CRISPR-Cas9 Screening Identifies Resistance Mechanisms to KRAS Inhibition in Pancreatic Cancer

KRAS inhibitors (KRASi) targeting various KRAS mutations have entered clinical trials for pancreatic cancer. Despite promising preliminary clinical responses, most patients relapse due to intrinsic and acquired resistance. Thus, combination treatments are essential to extend the efficacy of KRAS-targeted therapies. To further determine genetic mechanisms of KRASi resistance, we performed KRASi-anchored CRISPR-Cas9 loss-of-function screens in KRAS G12D-, KRAS G12R-, and KRAS Q61H-mutant PDAC cell lines, using six KRASi, to identify genes, that when lost, modulate sensitivity to KRAS inhibition. We pharmacologically validated several hits from our screens, including EGFR, CK2 p110 alpha and p110 gamma, and YAP, by combining targeted inhibitors with KRASi. We determined that KRAS Q61H-mutant PDAC cell lines are intrinsically less dependent on KRAS for survival than other KRAS mutational subtypes. Further, we found that EGFR inhibitor erlotinib synergized with the RAS(ON) multi-selective inhibitor RMC-7977 in KRAS Q61H-mutant PDAC cell lines, and in cell lines with highly active EGFR, by mitigating ERK rebound activity. We also developed KRASi-resistant cell lines and observed sustained ERK MAPK dependence in the KRASi-resistant cell lines despite decreased ERK activity. Our findings enhance the understanding of KRASi intrinsic and acquired resistance and identify therapeutic vulnerabilities that can potentially be exploited for KRASi combination therapies in patients with PDAC.