Identification of EphA4 and FGFR1 activation as therapeutic vulnerabilities in EGFR–TKI-resistant NSCLC

Both intrinsic and acquired resistance inevitably develop in non-small cell lung cancer (NSCLC) following treatment with epidermal growth factor receptor–tyrosine kinase inhibitors (EGFR–TKI), potentially attributable to functional redundancy among kinase signaling networks. This study aimed to identify kinase signaling pathways associated with EGFR–TKI resistance in NSCLC. Phospho-receptor tyrosine kinase (RTK) arrays were used to compare RTK phosphorylation profiles between EGFR–TKI-sensitive and EGFR–TKI-resistant NSCLC cells. Kinase inhibitor screening and siRNA-mediated knockdown were performed to investigate the functional role of EphA4 and FGFR1 activation in EGFR–TKI resistance. Immunofluorescence staining was performed to evaluate the phosphorylation levels of EphA4 and FGFR1 in clinical NSCLC specimens with different responses to EGFR–TKI therapy. Finally, subcutaneous xenograft models were established in nude mice to evaluate the in vivo anti-tumor efficacy of combined EphA4/FGFR1 inhibition. Sustained activation of EphA4 and FGFR1 was observed across multiple EGFR–TKI-resistant cell lines with distinct resistance mechanisms. Pharmacological and genetic inhibition of EphA4/FGFR1 activation reduced NF-κB phosphorylation and enhanced gefitinib sensitivity in resistant cells. Clinical analyses demonstrated elevated phosphorylation levels of EphA4 and FGFR1 in EGFR–TKI-resistant tumors compared with EGFR–TKI-sensitive tumors. Preclinical models demonstrated that targeting EphA4/FGFR1 phosphorylation suppressed the growth of resistant xenografts. Moreover, this strategy also showed activity against MET-mediated EGFR–TKI resistance, including resistance driven by MET amplification and HGF-induced MET activation. This study identifies EphA4/FGFR1–NF-κB signaling as an important pathway involved in EGFR–TKI resistance in NSCLC. Targeting this signaling network enhances EGFR–TKI sensitivity in resistant models. Phosphorylated EphA4 and FGFR1 may represent potential biomarkers associated with EGFR–TKI resistance and provide candidate therapeutic vulnerabilities for further investigation.