins (R0). Pathologic staging was T1N1. KRAS mutation was G12D. Patient completed 5 cycles of 5-FU with radiotherapy over 5 weeks followed by 6 cycles of adjuvant gemcitabine with capecitabine over 5 months (capecitabine stopped after 2 cycles for rash). After 8 months of his last cycle of chemotherapy, evidence of recurrence was observed. The research was conducted according to International Ethical Guidelines for Biomedical Research Involving Human Subjects. The mice were housed, maintained, and treated, and all the experiments were performed in accordance with our Institutional Animal Care and Use Committee procedures, guidelines, and regulations (Animal IACUC protocol AD10002523). Upon pancreatic tumor resection, we obtained fresh 2-mm tumor pieces, kept on ice and taken to the animal surgery suite for subcutaneous implantation into NOD. Cg-Prkdcscid I12rgtm 1 Wjl / SzJ (NSG) mice. A viable tumor piece was placed in the right flank subcutaneous tissue of anesthetized mice and the skin was closed (generation 1). Once tumors reached the end point (1.5 cm in diameter), tumors were harvested and divided into 3-4- mm pieces. Orthotopic implantation was then performed in NSG mice as described by us previously (Go KL et al, 2017). Starting on day 38 after implantation, tumorswere followed by abdominal ultrasound once a week using the LOGIQ™ e NextGen Ultrasound machine (ANTECH Sound Imaging) as described by us previously45, and continued to follow once a week. On day 86 after tumor implantation when tumors reached approximately 160-240 mm3, the mice were randomized into four groups and started treatment as described above for the xenograft models. Tumor measurements continued for 4 weeks until the experiment was terminated on day 114 after implantation.RESULTS FGTI-2734 and MRTX1133 interact synergistically to inhibit the viability and induce apoptosis of KRAS G12D pancreatic and colon cancer cell lines. Human KRAS G12D pancreatic (Panc0403, Panc0203, HPAC) and colon (LS174T) cancer cells were treated with various concentrations of MRTX1133 for 72 hours and were processed for cell viability by CellTiter-Glo® (CTG) assay to determine their IC50 values as described by us42. MRTX1133 inhibited the viability of Panc0203, Panc0403, HPAC, and LS174T cells with 1C50 values of 117 nM, 163 nM, 20 nM, and 6 nM, respectively (Figs. 1 A, E, I, M). We then determined whether FGTI-2734 can sensitize these cell lines to MRTX1133, and found that as the concentration of FGTI-2734 increased, the sensitivity to MRTX1133 of the cancer cell lines increased proportionally (Figs. 1 A, E, I, M). We then determined whether the two drugs interact synergistically by treating the cells with FGTI-2734 and MRTX1133 alone or in combination in a constant ratio to one another, and used CalcuSyn software to generate Combination Index-Fraction affected (CI-Fa) plots as described by us43. Figures 1 B, F, J, N show that all the CI values are less than 1, demonstrating that the combination of FGTI-2734 and MRTX1133 is synergistic in all four cell lines (CI values of less than 1, equal to 1 or greater than 1 indicate synergy, additivity or antagonism, respectively). To further evaluate the FGTI-2734 and MRTX1133 synergistic interaction, we also treated the four cell lines in a matrix format with different concentrations each of FGTI-2734 and MRTX1133 alone or in combination, and used the tool SynergyFinder to calculate synergy scores and to plot the corresponding 3D response surface plots as described by us42. Synergy scores above 0 and those below 0 indicate synergy and antagonism, respectively. Consistent with the CI-Fa plots of Figs. 1 B, F, J, N, for most concentrations the two drugs were synergistic at inhibiting the viability of the 4 KRAS G12D pancreatic and colon cancer cell lines (Figs. 1 C, G, K, O) with average synergy scores between 6 and 17 (Fig. 1 D, H, L, P). Therefore, FGTI-2734 was able to sensitize all four cell lines to MRTX1133 irrespective of their MRTX1133 sensitivitylevels with HPAC and LS174T cells being more sensitive than Panc0203 and Panc0403 cells to MRTX1133. Importantly, the ability of FGTI-2734 to sensitize cells to