Targeting Distinct Cell Cycle Nodes Overcomes RAS Inhibitor Resistance

Activating mutations in KRAS drive pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). Although mutant-selective KRAS inhibitors and pan-RAS inhibitors provide clinical benefits, the development of resistance limits durable responses. Here, transcriptomic and proteomic analyses revealed that, despite effective suppression of mutant KRAS signaling, resistant cells sustain cell cycle progression. Distinct orthogonal mitogenic pathways were engaged in a context-dependent manner to bypass KRAS inhibition. While these pathways could be broadly inhibited using the pan-RAS-ON inhibitor RMC-6236, cells remained capable of developing acquired resistance where cell proliferation was uncoupled from RAS signaling. Combinatorial drug screens and genome-wide CRISPR-Cas9 screens showed that perturbing cell cycle nodes via targeting cyclin dependent kinases CDK4/6 and CDK2 restored sensitivity to KRAS/RAS inhibitors. Co-targeting CDK4/6 induced G1 arrest and suppressed E2F-regulated proteins across all resistant models. In contrast, co-targeting CDK2 exerted a broader effect by impairing DNA replication, inducing G2 arrest, preventing mitotic entry, and yielding a more durable cytostatic response that delayed cellular outgrowth after drug withdrawal. Finally, concurrent inhibition of KRAS with either CDK4/6 or CDK2 yielded durable tumor control in vivo in xenografts derived from models with acquired resistance. In conclusion, these findings identify sustained cell cycle activity as a defining feature of resistance to KRAS-directed therapies and establish cell cycle co-targeting as an effective strategy to overcome KRAS/RAS inhibitor resistance.