COMBINATION THERAPY WITH DUAL FARNESYLTRANSFERASE AND GERANYLGERANYLTRANSFERASE-1 INHIBITOR AND SOTORASIB CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of United States provisional patent application 63/622,671, filed January 19, 2024, the contents of which are incorporated herein by reference. STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This 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 INVENTION The invention is generally related to a combination of FGTI-2734, a dual FT and GGT- 1 inhibitor, and sotorasib for the treatment of cancer. BACKGROUND OF THE INVENTION Lung cancer is a significant health concern in the United States with 238,340 new cases and 127,070 deaths in 2023,1 with non-small cell lung cancer (NSCLC) comprising approximately 85% of cases. Despite advances in diagnosis and treatment, the prognosis for NSCLC remains poor,2 emphasizing the urgent need for novel therapeutic strategies. Among the genetic alterations observed in NSCLC, mutations in KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) are key drivers of tumorigenesis. The three major RAS GTPase family members, KRAS, HRAS, and NRAS, function as molecular switches cycling between GDP (inactive) and GTP (active) states.3 This binary switch is regulated by GTPase- activating proteins (GAPs) that accelerate the intrinsic GTPase activity of RAS leading to inactive RAS-GDP state and guanine-nucleotide-exchange factors (GEFs) that promote formation of the active RAS-GTP state.4 RAS proteins are signal transducers that transfer biological information from external stimuli, such as growth factors, to multiple signaling pathways, such as Raf/Mek/Erk, PI3K/Akt, and RalGDS/Ral, that regulate gene expression, cell division, differentiation, and survival.5, 6 In human cancers, RAS often harbors mutations that lower RAS affinity for GAPs, thus reducing its intrinsic GTPase activity leading to
persistently active GTP-bound RAS. This constitutive activation of RAS triggers pleiotropic oncogenic events, including uncontrolled proliferation, resistance to apoptosis, sustained angiogenesis, immune evasion, invasion, metastasis, and drug resistance.6-9 KRAS is the most frequently mutated RAS isoform, followed by NRAS and HRAS. KRAS mutations are prevalent among the deadliest cancers with 90% in pancreatic ductal adenocarcinoma (PDAC), 40% in colorectal cancer (CRC), and 30% in NSCLC.7, 8 Whereas, KRAS G12D and G12V mutations are common in patients with PDAC and CRC, KRAS G12C mutations are more common in patients with NSCLC with a frequency of about 11-13%.10-12 Recent breakthroughs have led to the FDA approval of the KRAS G12C inhibitors sotorasib and adagrasib for the treatment of advanced KRAS G12C NSCLC in patients who have undergone prior systemic therapy.13-19 However, while sotorasib and adagrasib demonstrate disease control rates and clinical benefit in patients with advanced KRAS G12C NSCLC, the overall response rate (ORR) remains modest (37-43%), with limited and transient responses as evidenced by a progression-free survival (PFS) of 6.8-6.9 months.16, 17, 20 Furthermore, whether sotorasib as a single agent provides survival benefit to patients remains to be demonstrated.21 Together, these observations suggest the existence of additional genetic and genomic factors contributing to treatment resistance.14-20, 22-25 Clinically-relevant resistance mechanisms observed in tumors from patients treated with sotorasib or adagrasib are multifaceted and include amplification/overexpression of wild- type (WT) KRAS and WT receptor tyrosine kinases (RTKs) (e.g., MET, EGFR, HER2, FGFR2), RTK mutations (e.g., EGFR-A289V and RET-M918T), acquired secondary KRAS oncogenic mutations (e.g., G12D, G12V, G12R, G12W, G13D, Q61H), and acquired secondary mutations within the sotorasib switch II binding site in KRAS (e.g., R68S, Y96D, H95D, H95Q, H95R) 14-20, 22-25. Therapies that can enhance the efficacy of sotorasib treatment are needed. SUMMARY As described herein, FGTI-2734, a dual FT and GGT-1 inhibitor, overcomes sotorasib resistance by inhibiting RAS membrane localization and sotorasib-induced 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, comprising administering to the subject a therapeutically effective amount of
sotorasib or a pharmaceutically acceptable salt thereof and a therapeutically effective amount of FGTI-2734 or a pharmaceutically acceptable salt thereof. In some embodiments, the cancer is mediated by a KRAS G12C mutation. In some embodiments, the cancer is selected from lung, colorectal, or pancreatic cancer. In some embodiments, the cancer is resistant to sotorasib treatment alone. In some embodiments, the subject has previously been administered sotorasib. In some embodiments, the subject has not previously been administered sotorasib. The sotorasib or a pharmaceutically acceptable salt thereof and the FGTI-2734 or a pharmaceutically acceptable salt thereof may be administered simultaneously or sequentially. Another aspect of the disclosure provides a dosage composition comprising sotorasib or a pharmaceutically acceptable salt thereof; and FGTI-2734 or a pharmaceutically acceptable salt thereof, wherein said sotorasib and FGTI-2734 or pharmaceutically acceptable salts 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 sotorasib or a pharmaceutically acceptable salt thereof and a second dosage composition comprising FGTI-2734 or a pharmaceutically acceptable salt thereof. BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-P. FGTI-2734 and sotorasib are synergistic at inhibiting the viability of sotorasib-resistant (Calu-1 & LU99) and sotorasib-sensitive (H2122 & H358) human KRAS G12C lung cancer cells. (A) Calu1, (B) LU99, (C) H2122, and (D) H358 cells were treated with various concentrations of either sotorasib or FGTI-2734 for 72 hours and the cells were processed for viability. For synergy studies in (E) Calu1, (F) LU99, (G) H2122, and (H) H358 cells using the combination index method, the cells were treated with FGTI- 2734 and sotorasib 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 (the crosses are data points). For each cell line, the data is representative of 3 experiments. For the SynergyFinder method in (I) Calu1, (J) LU99, (K) H2122, and (L) H358 cells, the cells were treated in a matrix format with various concentrations each of FGTI-2734 and sotorasib alone or in combination, and SynergyFinder was used to calculate synergy scores and to plot the corresponding 3D response surface plots. Loewe and HSA scores in (M) Calu1, (N) LU99,
