LAUSR

KRAS G12C mutations in cancer can be targeted by G12C-specific inhibitors, but resistance develops rapidly. Preclinical studies suggested multiple drug combinations to improve efficacy, including CDK4/6, MEK, ERK, EGFR, and SHP2 inhibitors, collectively called vertical MAPK inhibition (“vMAPKi”). To identify mechanisms of resistance to vMAPKi, we treated the G12C-mutant colon cancer cell line SW837 with different vMAPKi triple combinations incorporating G12C, MEK, ERK, EGFR, SHP2, and/or S6K1 inhibitors until resistance developed. Notably, we identified two modes of resistance: fast growing colonies and slow growing or quiescent persisters. Unlike other fast growing resistant models that have pre-existing resistance mutations (e.g. EGFR-T790M for EGFR inhibitors), we did not identify any obvious resistance mutations by targeted exome sequencing in 6 separate colonies, each derived from a different vMAPKi combination. Instead, Reverse-Phase Protein Array revealed robust JNK pathway activation across all colonies, regardless of initial drug combination. Indeed, all colonies showed cross-resistance to all vMAPKi combinations, and sequence analysis revealed shared non-oncogenic mutations across colonies, suggesting that they pre-existed in the parental population. These data suggest that such pre-existing clones are fated for dominance, regardless of the vMAPKi combination used – implying that vertical pathway inhibition has intrinsic limitations. However, all colonies also gained exquisite sensitivity to single-agent JNK inhibition (“JNKi”) compared to parental controls (with identical results in a second cell line, SW480), suggesting a gained vulnerability. Indeed, brief high-dose JNKi treatment completely eliminated colonies, as they did not grow back when JNKi was switched back to vMAPKi. By contrast, JNKi had no effect on quiescent persisters. To understand the differences between colonies and persisters, we conducted RNA-seq on the isolated populations. Although persisters showed no evidence of upstream JNK activation, they showed high expression of FOS, FOSB, and FOSL1, the binding partners of JUN. Indeed, expression of any FOS paralog was sufficient to confer vMAPKi resistance. Thus, both colonies and persisters converge on FOS-JUN signaling, one through upstream JNK activation and the other through transcriptional upregulation of FOS family members. Interestingly, pre-treatment of parental SW837 with high-dose JNKi has no effect on later resistance development, contrasted to the elimination of colonies if JNKi is given after resistance developed. This implies that colonies must undergo a reprogramming step to gain sensitivity to JNKi. We are currently conducting longitudinal single cell and barcoding sequencing assays to understand how such reprogramming occurs and whether this can reveal earlier therapeutic vulnerabilities. Citation Format: Mohamad Karim Koleilat, Lawrence Kwong. KRAS G12C colon cancer resistance to vertical MAPK inhibition converges on FOS-JUN signaling through orthogonal mechanisms. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3893.