Abstract 561: Targeting mTOR/S6 signaling reverses acquired resistance to MTRX849 in KRASG12C-mutant lung adenocarcinoma cell lines

Abstract Background: KRAS is a commonly mutated oncogene long considered undruggable. Recent discoveries found compounds capable of binding to KRASG12C that inhibit KRAS-dependent signaling by locking the protein in its inactive state. However, clinical efficacy of these molecules has been limited by high rates of innate and acquired therapeutic resistance. Here, we developed cell line models of acquired resistance to the KRAS-G12C inhibitor MRTX849 (adagrasib) to study the mechanisms that underlie resistance which may help in the identification of novel therapeutic strategies to overcome resistance. Materials and Methods: KRASG12C-mutant H358 and H1373 lung cancer cell lines were treated through long-term exposure of increasing doses of the MRTX849 to generate the resistant clones, H358-R and H1373-R. Antiproliferative IC50s were determined by treating cells with serially diluted drug for 6 days and cells were quantified using CellVista® at Day 1 and Day 6. Resistant cells were genotyped by PCR amplification. Changes in protein signaling were determined by immunoblotting and changes in mRNA signaling were measured using RNA-seq analysis. Results: A 15-fold shift in IC50 was observed between H358-R and parental H358 (p-value = 0.029), and between H1373-R and parental H1373 (p-value = 0.022). Genotyping of the cell lines indicated that mutant KRASG12C was preserved in both resistant cell lines. RNA-seq analysis revealed a significant log-fold increase in RPS6 expression between H358-R and parental cells treated with MRTX849 (p-value = 6.06E-6). Treatment with MRTX849 inhibited downstream signaling of ERK1/2 and RSK in both parental and resistant cell lines. However, signaling downstream of mTORC1 through pS6 only remained active in the presence of MRTX849 in the resistant cells. Treatment with the mTORC1 inhibitor, RAD001, induced partial inhibition of pS6 in the resistant cells, whereas combined treatment with MRTX849 and RAD001 induced complete and sustained inhibition of resistant pS6 signaling. Combination treatment with MRTX849 and RAD001 also produced significantly improved inhibition of proliferation in both the acquired resistant clones relative to treatment with either single agent alone; H358-R (p-value = 0.0099) and H1373-R (p-value = 0.018). Discussion: In this study, we confirmed mTOR/S6 signaling as a potential escape pathway for KRAS-G12C inhibition. Targeting mTORC1 in combination with MRTX849 induced a sustained knockdown of S6-signaling and reversed resistance to MRTX849. Further genomic, transcriptomic and proteomic profiling of the generated resistant cell lines may identify additional mechanisms responsible for acquired KRAS-G12C resistance. Citation Format: Athena M. Madrid, Gabriel Choi, Jenny J. Hong, Sahel Hazeghsa, Jenny Z. Zhang, Kenny Castro, Neil A. O'Brien, Dennis J. Slamon. Targeting mTOR/S6 signaling reverses acquired resistance to MTRX849 in KRASG12C-mutant lung adenocarcinoma cell lines [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 561.