A saturation mutagenesis screen uncovers resistant and sensitizing secondary KRAS mutations to clinical KRAS(G12C) inhibitors

Mutant-specific inhibitors of KRAS(G12C), such as AMG510 (sotorasib) and MRTX849 (adagrasib), offer the unprecedented opportunity to inhibit KRAS, the most frequently mutated and heretofore undruggable oncoprotein. While clinical data are still limited, on-target mutations in KRAS(G12C) at position 12 and other sites are emerging as major drivers of clinical relapse. We identified additional mutations in KRAS(G12C) that impact inhibitor sensitivity through a saturation mutagenesis screen in the KRAS(G12C) NCI-H358 non–small-cell lung cancer (NSCLC) cell line. We also identified individuals in population genetic databases harboring these resistance mutations in their germline and in tumors, including a subset that co-occur with KRAS(G12C), indicating that these mutations may preexist in patients treated with KRAS(G12C) inhibitors. Notably, through structural modeling, we found that one such mutation (R68L) interferes with the critical protein–drug interface, conferring resistance to both inhibitors. Finally, we uncovered a mutant (S17E) that demonstrated a strong sensitizing phenotype to both inhibitors. Functional studies suggest that S17E sensitizes KRAS(G12C) cells to KRAS(G12C) inhibition by impacting signaling through PI3K/AKT/mTOR but not the MAPK signaling pathway. Our studies highlight the utility of unbiased mutation profiling to understand the functional consequences of all variants of a disease-causing genetic mutant and predict acquired resistant mutations in the targeted therapeutics.