(O) H2122, and (P) H358 cells were calculated using the SynergyFinder tool. For each cell line, the data are representative of 4 to 7 experiments. SynergyFinder p values ranged from p = 1.04e-03 to p = 1.29e-10. Figures 2A-G. FGTI-2734 inhibits KRAS, HRAS and NRAS prenylation and enhances the ability of sotorasib to induce apoptosis in KRAS G12C lung cancer LU99 and Calu1 cells. (A) LU99 and (B) Calu1 cells were treated for 24 and 48 hours with sotorasib (3 µM) and FGTI-2734 (20 µM) individually or in combination, and processed for Western blotting. P and U designate Prenylated and Un-prenylated, respectively. * designates KRAS G12C covalently modified with sotorasib. (C) Chemical structure of FGTI-2734. LU99 cells (D - vehicle) were treated (24 hours) with (E) sotorasib (3 µM) or (F) FGTI-2734 (20 µM) alone or (G) in combination and processed for Annexin V-FITC staining as a read out for apoptosis and DAPI to stain cell nuclei. Figures 3A-D. FGTI-2734 overcomes resistance and enhances the anti-tumor activity of sotorasib in resistant and sensitive KRAS G12C lung cancer xenografts and in a PDX derived from a KRAS G12C lung cancer patient. (A) LU99, (B) H358 and (C) H2122 cells were subcutaneously implanted in nude mice, and after the tumors were palpable, mice were orally treated daily with vehicle (n=4 [LU99], n=4 [H358], n=5 [H2122]), sotorasib (n=4 [LU99], n=4 [H358], n=5 [H2122]), FGTI-2734 (n=4 [LU99], n=4 [H358], n=5 [H2122]), or the combination (n=4 [LU99], n=4 [H358], n=5 [H2122]). High dose sotorasib (30 mpk) was used for sotorasib-resistant LU99 xenografts whereas 5 mpk sotorasib was used for the sensitive H358 and H2122 xenografts. FGTI-2734 was used at 100 mpk for all 4 xenografts. (D) The fresh biopsies derived from a KRAS G12C lung cancer patient were processed and subcutaneously implanted in NSG mice, and after the tumors were palpable, mice were orally treated daily with vehicle (n=5), sotorasib (n=5), FGTI-2734 (n=4), or the combination (n=5). The sotorasib and FGTI-2734 doses varied over time and were as follows: Day 0 to day 14 (sotorasib [30 mpk] and FGTI-2734 [100 mpk]); Day 15 to day 22 (sotorasib [15 mpk] and FGTI-2734 [150 mpk]); Day 23 to day 36 (mice were not treated with any drugs); Day 37 to day 47 (sotorasib [15 mpk] and FGTI-2734 [150 mpk]). Statistical analysis: Using repeated measures ANOVA (which combines all time points) followed by post hoc multiple pairwise t-tests with multiplicity adjustments via the FDR, it was observed that the group treated solely with FGTI-2734 significantly differed from the combination treatment group (FGTI-2734 + sotorasib) across all 3 xenografts (LU99, H358 and H2122) and the PDX, with FDR-adjusted
p-values < 0.05. Similarly, the group treated solely with sotorasib exhibited significant differences compared to the combination treatment group (FGTI-2734 + sotorasib) for all 3 xenografts (LU99, H358 and H2122) and the PDX, with FDR-adjusted p-values < 0.05. The bars represent standard errors. Figures 4A-C. FGTI-2734 blocks sotorasib-induced ERK adaptive reactivation in KRAS G12C lung cancer cells in vitro and in xenografts in vivo. (A) the KRAS G12C lung cancer Calu1 cells were treated with vehicle, FTI-2734, sotorasib alone or in combination for 2, 24, 48, and 72 hours and the cells were processed for Western blotting. (B) The tumors from the above PDX anti-tumor studies (Fig.3D) were collected 2 and 24 hours after drug treatment of mice on the last day of the study, lysed, and the lysates were subjected to Western blotting. (C) The tumors from the LU99 and H358 xenograft studies (Figs.3A & 3B) were collected 2 hours after drug treatment of mice on the last day of the study, lysed, and the lysates were subjected to Western blotting. Figure 5. Chemical structure of sotorasib. DETAILED DESCRIPTION Embodiments of the disclosure provide compositions and methods for a synergistic anti- cancer treatment comprising a combination of FGTI-2734 or a pharmaceutically acceptable salt thereof and sotorasib or a pharmaceutically acceptable salt thereof. FGTI-2734 or N-[2-[(4-cyano-2-fluorophenyl)[(1-methyl-1H-imidazol-5-yl)methyl] amino]ethyl]-N-(cyclohexylmethyl)-2-pyridinesulfonamide (chemical structure shown in Fig. 2C) is a dual farnesyltransferase (FT) and geranylgeranyltransferase-1 (GGT-1) inhibitor that inhibits membrane localization of WT and mutant KRAS, HRAS, and NRAS proteins regardless of mutation type. FGTI-2734 and other FGTIs that alternatively 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. Sotorasib or 6-fluoro-7-(2-fluoro-6-hydroxyphenyl)-(1M)-1-[4-methyl-2-(propan-2- yl)pyridin-3-yl]-4-[(2S)-2- methyl-4-(prop-2 enoyl)piperazin-1-yl]pyrido[2,3-d]pyrimidin- 2(1H)-one is an FDA-approved KRAS G12C inhibitor sold under the brand name LUMAKRAS® (chemical structure shown in Figure 5). Sotorasib selectively forms an irreversible covalent bond to the sulfur atom in the cysteine residue that is present in the mutated form of KRAS, but not in the normal form. It is contemplated that other KRAS G12C inhibitors
