Phosphoproteomic Analysis Identified Mutual Phosphorylation of FAK and Src as a Mechanism of Osimertinib Resistance in EGFR-Mutant Lung Cancer

Takehiro Tozuka; Rintaro Noro; Keisuke Yoshida; Satoshi Takahashi; Mariko Hirao; Kuniko Matsuda; Yasuhiro Kato; Shinji Nakamichi; Susumu Takeuchi; Masaru Matsumoto; Akihiko Miyanaga; Shinobu Kunugi; Kazufumi Honda; Jun Adachi; Masahiro Seike

doi:10.1016/j.jtocrr.2024.100668

Phosphoproteomic Analysis Identified Mutual Phosphorylation of FAK and Src as a Mechanism of Osimertinib Resistance in EGFR-Mutant Lung Cancer

JTO Clin Res Rep. 2024 Mar 21;5(4):100668. doi: 10.1016/j.jtocrr.2024.100668. eCollection 2024 Apr.

Authors

Affiliations

¹ Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.
² Department of Bioregulation, Graduate School of Medicine, Nippon Medical School, Tokyo, Japan.
³ Department of Analytic Human Pathology, Nippon Medical School, Tokyo, Japan.
⁴ Institution for Advanced Medical Science, Nippon Medical School, Tokyo, Japan.
⁵ Laboratory of Proteomics for Drug Discovery, Center for Drug Design Research, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan.

Abstract

Introduction: Osimertinib is a standard treatment for patients with EGFR-mutant 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.

Results: Phosphoproteomic analysis revealed 80 phosphorylation sites that were mutually up-regulated in PC-9OR and HCC827OR cells. The Kinase Enrichment Analysis version 3 analysis identified focal adhesion kinase (FAK) and proto-oncogene tyrosine-protein kinase Src (Src) as upstream kinases of these up-regulated phosphoproteins. The small-interfering RNA-mediated knockdown of FAK reduced Src phosphorylation and that of Src reduced FAK phosphorylation in both cell lines. Furthermore, FAK- or Src-specific small-interfering RNA 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.

Keywords: EGFR; FAK; Non–small cell lung cancer; Osimertinib; Resistant; Src.