MRTX1133 was highly selective for KRAS G12D cells as the H2122 lung cancer cells that harbor mutant KRAS G12C were resistant to MRTX1133 (as expected) and FGTI-2734 did not overcome this resistance and sensitize H2122 cells to MRTX1133 (data not shown). To determine whether FGTI-2734 can also enhance the ability of MRTX1133 to induce apoptosis, we exposed Panc0403 and Panc0203 cells for 24 hours to MRTX1133 and FGTI-2734 individually or in combination, and used Annexin V-FITC staining / DAPI nuclei staining44as a read-out for apoptosis. Single-agent treatments of Panc0403 and Panc0203 cells revealed little induction of apoptosis; however, combined treatment significantly increased apoptosis (Figs. 2A & 2C). To further confirm the ability of FGTI-2734 to enhance the ability of MRTX1133 to induce apoptosis, we exposed Panc0403 and Panc0203 cells for 48 hours to MRTX1133 and FGTI-2734 individually or in combination, and used FITC-Annexin V and Pl flow cytometry to quantify the percentage of Annexin V-FITC-positive apoptotic cells.Figure 2B shows that Panc0403 cells treated with vehicle DMSO, FGTI-2734, MRTX1133 and the combination contained 5%, 7%, 7%, and 32% apoptotic cells, respectively, further confirming the superior efficacy of the combination. Similarly, Panc0203 cells treated with vehicle, FGTI-2734, MRTX1133 and the combination contained 2%, 4%, 4%, and 16% apoptotic cells, respectively (Fig. 2D), indicating that the combination was more efficacious than either drug alone. These results coupled with the results of Figure 1 indicate that FGTI-2734 and MRTX1133 act synergistically at inhibiting viability and inducing apoptosis of KRAS G12D pancreatic cancer cells.FGTI-2734 prevents MRTX1133-induced adaptive reactivation of ERK and enhances MRTX1133-induced apoptosis in KRAS G12D pancreatic and colon cancer cells. Considering that MRTX1133 -induced ERK adaptive reactivation is a major resistance mechanism in KRAS G12D cancer cells and that ERK reactivation requires RAS membrane localization, which is inhibited by FGTI-2734 (see Introduction section and37), we reasoned that the ability of FGTI-2734 to significantly enhance MRTX1133 to inhibit cell viability and induce apoptosis could be due to FGTI-2734 preventing MRTX1133-induced ERK reactivation. To evaluate this possibility, Panc0403, Panc0203, HPAC, and LS174T cancer cells were treated with vehicle, MRTX1133, FGTI-2734 or the combination of MRTX1133 and FGTI-2734 for 2, 24 and / or 48 hours, lysed, and the lysates were subjected to Westernblotting as described by us37. In Panc0403 cells (Fig. 3A) and LS174T cells (Fig. 3B), MRTX1133 treatment suppressed the levels of P-ERK at 2 hours, but these levels rebounded at 24 and 48 hours suggesting that MRTX1133 induced an adaptive reactivation of ERK. Similarly, treatment with FGTI-2734 alone partially decreased P-ERK levels at 2 hours but not at 24 and 48 hours. While FGTI-2734 treatment alone had little effect on P-ERK levels after 24 and 48 hours of treatment, in combination with MRTX1133, it prevented MRTX1133-induced reactivation of ERK at 24 and 48 hours (Figs. 3A & 3B). Furthermore, the combination of MRTX1133 and FGTI-2734 was more effective than either drug alone at inducing apoptosis as determined by cleaved Caspase 3 and PARP (Figs. 3A & B). In Panc0203, both FGTI-2734 and MRTX 1133 suppressed the P-ERK levels at 2 hours (Fig.3C).However, in MRTX1133-treated Panc0203 cells, P-ERK levels progressively increased between 24 and 48 hours with a strong rebound at 48 hours. Furthermore, as was the case with Panc0403 and LS174T, while FGTI-2734 treatment alone had little effects on P-ERK levels after 24 and 48 hours of treatment, in combination with MRTX1133, it inhibited MRTX1133-induced reactivation of ERK at 24 and 48 hours, and enhanced the ability of MRTX1133 to induce apoptosis (Fig.3C). Similar results were observed with HPAC cells where MRTX1133 was able to inhibit P-ERK levels at two but not at 24 hours, and this rebound in P-ERK levels was inhibited by FGTI-2734, which was also paralleled by enhanced MRTX1133-induced apoptosis (Fig.2D). These results clearly demonstrate that the ability of MRTX1133 