may alternatively be used in the context of the disclosure such as adagrasib, garsorasib, etc. 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, 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 G12C KRAS, HRAS or NRAS mutation. In some embodiments the disclosure provides a method of treating cancer, wherein the method comprises determining if the subject has a KRAS, HRAS or NRAS G12C mutation and if the subject is determined to have the KRAS, HRAS or NRAS G12C 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 G12C 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 G12C KRAS, HRAS or NRAS mutations by real-time PCR. In real-time PCR, fluorescent probes specific for the KRAS, HRAS or NRAS G12C mutation are used. When a mutation is present, the probe binds and fluorescence is detected. In some embodiments, the KRAS, HRAS or NRAS G12C 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 G12C 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 some embodiments, 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 sotorasib (or other KRAS G12C inhibitor) treatment alone. In some embodiments, the subject has previously been administered sotorasib or another KRAS G12C inhibitor. In some embodiments, the subject has not previously been administered sotorasib or another KRAS G12C inhibitor. As described in the Example, FGTI-2734 enhances the anti-tumor activity of sotorasib in highly resistant and sensitive KRAS G12C cancer cells that have not previously been exposed to sotorasib, indicating that FGTI-2734 overcomes sotorasib intrinsic resistance. However, it is also contemplated that FGTI-2734 also overcomes sotorasib acquired resistance. Thus, FGTI- 2734 may enhance sotorasib anti-tumor efficacy in sotorasib-naïve patients, in addition to those who progressed while on sotorasib. 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 treatment described 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 1,500 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, or 1500 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 1500 mg of the active ingredient, in particular from 1 mg to about 250 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 100 mg/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 the viability of or inducing
apoptosis in sotorasib-resistant or sotorasib-sensitive lung cancer cells in vitro or in vivo comprising the step of exposing the lung 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, FGTI-2734 (or other FGTI) and sotorasib (or other KRAS G12C 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- 2734 (or other FGTI) and sotorasib (or other KRAS G12C 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 formed in 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, 19th ed.) 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, prior to 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, 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 Huls 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 individually indicated 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 KRAS G12C targeted therapies, such as sotorasib, represent a major breakthrough, but overall response rates and progression-free survival for patients with KRAS G12C lung cancer are modest due to the emergence of resistance mechanisms involving adaptive reactivation of ERK, which requires wild-type (WT) HRAS and NRAS membrane localization. Here, we demonstrate that the dual farnesyltransferase (FT) and geranylgeranyltransferase-1 (GGT-1) inhibitor FGTI-2734 inhibits WT RAS membrane localization and sotorasib-induced ERK feedback reactivation, and overcomes sotorasib adaptive resistance. The combination of FGTI-2734 and sotorasib is synergistic at inhibiting the viability and inducing apoptosis of KRAS G12C lung cancer cells, including those highly resistant to sotorasib. FGTI-2734 enhances sotorasib’s anti-tumor activity in vivo leading to significant tumor regression of a patient-derived xenograft (PDX) from a patient with KRAS G12C lung cancer as well as several xenografts from highly sotorasib-resistant KRAS G12C human lung cancer cells. Importantly, treatment of mice with FGTI-2734 inhibited sotorasib-induced ERK reactivation
in KRAS G12C PDX, and treatment of mice with the combination of FGTI-2734 and sotorasib were also significantly more effective at suppressing in vivo the levels of P-ERK in sotorasib- resistant human KRAS G12C lung cancer xenografts as well as a PDX. Our findings provide a foundation for overcoming sotorasib resistance and improving the treatment outcomes of KRAS G12C lung cancer. Materials and Methods Cells lines, cell culture, and reagents Human KRAS G12C lung cancer cell lines H358, H2122, H2030, HOP62, HCC1171, and Calu1 were obtained from American Type Culture Collection (ATCC). LU65 and LU99 cell lines were purchased from JCRB Cell Bank, Japan. All cell lines were cultured in RPMI- 1640 medium (Gibco, Thermo Fisher Scientific, USA). Culture media was supplemented with 10% heat-inactivated fetal bovine serum (FBS) (R&D Systems, USA), 1% penicillin- streptomycin (Sigma, USA). All cell lines were mycoplasma-free, monitored regularly with HEK-blue2 cells and Mycoplasma Detection Kit from InvivoGen (cat# rep-pt1). All cell lines were authenticated by University of Arizona Genetics Core. FGTI-2734 mesylate was synthesized as described previously.28, 53 Sotorasib (Cat# CT-AMG510) was purchased from ChemieTek, Indianapolis, IN, USA. Cell viability assay and synergy analysis Cell viability was determined by CellTiter-Glo® luminescent cell viability assay (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Briefly, cells (2000 cells/well) were seeded in 96-well plates, allowed to adhere overnight, treated in a matrix format with several concentrations of FGTI-2734 and sotorasib alone or in combination for 72 hours and the cell viability was determined by CellTiter-Glo® as described by us previously.38 Data were normalized to percentage of control, and IC50 values were determined using GraphPad Prism® software. The tool SynergyFinder was used to calculate synergy scores and to plot the corresponding 3D response surface plots. In addition to the tool synergy finder, Calcusyn software (Biosoft; Cambridge, UK) was also used to determine synergy using combination index method as described previously.38 Briefly, FGTI-2734 and sotorasib were combined at a constant ratio determined by their respective IC50 values. The resulting CTG viability data from the combination along with the viability data from single drug treatments was entered into Calcusyn to determine a combination index value (CI) for each combination point, which quantitatively defines additivity (CI=1), synergy (CI<1), and antagonism (CI>1).
