CRISPR Screens Reveal YAP/TEAD Axis as a Mediator of Drug-Tolerant Cells in EGFR- Mutant NSCLC

Abstract Despite initial responses, most patients with metastatic lung cancer—including those with EGFR mutations—ultimately develop resistance to targeted therapies. To systematically uncover mechanisms underlying this resistance, genome-wide CRISPR knockout and activation screens were conducted in EGFR-mutant lung cancer cell lines treated with EGFR inhibitors such as osimertinib and gefitinib. These screens highlighted a recurrent involvement of genes associated with the Hippo signaling pathway. Notably, a subset of tumor cells, termed 'persister' cells, survive initial osimertinib exposure by engaging non-genetic, transcriptional adaptation mechanisms that promote drug tolerance. Our studies, integrating both genetic and pharmacological approaches, identified Hippo pathway activation as a key driver of this drug-tolerant state. Importantly, co-inhibition of EGFR and the Hippo signaling axis led to a pronounced reduction in cell viability in both established cell lines and patient-derived organoids. These findings propose that dual targeting of EGFR and Hippo signaling may offer a promising therapeutic approach to overcome resistance in EGFR-mutant lung cancer.