to induce an adaptive ERK reactivation was blocked by FGTI-2734 leading to enhanced ability of MRTX1133 to induce apoptosis suggesting that the synergistic interaction between MRTX1133 and FGTI-2734 to inhibit viability (Fig. 1) and induce apoptosis as determined by FITC-Annexin V imaging and flow cytometry (Fig. 2) and Caspase 3 and PARP (Fig. 3) in KRAS G12D cancer cells may be due to, at least in part, to the ability of FGTI-2734 to inhibit RAS membrane localization and the subsequent reactivation of ERK, a major contributor to MRTX1133 resistance.FGTI-2734 enhances the in vivo anti-tumor activity of MRTX1133 in orthotopic patient-derived xenografts from a KRAS G12D pancreatic cancer patient as well as Panc0203 and Panc0403 pancreatic tumor xenografts. To evaluate the ability of FGTI-2734 to enhance MRTX1133 anti-tumor activity in vivo, we used orthotopic patient-derived xenografts (PDXs) from a patient with KRAS G12D pancreatic cancer as described by us previously43. On day 86 after orthotopic tumor implantation, the mice were treated daily withvehicle, MRTX1133, FGTI-2734 or the combination with the doses described in Fig.4 legend. Tumors were followed via abdominal ultrasound once a week from day 86 (corresponding to day 0 when drug treatment started) until the experiment was terminated on day 114 after implantation (corresponding to day 28 after start of treatment) as described by us45. Figure 4 shows average (A) Tumor Volumes and (B) % Change in Tumor Volume of the vehicle, FGTI-2734, MRTX1133, and the combination groups. Overall, the data show that while the single-agent treatments resulted in only partial tumor growth inhibition, the combination treatment with FGTI-2734 and MRTX1133 caused significant tumor regression in the orthotopic KRAS G12D pancreatic cancer PDX model (Fig. 4 and Fig. 5). Specifically, the average tumor volume from the vehicle-treated mice grew from the first to the last day of treatment by 247% (Fig. 4B) from 141+29 mm3to 490+184 mm3(Fig. 4A). The average tumor volume from the MRTX1133-treated mice grew by only 69% from 114±23 mm3to 192 ± 86 mm3. The average tumor volume from the FGTI-2734-treated mice grew by 182% from 127+28 mm3to 358+187 mm3. In contrast, the combination treatment was much more effective than single- agent treatment, where the average tumor volume from the MRTX1133 + FGTI-2734 combination-treated mice actually regressed by 59% from 121+42 mm3to 50±20 mm3(Figs. 4A&4B). The combination treatment with FGTI-2734 and MRTX1133 showed a statistically significant difference compared to the single-agent MRTX1133 treatment on days 14, 21, and 28, with p values of 0.06, 0.02, and 0.02, respectively. Figure 5 shows ultrasound images of representative tumors captured at the start of treatment (day 0) and after 28 days of treatment. The orthotopic implantation produced tumors of varying volumes at day 0, with Fig.5A depicting representative ultrasound images of larger tumors at day 0 (171 mm3to 283 mm3), and Fig. 5B showing representative ultrasound images of smaller tumors at day 0 (86 mm3to 121 mm3). Regardless of the initial tumor size, Figures 5A& B show that the MRTX1133 and FGTI-2734 combination but not single-agent treatment causes tumor regression in this orthotopic PDX from a patient with KRAS G12D pancreatic cancer.For Panc0403 xenografts, the mice were treated with vehicle MRTX1133, FGTI-2734, or the combination with the doses described in the Fig. 4 legend. The average tumor volume of the mice treated with vehicle increased from day 0 to day 21 by 327% from 172 + / - 33 mm3to 734 + / - 48 mm3(Figs. 4C&4D). The average tumor volume from the MRTX1133 group increased 34% from 171 + / -25 mm3to 229 + / - 47 mm3.' The average tumor volume from theFGTI-2734 group increased by 225% from 173 + / - 31 mm3to 563 + / - 39 mm3. In contrast, the average tumor volume from the combination-treated mice did not increase and actually regressed by -48% from 171 + / - 26 mm3to 89 + / - 15 mm3(Figs. 4C&4D). Therefore, while MRTX1133 and FGTI-2734 alone inhibited only partially tumor growth, the combination treatment with the two drugs caused significant tumor growth