The resulting values were used to construct a plot of CI values over a range of fractions affected (Fa-CI plot) as described previously.39 Western blot analysis for cell lines and tumor xenografts Following drug treatment, human KRAS G12C lung 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 inhibitor cocktail (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 previously (37, 38). Lysates were cleared by centrifugation at 12,000g for 15 minutes, and the supernatants were collected as whole cell extracts. Protein concentrations were determined using the BCA protein assay kit. Proteins were separated by SDS-PAGE and transferred to PVDF membranes, which were then blotted with antibodies specific for HRAS (no. sc-53959), NRAS (no. sc-31), (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Other antibodies included phospho-ERK (no. 4370S), ERK (no.8727S), cleaved-CASP-3 (no. 9661S), cleaved-PARP (no. 9541S), and Vinculin (no. 13901) from Cell Signaling (Danvers, MA, USA) and anti-KRAS (no. 703345) from Invitrogen (Waltham, MA USA 02451) For xenografts, tumor tissue samples were lysed in tissue protein extraction reagent (product no. 78510, Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (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 previously.44 The automatic hand-operated OMNI-TIPTM homogenizer (Omni International, Inc. Kennesaw, GA, USA) was used to homogenize the tumor tissues. Tumor homogenates were cleared by centrifugation at 12,000g for 15 minutes, and the supernatants were collected as tumor cell extracts. Protein concentrations were determined using the BCA protein assay kit. 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 previously.44 Annexin V-FITC staining H2122, H358, Calu1, and LU99 cells were cultured in RPMI 1640 medium
supplemented with 10% FBS, and 100 U/mL of penicillin and 100 mg/mL of streptomycin. Cells were seeded in chamber slides and allowed to adhere overnight. The next day, the cells were treated for 24 hours either with 0.1% DMSO vehicle control, sotorasib, FGTI-2734 or the combination of sotorasib and FGTI-2734. The sotorasib concentration used for the sotorasib-sensitive H2122 and H358 cells was 0.1 µM and for the sotorasib-resistant LU99 and Calu1 cells was 3 µM sotorasib. The FGTI-2734 concentration used was 20 µM for all 4 cell lines. After 24 hours, the cells were washed with ice cold PBS and suspended in 100 µL of 1X binding buffer (sterile 0.1M Hepes (pH 7.4), 1.4M NaCl, and 25 mM CaCl2 solution) with 5 µL of Annexin V conjugated FITC dye [BD Pharmingen cat # 556420] and incubated for 20 min in the dark at RT. Then cells were washed with 1x binding buffer to remove excess dye and mounted with DAPI. Apoptotic cells were identified by direct visualization of green- colored staining under a fluorescent confocal microscopy by using 20x objective (Keyence BZ-X810 and LSM 700). Anti-tumor studies of human tumor xenografts in mice Six-week-old female athymic nude mice (Crl:NU(NCr)-Foxn1nu) were purchased from Charles River Laboratories, Wilmington, MA, USA and were maintained and treated in accordance with VCU’s Institutional Animal Care and Use Committee procedures and guidelines (IACUC Protocol Number AD10002149). Exponentially growing LU99, Calu1, H358 and H2122 cells were harvested via trypsinization, pelleted at 300g for 5 minutes, resuspended in sterile Dulbecco’s PBS (DPBS: Gibco) (Thermo Fisher Scientific, USA) at 10 × 106 cells (H358), 5 × 106 cells (LU99, Calu1, and H2122) per 100 μL, and injected into the right flank of each mouse. Tumor volume was calculated using the formula: volume (v) = (L2W)/2, where L = length (smallest measurement) and W = width (largest measurement) as described previously.28 When the tumors reached approximately 200 mm3, the animals were randomized to the following treatment groups: (1) vehicle control, (2) FGTI-2734, (3) sotorasib, and (4) FGTI-2734 and sotorasib 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; FGTI-2734 was dissolved in the above vehicle, and sotorasib was dissolved in 40% PEG300 (Sigma-Aldrich (Millipore), USA, Cat# 202371) + 10% DMSO (Sigma-Aldrich (Millipore), USA, Cat# 472301) + 1% Tween®- 80 (Sigma-Aldrich (Millipore), USA, Cat# P1754) + 49% sterile saline (0.9% NaCl). All mice were treated orally once daily via 100 μL with the doses as described herein. For all 4 groups,
animals showed no evidence of gross toxicity (weight loss, decreased activity, decreased food intake). Per the IACUC Protocol (# AD10002149) criteria, mice with tumors that reached 2000 mm3 were euthanized. Anti-tumor efficacy studies of a PDX of from a patient with KRAS G12C lung cancer Fresh tumor biopsies were obtained from a KRAS G12C lung cancer patient (VCU, IRB protocol HM20021072) with written consent from the patient. The research was conducted according to International Ethical Guidelines for Biomedical Research Involving Human Subjects. Fresh 2-mm tumor pieces were obtained from lung cancer resection and transported on ice to the animal surgery suite for subcutaneous implantation into NOD.Cg- Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. The mice were housed, maintained, and treated in accordance with VCU’s Institutional Animal Care and Use Committee procedures and guidelines (Animal IACUC protocol AD10002523). A viable tumor piece was then implanted s.c. on the right flanks of anesthetized NSG mice (generation 1) and passaged for 2 more generations as described previously.28, 44, 54 When the tumors of generation 3 mice reached approximately 100-200 mm3, the mice were randomized into 4 groups as described above for the xenograft models. Statistical analysis Differences between treatment groups at specific time-points were assessed using unpaired t-tests. To examine the differences between the 4 treatment groups (vehicle, sotorasib