Adaptive and acquired mechanisms underlying RAS-mutant tumor response to mutant and state selective RAS inhibitors

RAS mutations drive over 20% of human cancers and often correlate with poor clinical outcomes. Mutant RAS (RAS-MUT) maintains elevated GTP binding, biasing the protein toward its active (ON) state and driving hyperactivated signaling through effector pathways (MAPK and PI3K, among others), leading to uncontrolled cell growth and resistance to apoptosis. Thus, targeting RAS with small-molecule inhibitors presents a promising therapeutic strategy for RAS-MUT cancers. Recent advances in drug discovery have enabled the development of the pan-RAS(ON) inhibitor RMC6236, which uses a tricomplex strategy, as well as several KRAS-MUT-specific inhibitors (such as Adagrasib, Sotorasib, and MRTX1133) that target the OFF state. However, the antitumor efficacy of these direct RAS inhibitors varies across cancer types and is limited by a “therapeutic ceiling,” marked by stable disease and transient responses for most patients. Thus, a deeper understanding of mechanisms underlying variability in response and development of acquired resistance in RAS-MUT cancers is urgently needed. By analyzing signaling and cell growth inhibition in response to distinct direct RAS inhibitors, we observed similar sensitivity patterns across cell line models, suggesting that the antitumor activity of these drugs is largely confined to a shared subset of RAS-MUT cancers. Notably, we found that a subset of insensitive cells could regain sensitivity to direct RAS inhibitors through serum starvation or cotreatment with an SHP2 inhibitor, indicating hyperactive upstream Receptor Tyrosine Kinase (RTK) signaling contributing to RAS inhibitor resistance. Moreover, we found that resistant cell lines show a greater contribution of wild-type RAS (RAS-WT) to overall RAS activity than sensitive lines, suggesting that resistance may arise from RAS-WT-dependent compensation upon RAS-MUT inhibition. To model acquired resistance, we exposed KRAS-MUT cell line models to high concentrations of various RAS inhibitors individually and derived resistant models that displayed markedly reduced sensitivity compared with parental lines. No evidence of acquired secondary KRAS mutations was observed. However, we detected upregulation of the RTK-RAS-MAPK pathway along with elevated KRAS-MUT protein expression in resistant models. This increased RAS-MUT expression reduced inhibitor effectiveness and impaired suppression of RAS-GTP cycling. We also identified multiple cross-resistance patterns across different direct RAS inhibitors, highlighting the need for better mechanistic understanding to optimize RAS-inhibitor treatments for maximal therapeutic benefit. Together, our results provide mechanistic insight into resistance to direct RAS inhibitors in RAS-MUT cancers and emphasize the urgent need for improved strategies to treat patients who do not respond to current RAS-targeted therapies.