regression. (Figs. 4C & 4D).The combination treatment with FGTI-2734 and MRTX1133 showed a highly statistically significant difference compared to the single-agent MRTX1133 treatment throughout the treatment period with p values ranging from 0.018 to 0.0006. For Panc0203 xenografts, the mice were treated with vehicle, MRTX1133, FGTI-2734 or the combination with the doses described in Fig. 4 legend. The average tumor volume of the mice treated with vehicle increased from day 0 to day 22 by 814% from 181 + / - 14 mm3to 1654+ / - 153 mm3(Figs.4E&4F) (vehicle-treated mice were euthanized on day 22 to comply with IACUC protocol tumor size limits). Over the same period of time (day 0 to day 22), average tumor volume from the FGTI-2734, MRTX1133 and the combination-treated mice increased by only 420%, 301%, and 133%, respectively, demonstrating that the combination is significantly more efficacious than single treatments (Figs. 4E&4F). To further evaluate whether the combination superior efficacy is maintained, we continued the anti-tumor efficacy study by treating the mice with the doses described in the Fig. 4 legend. As such, the average tumor volume from the FGTI-2734 group increased from day 0 to 30 days by 738% from 185 + / - 20 mm3to 1550 + / - 338 mm3(FGTI-2734-treated mice were sacrificed on day 30 to comply with IACUC protocol tumor size limits). Over the same period of time (day 0 to day 30), average tumor volume from the MRTX1133- and the combination-treated mice increased by only 348% and 145%, respectively, further confirming that the combination is significantly more efficacious than single treatments (Figs. 4E&4F). Finally, to determine whether the combination is more efficacious than single-agent MRTX1133, we continued treatment until day 39 as described in the Fig. 4 legend. The average tumor volume from the MRTX1133 group increased from day 0 to 39 days by 531% from 182 + / - 16 mm3to 1149 + / - 108 mm3. The average tumor volume from the combination-treated mice increased from day 0 to day 39 days by only 201% from 180 + / - 10 mm3to 542 + / - 77 mm3(Figs. 4E&4F), further demonstrating that over time FGTI-2734 significantly enhances the anti-tumor efficacy of MRTX1133. The combination treatment with FGTI-2734 and MRTX1133 showed a highly statistically significant difference compared to the single-agent MRTX1133 treatment startingon day 2 and continuing throughout the entire treatment period with p values ranging from 0.02 to 0.0001.FGTI-2734 suppresses MRTX1133-induced ERK adaptive reactivation in KRAS G12D pancreatic cancer xenografts in vivo. Next, we evaluated the ability of FGTI-2734 to block MRTX 1133 -induced ERK reactivation in KRAS G12D pancreatic cancer xenograft models in vivo. To this end, tumors from the Panc0203 xenograft studies (Fig. 4) were collected 2 and 24 hours after drug treatment of mice on the last day of treatment, lysed, and the lysates were subjected to Western blotting as described in Methods and previously by us37,45. Treatment of mice with MRTX1133 for 2 hours led to a decrease of the levels of P-ERK in the KRAS G12D Panc0203 tumor xenografts (Figs. 3 E & 3F). However, after 24 hours of MRTX1133 treatment, the levels of P-ERK strongly rebounded in two out of three mice (Figs. 3 E & 3F), consistent with a MRTX1133-induced adaptive reactivation of ERK, similar to the cell culture results (Figs. 3 A, B, C, D). As seen in cell culture, in combination with MRTX1133, FGTI-2734 inhibited the MRTX1133-induced reactivation of ERK at 24 hours. Taken together, our in vitro and in vivo results suggest that FGTI-2734 enhances MRTX1133 anti-tumor activity by inhibiting MRTX1133-induced adaptive reactivation of ERK.DISCUSSIONAlthough the discovery of KRAS inhibitors, such as the KRAS G12C binders sotorasib and adagrasib and the KRAS G12D binders such as MRTX1133, have the potential to transform the treatment landscape of KRAS G12C- and KRAS G12D-driven cancers, resistance remains a significant challenge21,27. Adaptive ERK reactivation through feedback mechanisms has emerged as a critical and common driver of resistance to inhibitors of the RTK-RAS-RAF-MEK-ERK signaling pathway, particularly