Abstract LB335: Silent KRAS mutations confer altered sensitivity to targeted KRAS inhibition

Abstract KRAS is mutated in over 90% of pancreatic adenocarcinomas (PDAC) and is one of the most recalcitrant therapeutic targets. Activating missense mutations at G12, G13, and Q61 lock KRAS in a state of persistent signaling, driving PDAC growth. Synonymous, or silent, mutations are often disregarded clinically as they do not alter the encoded amino acid and are presumed functionally insignificant. For example, the FDA-approved cobas® KRAS Mutation Test is specifically designed to ignore KRAS G12G and G13G silent mutations, to prevent them from inadvertently being reported. Despite this, silent mutations cluster in KRAS with low frequency at G12, G13, and G60, at or adjacent to the hotspot activating mutation positions in cancer patients. KRAS is evolutionarily constrained to contain rare codons or codon pairs at these positions, and we have previously shown that any silent mutation at these positions increases KRAS protein expression in accordance with glycine codon usage frequency and drives increased proliferation and tumorigenic phenotypes. Furthermore, among cancer patients with wild-type (WT) KRAS tumors, high expression of KRAS is associated with a poor prognosis in a pan-cancer cohort. Here, utilizing KRAS inhibitors that target WT and mutant KRAS, we examine how NIH3T3 cells containing synonymous KRAS variants respond to targeted KRAS inhibition, relative to the native KRAS nucleotide sequence and the oncogenic G12V mutation. NIH3T3 cells overexpressing silent G12G, G13G, and G60G KRAS mutations exhibited marked growth inhibition in response to three KRAS inhibitors with different mechanisms of action, comparable to the oncogenic KRAS G12V-variant expressing cells and in contrast to the native KRAS nucleotide sequence. These silent mutant variants also demonstrated increased KRAS expression and altered ERK MAPK and PI3K signaling as assessed by immunoblotting, as well as differential pathway suppression following KRAS inhibition. Together these data demonstrate that KRAS silent mutants may confer oncogenic-like signaling and therapeutic sensitivity and challenge the paradigm that all synonymous variants are biologically inert. Our data suggest that cancer cells containing silent KRAS mutations should not be ignored and may represent a therapeutic target. Further studies will define the mechanisms underlying these effects and clarify their clinical relevance. Ongoing and planned work includes engineering silent KRAS mutations at the endogenous locus in non-transformed human cells to directly assess their impact on KRAS expression, downstream signaling, and therapeutic response in a physiologically relevant setting. Citation Format: Megan A. Satyadi, Rachel Bridgens, Haley Todd, Grace Goodhart, Syed A. Ahmad, Andrew M. Waters. Silent KRAS mutations confer altered sensitivity to targeted KRAS inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB335.