alone, FGTI-2734 alone, and FGTI-2734 + sotorasib) for tumor volume (a continuous measure), combining all time points separately for the cell lines, we conducted mixed (repeated) measures ANOVA (F-tests), followed by post hoc pairwise F-tests with multiplicity adjustments via the false discovery rate (FDR).55 Necessary checks for no significant outliers, normality assumptions, and assumptions of sphericity were conducted, with automatic applications of the Greenhouse-Geisser sphericity corrections. All throughout, the tests considered were 2-sided, with significance level set to 5%. Analysis was conducted using R version 4.4.0. The tests comparing different treatments (vehicle, sotorasib, FGTI-2734, combination) for all 5 xenografts (LU99, H358, H2122, Calu1, PDX) show that the type of treatment given is important, and that the treatments have varying effects (p < 0.01) when looking at all time points together. The tests examining changes within the same subjects (xenograft) over time show a significant effect of time (p < 0.001), meaning that the effects of the treatments change
over time for all 5 xenografts. Additionally, the interaction between time and treatment is significant (p < 0.01) for all 5 xenografts, indicating that the rate at which treatments change over time is varying for each treatment. Results FGTI-2734 inhibits KRAS, NRAS, and HRAS membrane localization and interacts synergistically with sotorasib to inhibit the viability and induce apoptosis of KRAS G12C lung cancer cell lines. To determine whether FGTI-2734 can enhance the anti-tumor activity of sotorasib, we first determined the sotorasib sensitivity/resistance of 8 human KRAS G12C NSCLC cell lines (H358, H2122, H2030, HOP62, HCC1171, LU65, LU99, Calu1) by treating them with various concentrations of sotorasib for 72 hours and processed the cells for viability by CellTiter-Glo® (CTG) assay as described previously.38 We found H358, HCC1171, LU65, and H2122 cells to be sensitive to sotorasib (IC50s of 0.016, 0.019 0.030, and 0.048 µM, respectively), and LU99, H2030, HOP62, and Calu1 cells to be highly resistant (IC50s of 25.23, 33.8, 49.2, and 54.20 µM, respectively) (Fig. 1A-1D and data not shown). Thus, just as in patients, although all 8 NSCLC cell lines harbor KRAS G12C, some are sensitive and others are resistant to sotorasib. We then determined whether FGTI-2734 can sensitize these cell lines to sotorasib, and found that as the concentration of FGTI-2734 increased the sensitivity to sotorasib of all 4 cell lines increased proportionally (Fig. 1A-1D and data not shown). We then determined whether the 2 drugs interact synergistically by treating the cells with FGTI- 2734 and sotorasib 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 previously.39 Fig. 1E-1H show that all the CI values are less than 1, demonstrating that the combination of FGTI-2734 and sotorasib is synergistic in all 8 cell lines (CI values of less than 1 indicate synergism whereas CI values equal to 1 or greater than 1 indicate additivity or antagonism, respectively). To further evaluate the FGTI-2734 and sotorasib synergistic interaction, we also treated the 8 cell lines in a matrix format with various concentrations each of FGTI-2734 and sotorasib alone or in combination, and used the tool SynergyFinder to calculate synergy scores and to plot the corresponding 3D response surface plots as described previously.38 Synergy scores above 0 and those below 0 indicate synergy and antagonism, respectively. Consistent with the CI-Fa plots of Fig.1E-1H, the 2 drugs were synergistic at inhibiting the viability of the 4 KRAS G12C lung cancer cell lines (Fig. 1I-1L and data not shown) with average synergy scores between 5 and 9 (Fig. 1M-1P and data not shown).
To determine whether FGTI-2734 can inhibit KRAS, HRAS and NRAS prenylation and membrane localization and enhance the ability of sotorasib to induce apoptosis, we exposed the cells for 24 and 48 hours to sotorasib (3 µM for LU99, Calu-1, HOP62, and H2030 cells and 0.1 µM for H358, LU65, HCC1171, and H2122 cells) and FGTI-2734 (20 µM for all cell lines) individually or in combination, and processed the cells for Western blotting. Figures 2A&B show that treatment of LU99 and Calu1 cells with FGTI-2734 resulted in slow migrating KRAS, HRAS, and NRAS bands indicative of inhibition of the prenylation of these proteins (prenylated proteins migrate faster in SDS-PAGE gels 27-29, 36, 40-42. Prenylation of KRAS, HRAS, and NRAS is required for their membrane localization27-29, and therefore, FGTI-2734 treatment resulted in inhibition of RAS membrane localization consistent with our previous publication using cytosol/membrane fractionation and immuno-fluorescence.28 Figures 2A&B also show that treatment with sotorasib resulted in covalent modification of KRAS G12C as evidenced by the upward band shift (see * next to shifted bands) as documented previously.14 As expected, sotorasib binds KRAS G12C but not WT NRAS and WT HRAS and as such did not induce an upward band shift of NRAS and HRAS (Figs. 2A&B). We next determined whether FGTI-2734 enhances the ability of sotorasib to induce apoptosis as evidenced by Caspase 3 activation and PARP cleavage. Figures 2A&B show that treatment with the combination of FGTI-2734 and sotorasib was much more effective than single-agent treatment at inducing Caspase 3 activation and PARP cleavage, consisting with the synergistic interaction of these 2 compounds to inhibit cell viability (Fig.1). Similar results were obtained with the other 6 KRAS G12C cell lines, HOP62, H2030, H358, H2122, LU65, and HCC1171 (data not shown). To confirm the FGTI-2734 and sotorasib synergistic interaction to induce apoptosis, we treated the cells for 24 hours with sotorasib (3 µM for LU99 and Calu-1 cells, and 0.1 µM for H358 and H2122 cells) and FGTI-2734 (20 µM for all cell lines) individually or in combination, and processed the cells for Annexin V-FITC staining as a read out for apoptosis and DAPI to stain cell nuclei.43 Single-agent treatments of LU99 cells revealed some induction of apoptosis; however, combined treatment significantly increased apoptosis (Fig. 2D-G) consistent with the Caspase 3 and PARP findings (Fig. 2A). Similar results were observed with the other 3 KRAS G12C lung cancer cell lines (data not shown). FGTI-2734 enhances the in vivo anti-tumor activity of sotorasib in KRAS G12C lung cancer xenografts. To evaluate the ability of FGTI-2734 to enhance sotorasib anti-tumor
