AXL phosphosite mapping links FAK1 and YAP1 signaling to erlotinib resistance in EGFR-mutant lung cancer.

Targeted therapies for receptor tyrosine kinases are effective but invariably limited by drug resistance. In EGFR-mutant lung cancer, AXL activation drives resistance to erlotinib by restoring cell survival and migration. To map these signaling mechanisms, we generated a panel of lung adenocarcinoma PC9 cell lines with phenylalanine substitutions at each intracellular AXL tyrosine residue. By integrating phosphorylation data with phenotypic changes via multivariate modeling, we found that AXL signaling organizes into two clusters enriched for Abl1 and SFK substrate motifs. A peptide specificity screen identified FAK1 as a top proximal substrate of AXL. Downstream, AXL-mediated YAP1 activation was found to sustain drug tolerance, while combined inhibition eliminated persister cells in vitro. These AXL and YAP pathways correlate with disease progression and poor clinical outcomes in erlotinib-treated patients. Collectively, this study dissects the specific signaling regulators by which AXL drives erlotinib resistance.