Agnieszka K. Witkiewicz

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. In this study, 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. Although these pathways could be broadly inhibited using the pan–RAS-ON inhibitor RMC-6236, cells remained capable of developing acquired resistance, in which 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. Cotargeting CDK4/6 induced G1 arrest and suppressed E2F-regulated proteins across all resistant models. In contrast, cotargeting 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 cotargeting as an effective strategy to overcome KRAS/RAS inhibitor resistance. Significance: Acquired resistance to KRAS/RAS inhibitors limits clinical benefit. We identify active cell cycle machinery as a defining feature of resistance and demonstrate distinct combinatorial strategies to target cell cycle progression.

RB-independent activity of CDK2 inhibition in CDK4/6 inhibitor-resistant models

Quantitative imaging analysis of tumor biopsies

CDK2 inhibition does not activate RB

Phosphoproteomic analysis after CDK2 inhibitor treatment

On-target and p53-dependent activity of CDK2 inhibition

Efficacy of CDK2 inhibition in patient-derived CDK4/6 inhibitor-resistant cell lines

Gene amplification profile of the MGH321.4 model

Genomic alterations in models with acquired resistance to CDK4/6i

Expression of cell cycle genes in paired CDK4/6i-sensitive and -resistant models