activity in vivo, we subcutaneously implanted nude mice with LU99, H358, H2122 and Calu- 1 cells, and after the tumors were palpable, mice were orally treated daily with vehicle, sotorasib, FGTI-2734, or the combination. High doses of sotorasib (30 mpk) inhibited only partially LU99 tumor growth (Fig.3A) whereas the much lower 5 mpk dose suppressed H358 tumor growth (Fig.3B), demonstrating that LU99 and H358 tumors are resistant and sensitive to sotorasib, respectively. While FGTI-2734 (100 mpk) alone had little effect, it enhanced the ability of sotorasib to cause tumor regression of LU99 tumors from an average tumor volume of 205 +/- 11 mm3 at day 0 to 172 +/- 61 mm3 at day 18, corresponding to a 16% tumor regression (Fig.3A). Sotorasib alone did not induce LU99 tumor regression and only inhibited tumor growth partially (Fig. 3A). In H358 xenografts, FGTI- 2734 (100 mpk) alone inhibited tumor growth partially, but in combination with sotorasib (5 mpk), the 2 drugs caused significant tumor regression as early as day 2 and continued throughout the treatment period decreasing the average tumor volume from 187 +/- 22 mm3 at day 0 to 24 +/- 3 mm3 at day 29, corresponding to 87% tumor regression on day 29 (Fig. 3B). In H2122 xenografts, low- dose sotorasib (5 mpk) inhibited tumor growth partially (Fig. 3C). While FGTI-2734 (100 mpk) alone had little effect on H2122 tumor growth, in combination with sotorasib (5 mpk) it significantly enhanced sotorasib anti-tumor activity causing significant growth inhibition at day 2 of treatment and continuing throughout the treatment period (Fig. 3C). In Calu-1 xenografts, while high dose of sotorasib (30 mpk) or FGTI-2734 (100 mpk) as single agents inhibited tumor growth only partially, the combination caused tumor regression as early as day 2 and continued throughout the treatment period with 82% tumor regression on day 29 (data not shown). It is important to note that the LU99 and H2122 xenograft studies had to be stopped on days 18 and 25, respectively, due to reaching animal protocol tumor volume limits in the vehicle-treated mice. FGTI-2734 enhances the in vivo anti-tumor activity of sotorasib in a KRAS G12C lung cancer patient- derived xenograft (PDX). We next evaluated the ability of FGTI-2734 to enhance sotorasib anti-tumor activity in vivo in a PDX model from a patient with KRAS G12C NSCLC. To this end, we subcutaneously implanted KRAS G12C lung patient tumor specimen in nude mice, and after the tumors were palpable, mice were orally treated daily with vehicle, sotorasib, FGTI-2734, or the combination. Overall, the combination was significantly more effective than single FGTI-2437 and sotorasib treatments at causing significant tumor regression starting at day 5, which was sustained throughout the entire treatment period (Fig.
3D). Specifically, during the first 8 days of treatment, tumors treated with sotorasib alone (30 mpk) and FGTI-2734 alone (100 mpk) grew whereas tumors treated with the combination did not grow and on day 5 started to shrink. On day 8, tumors treated with FGTI-2734 alone continued to grow whereas those treated with sotorasib alone started to regress but significantly less than the regression caused by the combination treatment (Fig. 3D). Starting on day 15 until day 22, tumors from mice treated with sotorasib (15 mpk) alone and FGTI- 2734 (150 mpk) alone grew whereas those from mice treated with the combination regressed significantly. The treatment was re-started on day 37 with sotorasib at 15 mpk and FGTI-2734 at 150 mpk. Here again, the tumors treated with single-agent FGTI-2734 or sotorasib continued to grow whereas those treated with the combination continued to significantly regress throughout the treatment period (Fig. 3D). Specifically, the average tumor volume from the mice treated with vehicle, FGTI-2734, and sotorasib alone grew from 118 ± 27, 129 ± 26, and 113 ± 27 at day 0 to 1573 ± 440, 726 ± 200, and 339 ± 142 at day 47, respectively (Fig. 3D), corresponding to 1333%, 563%, and 300% increase in tumor growth. In contrast, the average tumor volume from the mice treated with the combination decreased from 110 ± 28 mm3 at day 0 to 41 ± 12 mm3 at day 47 (Fig.3D), corresponding to -62% tumor regression on day 47. Therefore, while single-agent treatments only inhibited partially tumor growth, the combination treatments caused significant tumor regression in this PDX from a patient with KRAS G12C NSCLC. FGTI-2734 blocks sotorasib-induced ERK adaptive reactivation in KRAS G12C lung cancer cells in vitro and in xenografts in vivo. We next determined whether the ability of FGTI-2734 to significantly enhance the sotorasib inhibition of KRAS G12C lung cancer cell viability, induction of apoptosis and inhibition of xenografts and PDX anti-tumor activity is due at least in part to FGTI-2734 inhibiting sotorasib-induced ERK reactivation. To this end, we treated the KRAS G12C lung cancer Calu1 cells with vehicle, FTI-2734, sotorasib alone or in combination for 2, 24, 48, and 72 hours and processed the cells for Western blotting. After 2 hours of treatment, sotorasib potently decreased P-ERK levels (Fig. 4A). However, between 24 and 72 hours, sotorasib treatment induced a gradual increase in the P-ERK levels in a time-dependent manner (Fig. 4A), consistent with a sotorasib-induced adaptive reactivation of ERK. While FGTI-2734 treatment alone slightly increased P-ERK levels, in combination it blocked the ability of sotorasib to induce P-ERK levels over time (Fig. 4A). The ability of FGTI-2734 to block sotorasib-induced adaptive reactivation of ERK resulted in
