Acquired Resistance Mechanism of EGFR Kinase Domain Duplication to EGFR TKIs in Non–Small Cell Lung Cancer

PURPOSE: Epidermal growth factor receptor kinase domain duplication (EGFR-KDD) is a rare and poorly understood oncogenic mutation in non–small cell lung cancer (NSCLC). We aimed to investigate the acquired resistance mechanism of EGFR-KDD against EGFR-TKIs. MATERIALS AND METHODS: We identified EGFR-KDD in tumor tissue obtained from a patient with stage IV lung adenocarcinoma and established the patient-derived cell line SNU-4784. We also established several EGFR-KDD Ba/F3 cell lines: EGFR-KDD wild type (EGFR-KDD(WT)), EGFR-KDD domain 1 T790M (EGFR-KDD(D1T)), EGFR-KDD domain 2 T790M (EGFR-KDD(D2T)), and EGFR-KDD both domain T790M (EGFR-KDD(BDT)). We treated the cells with EGFR tyrosine kinase inhibitors (TKIs) and performed cell viability assays, immunoblot assays, and ENU (N-ethyl-N-nitrosourea) mutagenesis screening. RESULTS: In cell viability assays, SNU-4784 cells and EGFR-KDD(WT) Ba/F3 cells were sensitive to 2nd generation and 3rd generation EGFR TKIs. In contrast, the T790M-positive EGFR-KDD Ba/F3 cell lines (EGFR-KDD(T790M)) were only sensitive to 3rd generation EGFR TKIs. In ENU mutagenesis screening, we identified the C797S mutation in kinase domain 2 of EGFR-KDD(BDT) Ba/F3 cells. Based on this finding, we established an EGFR-KDD domain 1 T790M/domain 2 cis-T790M+C797S (EGFR-KDD(T/T+C)) Ba/F3 model, which was resistant to EGFR TKIs and anti-EGFR monoclonal antibody combined with EGFR TKIs. CONCLUSION: Our study reveals that the T790M mutation in EGFR-KDD confers resistance to 1st and 2nd generation EGFR TKIs, but is sensitive to 3rd generation EGFR TKIs. In addition, we identified that the C797S mutation in kinase domain 2 of EGFR-KDD(T790M) mediates a resistance mechanism against 3rd generation EGFR TKIs.