Phosphoproteomic analysis identified mutual phosphorylation of FAK and Src as a mechanism of osimertinib resistance in EGFR-mutant lung cancer

Background Osimertinib is a standard treatment for patients with EGFR-mutant non-small cell lung cancer (NSCLC). Although some osimertinib resistance mechanisms have been identified, nearly 50% of the mechanisms remain to be elucidated. This study was aimed at identifying non-genetic mechanisms underlying osimertinib resistance. Methods We established two osimertinib-resistant cell lines from EGFR mutation-positive PC-9 and HCC827 NSCLC cell lines (PC-9OR and HCC827OR, respectively) using a stepwise method. We compared the phosphoproteomic profiles of the osimertinib-resistant and parental cells using mass spectrometry. Upstream kinases were identified using the application Kinase Enrichment Analysis version 3 (KEA3). Results Phosphoproteomic analysis revealed 80 phosphorylation sites that were mutually upregulated in PC-9OR and HCC827OR cells. KEA3 analysis identified focal adhesion kinase (FAK) and proto-oncogene tyrosine-protein kinase Src (Src) as upstream kinases of these upregulated phosphoproteins. The siRNA-mediated knockdown of FAK reduced Src phosphorylation and that of Src reduced FAK phosphorylation in both cell lines. Furthermore, FAK- or Src-specific siRNA treatments restored EGFR phosphorylation in PC-9OR and HCC827OR cells. The combination of FAK and Src inhibitors inhibited PC-9OR and HCC827OR cell proliferation in vitro and suppressed tumor growth in a xenograft mouse model. Immunohistochemistry of tumors from patients with EGFR-mutant NSCLC suggested that phosphorylated FAK and Src are involved in initial and acquired resistance to osimertinib. Conclusions Phosphoproteomic analysis may help elucidate the mechanisms of resistance to molecular-targeted therapies in lung cancer. Mutual phosphorylation of FAK and Src is involved in osimertinib resistance. Thus, FAK and Src inhibition may be novel treatment strategies for osimertinib-resistant NSCLC.