a significant decrease in P-ERK levels at 24, 48, and 72 hours (Fig. 4A), and induction of apoptosis as evident from the much more potent activation by the combination of Caspase-3 and PARP cleavage at 24, 48, and 72 hours (Fig. 4A). Next, we evaluated the ability of FGTI-2734 to block sotorasib-induced ERK reactivation in a KRAS G12C lung cancer PDX in vivo. To this end, tumors from the above PDX anti-tumor studies (Fig. 3D) were collected 2 and 24 hours after drug treatment of mice on the last day of the study, lysed, and the lysates were subjected to Western blotting as described previously28, 44 Treatment of mice with sotorasib for 2 hours led to a decrease of the levels of P-ERK in the KRAS G12C lung cancer PDX tumors (Fig. 4B). However, after 24 hours of sotorasib treatment of mice, the levels of P-ERK rebounded (Fig.4B), consistent with a sotorasib-induced adaptive reactivation of ERK. Similar to the cell culture results (Fig.4A), FGTI-2734 treatment, in combination with sotorasib, blocked the ability of sotorasib to induce P-ERK at 24 hours in this KRAS G12C lung cancer PDX model (Fig. 4B). Because FGTI-2734 was able to enhance sotorasib anti-tumor activity of both sotorasib-resistant and sotorasib-sensitive xenografts (Fig.3), we next determined whether the combination of FGTI-2734 and sotorasib is more effective at suppressing P-ERK in both resistant and sensitive xenografts in vivo. To this end, tumors from the LU99 and H358 xenograft studies (Figs. 3A & 3B) were collected 2 hours after drug treatment of mice on the last day of the study, lysed, and the lysates were subjected to Western blotting as described previously.28, 44 Sotorasib treatment of mice decreased the levels of P-ERK by 75% in sotorasib-sensitive H358 tumors, and by only 36% in the sotorasib-resistant LU99 tumors (Fig. 4C). While FGTI-2734 alone had little effect, the combination of FGTI-2734 and sotorasib suppressed, to a much greater extent than sotorasib alone, the levels of P-ERK by 94% in H358 tumors (Fig. 4C), and by 84% in LU99 tumors (Fig. 4C). The ability of FGTI-2734 to enhance the ability of sotorasib to suppress P-ERK levels and sotorasib anti-tumor efficacy in sensitive KRAS G12C lung tumors suggests that these tumors harbor intrinsic sotorasib resistance mechanisms that require RAS membrane localization. Discussion In this Example, we have developed a highly innovative approach to block sotorasib- induced ERK reactivation, overcome sotorasib resistance, and significantly enhance its ability to cause tumor regression in KRAS G12C human lung cancer cell lines, in vivo in tumor xenografts, and in a PDX. This approach is highly effective as it uses FGTI-2734 to prevent
WT HRAS and NRAS from localizing to the membrane where they must be in order to be activated and to subsequently mediate ERK reactivation. Our findings that FGTI-2734 blocks sotorasib-induced ERK reactivation in vitro and in vivo, enhances sotorasib anti-proliferative and pro-apoptotic activities in cell culture and enhances its anti-tumor efficacy in xenografts and a PDX in vivo are highly significant as ERK reactivation is a major resistance mechanism that cancer cells use against many anticancer drugs including inhibitors of RTKs, RAS, RAF, and MEK.25, 26, 45, 46 Patients with KRAS G12C NSCLC who relapse while being treated with the KRAS G12C inhibitors sotorasib or adagrasib acquire several mechanisms of resistance that are mediated by adaptive feedback activation of RTK leading to ERK reactivation.14, 18, 19, 22, 24, 25 For example, some of these clinically-relevant resistance mechanisms involve overexpression of WT RTKs (e.g., MET, EGFR, HER2, FGFR2), which require activation of WT HRAS and NRAS and ERK reactivation. Further support for this was provided by Ryan et al25 who showed that sotorasib treatment initially suppresses P-ERK levels but overtime these levels increased, and that this ERK reactivation is paralleled by a KRAS G12C-independent feedback activation of WT HRAS and NRAS, limiting sotorasib efficacy. Our studies show that treatment with sotorasib of KRAS G12C cells suppressed P-ERK levels after 2 hours but the levels of P-ERK rebounded starting at 24 hours and continued to increase until 72 hours, consistent with sotorasib-induced ERK reactivation. Importantly, FGTI-2734 by inhibiting HRAS and NRAS membrane localization, which is required for RAS activation, blocked sotorasib-induced ERK reactivation, leading to suppression of P-ERK levels and substantial induction of apoptosis. Significantly, in vivo treatment with sotorasib of mice bearing a PDX from a patient with KRAS G12C NSCLC suppressed P-ERK levels after 2 hours but these levels rebounded after 24 hours of treatment. This in vivo ERK reactivation was suppressed by combination treatment of mice with sotorasib and FGTI-2734. Furthermore, combination treatment, but not treatments with sotorasib and FGTI-2734 alone, led to significant tumor regression of this KRAS G12C PDX. Similarly, the combination but not single-agent treatment of mice bearing KRAS G12C lung cancer xenografts led to significant suppression of P-ERK and tumor regression. Together, these findings suggest that by inhibiting RAS membrane localization, FGTI-2734 blocked sotorasib-induced ERK reactivation, overcoming a major resistance mechanism, and as such enhanced significantly the anti-tumor efficacy of sotorasib.