KRAS G12C and KRAS G12D inhibitors6,34,46,47. Consistent with this, recent studies have shown that MRTX1133 treatment of KRAS G12D cancer cells initially suppressed P-ERK levels, but over time led to MRTX1133-induced feedback increases in WT HRAS-GTP and N-RAS-GTP binding, and subsequent ERK reactivation, ultimately resulting in adaptive resistance28— an outcome similarly observed in other studies across KRAS G12D pancreatic and colon cancer cell lines25,29-31. Here, we have developed an innovative approach to mitigate this ERK reactivation-driven resistance by using FGTI-2734 to prevent RAS proteins from localizing to the membrane37, where they must be to be activated and to subsequently mediate ERKactivation36. Our findings demonstrate that FGTI-2734 prevents MRTX1133 -induced ERK reactivation overcoming resistance and significantly enhancing MRTX1133 anti-tumor activity in KRAS G12D pancreatic cancer cells and causing regression of cancer tumor xenografts and orthotopic xenografts from a KRAS G12D pancreatic cancer patient who relapsed after radiation and chemotherapy. Several studies have shown that amplification / overexpression and activation of RTKs such as EGFR and HER2 are responsible for MRTX1133 resistance in in vitro and in vivo models25,27’31. This overexpression and activation of RTKs such as EGFR and HER2 also leads to activation of WT RAS, which leads to ERK reactivation, and as such FGTI-2734 is anticipated to overcome this resistance. Recent studies27have demonstrated that in KRAS G12D pancreatic cancer cells and organoids that MRTX1133 resistance is associated with activation of the PI3K-AKT-mTOR signaling pathway. Since RAS is known to activate this pathway it is plausible that FGTI-2734 has the potential to overcome PI3K-AKT-mTOR-driven resistance in clinical settings. Furthermore, MRTX1133-resistant tumors from MRTX1133-treated KRAS G12D / p53 KPC mice as well as cell lines with acquired resistance to MRTX1133 had a high- level amplification of Cdk627. Since RAS is known to activate cyclin DI which activates CDK4 / 6, it is plausible that FGTI-2734 may also overcome CDK6-driven resistance in pancreatic cancer.In summary, our novel combination therapeutic strategy disrupts compensatory signaling pathways that allow tumors to evade MRTX1133 monotherapy. Our discovery reveals that combining FGTI-2734 with MRTX1133 effectively prevents ERK reactivation and enhances MRTX1133’s therapeutic efficacy across a range of KRAS G12D pancreatic cancer models. This includes not only human pancreatic cancer cell lines and their in vivo xenografts but also an orthotopic PDX derived from a KRAS G12D pancreatic cancer patient who relapsed after chemotherapy and radiation, suggesting that this combination could provide a treatment option for both early-stage and advanced pancreatic cancer.EXAMPLE 2. Prenylation Blockade with FGTI-2734 Enhances the Activity of Pan-RAS inhibitors RMC-6236 and RMC-7977 in KRAS-Mutant CancersKRAS-driven cancers remain largely refractory to current targeted therapies, underscoring the need to identify drug combinations that prevent sustained tumor growth and therapeutic resistance. Here, we identify a potent and cooperative interaction between FGTI-2734 and the KRAS inhibitors RMC-6236 and RMC-7977 across multiple KRAS-mutant cancer models. Co-treatment with FGTI-2734 and either RMC compound resulted in markedly greater suppression of tumor cell viability than either agent alone, demonstrating a strong synergistic anti-proliferative effect. Mechanistically, the combination induced a robust apoptotic response, as evidenced by increased caspase 3 activation, PARP cleavage, and Annexin V positivity. These findings establish that FGTI-2734 significantly enhances the antitumor activity of both RMC-6236 and RMC-7977, defining a rational combination strategy for more effective targeting of KRAS-driven cancers.RESULTS FGTI-2734 synergizes with the pan-RAS inhibitor RMC-6236 to suppress the viability of KRAS G12C lung, KRAS G12D pancreatic and KRAS G13D colon cancer cells To evaluate whether FGTI-2734 cooperates with the pan-RAS inhibitor RMC-6236 to inhibit cancer cell viability, we