Concurrent loss of RB1 facilitates EGFR-TKI resistance in EGFR-mutant lung cancer by modulating histone crotonylation

Lung cancer harboring epidermal growth factor receptor ( EGFR ) mutations exhibits marked heterogeneity in clinical responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs), indicating that co-occurring genetic alterations critically influence therapeutic outcomes. However, the underlying mechanisms remain incompletely understood. Here, we modeled frequent EGFR co-alterations using CRISPR-mediated gene knockout and Tet-On-based gene overexpression in two EGFR -mutant cell lines. Functional screening across cell lines and patient-derived organoids revealed that concurrent loss of retinoblastoma 1 ( RB1 ) significantly reduced sensitivity to EGFR-TKIs. Integrated transcriptomic and metabolomic analyses further demonstrated that RB1 deficiency induced extensive metabolic reprogramming, leading to decreased intracellular crotonyl-CoA levels and reduced histone crotonylation. Among multiple crotonylation sites, histone H3 lysine 23 crotonylation (H3K23cr) emerged as a critical determinant of EGFR-TKI responsiveness. Mechanistically, decreased H3K23cr was associated with transcriptional repression of PEBP1 and reduced expression of the PEBP1 -encoded Raf kinase inhibitor protein (RKIP), thereby sustaining RAF/MEK/ERK pathway activation and attenuating EGFR-TKI efficacy. Importantly, pharmacological restoration of histone crotonylation or inhibition of MAPK signaling effectively resensitized EGFR/RB1 co-mutant models to EGFR-TKIs. Taken together, our findings identify a metabolic-epigenetic mechanism linking RB1 loss to reduced histone crotonylation and EGFR-TKI resistance, highlighting therapeutically actionable vulnerabilities in EGFR/RB1 co-mutant lung cancer.