Because the majority of the patients that become resistant to KRAS G12C inhibitors acquire alterations that re-establish RTK-SHP2-SOS-RAS–RAF-MEK-ERK signaling pathways, several clinical trials are ongoing using combinations designed to overcome these resistance mechanisms.47 For example, in clinical trial CodeBreak 101 Ib/II (NCT04185883), sotorasib is being evaluated in combination with a) the EGFR/HER2/4 inhibitor afatanib, b) the EGFR antibody panitumumab, c) SHP2 inhibitors RMC-4630 and TNO155, d) the SOS1 inhibitor BI 1701963, e) the MEK1/2 inhibitor trametinib, and f) the CDK4/6 inhibitor Palbociclib.47 Similarly, in clinical trial KRYSTAL I/II, adagrasib is being evaluated in combination with a) the EGFR antibody cetuximab and b) the EGFR/HER2/4 inhibitor afatanib (NCT03785249), c) SHP2 inhibitors TNO155 (NCT04330664) and RMC-4630 (NCT04418661), d) the SOS1 inhibitors BI 1701963 (NCT04975256) and MRTX0902 (NCT05578092), e) the MEK/RAF inhibitor VS-6766 (avutometinib) and f) the CDK4/6 inhibitor palbociclib (NCT05178888).47 While some of these clinical trials are awaiting final reports, so far available data show that with most of the above combinations, ORR remains modest (17%-30%).47-50 Furthermore, recently RMC-7977, a RAS(ON) multi-selective inhibitor was reported to be highly efficacious against tumors with various RAS genotypes, particularly with KRAS codon 12 mutations.51 Preclinical data in KRASG12C cancer models suggests that RMC-7977 may be able to overcome KRAS G12C inhibitor clinically-relevant resistance mechanisms that lead to reactivation of RAS pathway signaling. However, results from an ongoing clinical trial (NCT05379985) with a RMC-7977-related RAS(ON) multi- selective inhibitor, RMC-6236, show that among the 40 patients with NSCLC and the 46 patients with PDAC, the ORR is only 38% and 20%, respectively, suggesting that resistance remains a challenge that will need to be addressed with RAS(ON) multi-selective inhibitors as well.52 FGTI-2734 enhances the anti-tumor activity of sotorasib in highly resistant and sensitive KRAS G12C lung cancer cell lines that have not previously been exposed to sotorasib, indicating that FGTI-2734 overcomes sotorasib intrinsic resistance. However, FGTI-2734 is anticipated to also overcome sotorasib acquired resistance. Indeed, in addition to overcoming sotorasib resistance mechanisms mediated by feedback RTK activation and subsequent ERK reactivation, FGTI-2734 could also enhance the anti-tumor activity of sotorasib in the context of other clinically-relevant resistance mechanisms that are dependent on RAS membrane localization including those involving patients whose tumors acquired
RTK mutations such as EGFR-A289V and RET-M918T, fusions involving ALK, RET and FGFR3, mutations in NRAS, and loss-of-function mutations in NF1 while on KRAS G12C inhibitors.19, 24, 47 Because these genetic alterations also require RAS membrane localization to activate the MAPK pathway, FGTI-2734 is anticipated to also block ERK reactivation in tumors that harbor these genetic alterations. Furthermore, patients treated with KRAS G12C inhibitors also present clinically-relevant mechanisms of resistance involving acquired secondary KRAS oncogenic mutations (e.g., G12D, G12V, G12R, G12W, G13D, Q61H), as well as acquired secondary mutation within the sotorasib switch II binding site in KRAS (e.g., R68S, Y96D, H95D, H95Q, H95R).19, 24, 47 Here again, because these mutant KRAS with acquired secondary mutations require RAS membrane localization to activate the MAPK pathway, FGTI-2734 is anticipated to also block ERK reactivation and enhance sotorasib anti- tumor activity in tumors that harbor mutant KRAS with these secondary mutations. However, other acquired genetic alterations such as mutations in BRAF and oncogenic fusions involving BRAF and RAF1 will hyperactivate ERK independently of RAS membrane localization because BRAF and RAF1 are downstream of RAS and hence do not need RAS to activate ERK, and as such, we do not anticipate that FGTI-2734 will overcome mt BRAF and RAF1- dependent sotorasib resistance. Similarly, loss-of-function mutations in PTEN are not expected to hyperactivate ERK and do not need RAS membrane localization, and therefore, we do not anticipate that FGTI-2734 will overcome mutant PTEN-dependent sotorasib resistance. Finally, the demonstration that FGTI-2734 and sotorasib combination was significantly more effective than monotherapy to inhibit P-ERK levels and induce tumor regression, not only in sotorasib-resistant xenografts but also in sotorasib-sensitive xenografts (Figs. 3 and 4) suggests that intrinsic (not acquired) resistance mechanisms may be operative in tumors from KRAS G12C lung cancer patients who have not been treated with sotorasib. This raises the possibility that FGTI-2734 may also enhance sotorasib anti-tumor efficacy in sotorasib-naïve patients, in addition to those who progressed while on sotorasib. In summary, our findings demonstrate that the combination of FGTI-2734 with sotorasib not only blocks ERK reactivation but also enhances the overall efficacy of sotorasib in various KRAS G12C lung cancer models including human cancer cell lines and their corresponding in vivo xenografts as well as a PDX from a patient with KRAS G12C NSCLC. This combinatorial approach is scientifically grounded and rational, as it disrupts the
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