81P - EPHA2/MAPK pathway confers acquired resistance of afatinib in gastric cancer

Abstract

Background: We explored antitumor activity of afatinib in vitro and in vivo in gastric cancer (GC). In addition, underlying antitumor and acquired resistant mechanisms of afatinib were also investigated. Methods: Four GC cell lines were used to evaluate cell viability, cell cycle and apoptosis using cell proliferation assay and flow cytometry, respectively. Nine patient-derived xenograft (PDX) models were established to investigate the antitumor activity of afatinib in vivo. Immunohistochemistry and western blot were used to detect the expression level of ErbB family and downstream PI3K/AKT and MAPK pathway in cell lines and PDX models after afatinib treatment. In addition, one afatinib-resistant PDX model was established, and we detected the gene expression alterations between parental, sensitive and resistant PDX models via RNA sequencing and immunoblot. Furthermore, potential mechanisms of acquired resistance and corresponding reverse strategy were explored. Results: Afatinib significantly inhibited the proliferation of NCI-N87 cell line (IC50 = 2nM) and induced tumor growth regression or inhibition in EGFR-amplified/overexpressed and HER2-positive PDX models (TGI: 61.9% -116%, p < 0.05). However, in PDX models without ErbB family molecular variations, the anti-tumor activity of afatinib were weak (TGI: 25% -38%, p > 0.05). The effective treatment of afatinib induced cell cycle arrest at G1 (p < 0.05) and obvious apoptosis (p < 0.05) in NCI-N87 cells. Afatinib exerted its antitumor effect by inhibiting the phosphorylation of ErbB family and activation of PI3K/AKT and MAPK signaling pathway. We observed the overexpression and phosphorylation of EPHA2, and reactivation of MAPK pathway in the afatinib-resistant PDX model. The EPHA2 inhibitor ALW-II-41-37 could restore the sensitivity to afatinib by monotherapy (TGI: 60%, p < 0.001) or in combination with afatinib (TGI: 81%, p < 0.001). Conclusions: Afatinib exerted selective antitumor activity against GC cell lines and PDX models via inducing cell apoptosis and cell cycle arrest at G1 phase, and suppressing phosphorylation of ErbB family, PI3K/AKT and MAPK signaling pathway. EPHA2 conferred the acquired resistance of afatinib against GC and the inhibition of EPHA2 could reverse the resistance of afatinib. Legal entity responsible for the study: Zuhua Chen. Funding: The National Key Research and Development Program of China. Disclosure: All authors have declared no conflicts of interest.