treated KRAS-mutant human lung (KRAS G12C: LU99, H358, H2122), pancreatic (KRAS G12D: Panc0203, Panc0403), and colon (KRAS G13D: HCT116) cancer cell lines with increasing concentrations of each drug for 72 hours and assessed viability using the CellTiter-Glo® (CTG) assay. As single agent, RMC-6236 reduced the viability of LU99, H358, H2122, Panc0403, Panc0203, and HCT116 cells with IC50values of 10, 3, 60, 70, 10, and 20 nM, respectively (Figs. 6A-6F). FGTI-2734 alone inhibited viability in the same panel with IC50values of 9.4, 12.2, 18.8, 16.7, 13.5, and 11.11 μM, respectively. Across all six cell lines, irrespective of tumor origin or KRAS mutation isoform, FGTI-2734 markedly enhanced the ability of RMC-6236 to suppress cell viability (Figs. 6A-6F and Figs. 6G-6L). To quantify drug-drug synergistic interactions, we next performed matrix-format treatments using a range of FGTI-2734 (0.03-100 pM) and RMC-6236 (0.03— 3000 nM) concentrations alone or in combination for 72 hours. Synergy was assessed using the SynergyFinder tool, and corresponding 3D synergy surface plots were generated as previously described by us. Synergy scores >0 indicate synergy, whereas scores <0 indicate antagonism. Representative 3D plots demonstrated robust synergy between FGTI-2734 and RMC-6236 in all six KRAS-mutant cell lines (Figs.6M-6R). To further validate this synergy, each cell line was treated with FGTI-2734 and RMC-6236 at constant drug ratios, and Combination Index / Fraction affected (CI-Fa) plots were generated using Calcusyn software. CI values were <1, confirming that the FGTI-2734-RMC-6236 combination is synergistic in KRAS G12C lung, KRAS G12D pancreatic and KRAS G13D colon cancer cells (Figs. 6S-6X). (CI values <1 denote synergy, values =1 indicate additivity, and values >1 indicate antagonism).Similar results were obtained with RMC-7977, the preclinical analogue of the clinical candidate RMC-6236. LU99, H2122, Panc0203, and Panc0403 cells were treated with increasing concentrations of RMC-7977 and FGTI-2734, alone or in combination, for 72 hours. As with RMC-6236, FGTI-2734 significantly potentiated the ability of RMC-7977 to inhibit cell viability in all these KRAS-mutant lines (Figs. 7A-7D and Figs. 7E-7H). To quantify the interaction between the two agents, we applied both the SynergyFinder matrixbased analysis and the Calcusyn Combination Index approach. In each assay, FGTI-2734 and RMC-7977 displayed strong and consistent synergy across LU99, H2122, Panc0203, and Panc0403 cells, with positive synergy scores in SynergyFinder (Figs. 7I-7L) and CI values <1 in Calcusyn (Figs. 7M-7P), confirming robust cooperative efficacy of the FGTI-2734 / RMC-7977 combination.FGTI-2734 strongly enhances RMC-6236-induced apoptosisTo determine whether the combination of FGTI-2734 and RMC-6236 induces apoptosis, LU99, Panc0403, and HCT116 cells were stained with Annexin V-FITC to detect apoptotic cells and DAPI to visualize nuclei, followed by fluorescence microscopy. In all three cell lines, single-agent treatment with either FGTI-2734 or RMC-6236 produced only a modest increase in Annexin V-positive cells; however, the combination treatment led to a marked increase in apoptotic cells (Fig. 8A, D, G).We next quantified apoptosis using Annexin V-FITC and propidium iodide (PI) staining followed by flow cytometry after 48 hours of treatment. In LU99 cells, DMSO, FGTI-2734, and RMC-6236 produced early+late apoptosis rates of 2%, 3%, and 12%, respectively, whereas the combination increased apoptosis to 36% (Fig. 8B, C). In HCT116 cells, DMSO, FGTI-2734, and RMC-6236 induced apoptosis rates of 6%, 17%, and 27%, respectively, while the combination produced a substantial increase to 52% (Fig. 8E, F). In Panc0403 cells, apoptosis levels were 3% (DMSO), 4% (FGTI-2734), and 6% (RMC-6236) as single agents, increasing to 13% with the combination (Fig.8H, I). 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Journal of Medicinal Chemistry 53, 6867-6888 (2010).While the invention has been described in terms of its preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments as described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

