Abstract 5512: Combining RAS(ON) G12C-selective and RAS(ON) multi-selective inhibitors overcomes sotorasib resistance driven by<i>KRASG12C</i> amplification or <i>NRASG13R</i> mutation

Abstract Amplification of the KRASG12C mutant allele and acquisition of NRAS mutations have been reported as resistance mechanisms in tumors progressing on sotorasib and adagrasib. We also identified these alterations in resistance models generated in vitro and in vivo utilizing H358 cells. We evaluated the activity of the RAS(ON) multi-selective noncovalent tri-complex inhibitor RMC-7977, both alone and in combination with the RAS(ON) G12C-selective covalent inhibitor RMC-4998. We generated five sotorasib-resistant cell lines from relapsed tumors of sotorasib-treated H358 xenografts (MR1-MR5) and two vehicle-treated tumors (MV1 and MV2). We created an additional isogenic cell line model, H358-MX1, by treating H358 cells with sotorasib and a SHP2 inhibitor, RMC-4550. Whole exome sequencing (WES) and digital polymerase chain reaction (dPCR) were performed to study mutant allelic fractions. Taqman copy number analysis and fluorescence in situ hybridization (FISH) were performed to assess copy number alterations. The anti-proliferative activity of RMC-7977 alone and in combination with RMC-4998 was evaluated. We identified increased allelic frequency of KRASG12C in MR2 and acquisition of a secondary NRASG13R mutation in H358-MX1 models. Copy number analysis revealed nearly 20 KRAS copies in MR2 compared to parental controls. KRAS amplification in MR2 was confirmed by dPCR and FISH. RAF-RBD pull down assays confirmed elevated KRAS-GTP and pan-RAS-GTP levels in MR2 cells. A small molecule screen using a customized library revealed that sotorasib had synergistic anti-proliferative activity in vitro with agents targeting the MAPK pathway. We hypothesized that simultaneous suppression of SHP2 and KRASG12C could elicit more pronounced inhibition of MAPK signaling and tumor growth. Dual inhibition of KRASG12C and SHP2 provided effective tumor control in vivo for 2 weeks before tumors started to regrow. Treatment with either RMC-4998, RMC-7977, or the combination of RMC-4998 + RMC-4550 did not sustain long-term tumor growth inhibition in this model. However, the RMC-4998 + RMC-7977 combination drove sustained, significant regressions compared to single-agent treatments or other combination regimens. In H358-MX1 model, siRNA-based knockdown of NRAS in combination with sotorasib reduced ERK phosphorylation and inhibited cell proliferation more effectively than NRAS knockdown or KRASG12C inhibition alone. In H358-MX1 mouse xenografts, RMC-7977 + RMC-4998 significantly inhibited tumor growth compared to single-agent treatments or other combination regimens. These preclinical data suggest that combination of RAS(ON) mutant-selective and RAS(ON) multi-selective inhibitors overcome resistance to KRASG12C (OFF) inhibitors driven by KRASG12C amplification and secondary NRASG13R mutation in KRASG12C mutated lung cancer models. Citation Format: Hitendra Singh Solanki, Denis Imbody, Bina Desai, Daria Miroshnychenko, Bhaswati Sarcar, Anurima Majumder, Yonghong Zhang, Uwe Rix, Andriy Marusyk, Mallika Singh, Ida Aronchik, Eric B. Haura. Combining RAS(ON) G12C-selective and RAS(ON) multi-selective inhibitors overcomes sotorasib resistance driven byKRAS G12C amplification or NRAS G13R mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5512.