Non-small cell lung cancer (NSCLC), which constitutes 85% of all lung cancer cases, is often driven by oncogenic mutations within the kinase domain of the epidermal growth factor receptor (EGFR). Osimertinib, a third-generation EGFR tyrosine kinase inhibitor (EGFR-TKI), has revolutionized the treatment of EGFR-mutant non-small cell lung cancer (NSCLC). However, acquired resistance remains a major challenge, with non-genetic mechanisms increasingly recognized as key drivers. Here, we employ a multi-omics approach to characterize genome-wide chromatin accessibility and transcriptional landscapes between osimertinib-sensitive and -resistant EGFR-mutant NSCLC cells. Our findings reveal a strong correlation between epigenetic remodeling and transcriptomic alterations that define the resistant state.

Osimertinib-resistant subclones of HCC827, PC9, and H1975 cells, lacking secondary EGFR mutations, were generated using a dose-escalation approach. CRISPR/Cas9-based functional genomics screen targeting epigenetic regulators and transcription factors identifies critical resistance-associated regulatory networks, prominently featuring the NuRD and PRC2 complexes. Notably, among the top hits, FOSL1 and JUN—core components of the AP-1 transcription factor—emerge as central mediators of resistance. Mechanistically, we demonstrate that cis-regulatory elements with altered chromatin accessibility in resistant cells are enriched for AP-1 binding motifs, allowing AP-1 to drive a gene expression program that sustains the druggable MEK/ERK signaling axis, thereby enhancing cell viability and fitness.

Crucially, genetic depletion or pharmacological inhibition of AP-1 restores osimertinib sensitivity and reverses resistance-associated phenotypes, including epithelial-to-mesenchymal transition, upon anti-EGFR rechallenge. This effect is mediated through suppression of AKT and ERK signaling, underscoring AP-1 as a key vulnerability in the resistant state.

These findings provide novel insights into the epigenetic and transcriptional regulation of osimertinib resistance in EGFR-mutant NSCLC. Targeting AP-1 represents a promising therapeutic strategy to overcome osimertinib resistance and enhance the efficacy of EGFR-TKIs in NSCLC.