Claims

1. CLAIMSWe claim:

1. A method of treating cancer in a subject in need thereof, wherein the cancer is mediated by a KRAS mutation, comprising administering to the subjecti) a therapeutically effective amount of a dual farnesyltransferase and geranylgernayltransferase- 1 inhibitor (FGTI) or a pharmaceutically acceptable salt thereof; andii) a therapeutically effective amount of a KRAS inhibitor or a pharmaceutically acceptable salt thereof,wherein administration of the FGTI and the KRAS inhibitor provides a synergistic enhancement of anti-tumor activity compared to administration of either agent alone.

2. The method of claim 1, wherein the cancer is selected from the group consisting of pancreatic cancer, lung cancer, colon cancer, and breast cancer.

3. The method of claim 1, wherein the KRAS mutation is selected from the group consisting of G12D, G12C, G13D, and G12V mutations.

4. The method of claim 1, wherein the KRAS inhibitor is a KRAS G12D inhibitor.

5. The method of claim 4, wherein the KRAS inhibitor is MRTX1133.

6. The method of claim 5, wherein the FGTI is FGTI-2734.

7. The method of claim 1, wherein the KRAS inhibitor is a pan RAS inhibitor.

8. The method of claim 7, wherein the KRAS inhibitor is RMC-6236 or RMC-7977.

9. The method of claim 8, wherein the FGTI is FGTI-2734.

10. The method of claim 1, wherein the KRAS inhibitor is not a KRAS G12C inhibitor.

11. The method of claim 1, wherein the cancer is resistant to treatment with the KRAS inhibitor alone.

12. The method of claim 1, wherein the synergistic anti-tumor activity comprises increased Annexin V positivity relative to single-agent treatment.

13. The method of claim 1, wherein the synergistic anti-tumor activity comprises enhanced activation of caspases relative to single-agent treatment.

14. The method of claim 1, wherein the synergistic anti-tumor activity comprises increased PARP cleavage relative to single-agent treatment.

15. 1'he method of claim 1, wherein the FGTI or pharmaceutically acceptable salt thereof and the KRAS inhibitor or pharmaceutically acceptable salt thereof are administered simultaneously.

16. The method of claim 1, wherein the FGTI or pharmaceutically acceptable salt thereof is administered prior to the KRAS inhibitor or pharmaceutically acceptable salt thereof.

17. A method of inhibiting tumor cell viability of or inducing apoptosis in KRAS-mutant cancer cells, comprising exposing the KRAS-mutant cancer cells toi) a dual famesyltransferase and geranylgemayltransferase- 1 inhibitor (FGTI) or a pharmaceutically acceptable salt thereof; andii) a KRAS inhibitor or a pharmaceutically acceptable salt thereof.

18. The method of claim 17, wherein the cancer cells are pancreatic cancer cells, lung cancer cells, colon cancer cells, or breast cancer cells.

19. The method of claim 17, wherein the cancer cells have a KRAS mutation selected from the group consisting of G12D, G12C, G13D, and G12V mutations.

20. A dosage composition comprising:a dual farnesyltransferase and geranylgernayltransferase-1 inhibitor (FGTI) or a pharmaceutically acceptable salt thereof, anda KRAS inhibitor or a pharmaceutically acceptable salt thereof,wherein the FGTI and KRAS inhibitor or pharmaceutically acceptable salts thereof are present together in a single dosage form.

21. The dosage composition of claim 20, wherein said single dosage form is selected from the group consisting of a tablet, dragee, liquid, drop, capsule, caplet, and gelcap.

22. A kit, comprising:a first dosage composition comprising a dual farnesyltransferase and geranylgemayltransferase-1 inhibitor (FGTI) or a pharmaceutically acceptable salt thereof; anda second dosage composition comprising a KRAS inhibitor or a pharmaceutically acceptable salt thereof.

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