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

Abstract

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-KDDWT), EGFR-KDD domain 1 T790M (EGFR-KDDD1T), EGFR-KDD domain 2 T790M (EGFR-KDDD2T), and EGFR-KDD both domain T790M (EGFR-KDDBDT). 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-KDDWT 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-KDDT790M) were only sensitive to 3rd generation EGFR TKIs. In ENU mutagenesis screening, we identified the C797S mutation in kinase domain 2 of EGFR-KDDBDT Ba/F3 cells. Based on this finding, we established an EGFR-KDD domain 1 T790M/domain 2 cis-T790M+C797S (EGFR-KDDT/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-KDDT790M mediates a resistance mechanism against 3rd generation EGFR TKIs.

Keywords: Acquired resistance; EGFR C797S mutation; EGFR T790M mutation; EGFR kinase domain duplication; Non–small cell lung carcinoma.

Fig. 1

Clinical history and genomic features for a patient with EGFR KDD. (A) Clinical history of a patient with EGFR-KDD. Numbers beneath the line represent months after diagnosis. Patient’s computerized tomography images indicated tumor masses pre-erlotinib treatment (first), post-erlotinib with partial response (second), post-erlotinib with progressive disease (third), post-osimertinib with partial response (fourth). A patient-derived cell line SNU-4784 was established with pleural effusion upon emerging erlotinib resistance, but without the EGFR T790M mutation. (B) EGFR-KDD breakpoint in a patient cDNA from pleural effusion. (C) The EGFR T790M mutation confirmed by droplet digital polymerase chain reaction (orange dots, blank; green dots, wild type; blue dots, T790M; red dots, T790M plus WT). EGFR-KDD, epidermal growth factor receptor kinase domain duplication.

Fig. 2

Characteristics of the patient-derived EGFR-KDD cell line SNU-4784. (A) Cell viability assay in SNU-4784 cell line. Cells were exposed to erlotinib, afatinib, and osimertinib for 72 hours. (B) Immunoblot assay of SNU-4784 cell line. Cells were exposed to erlotinib, afatinib, or osimertinib for 4 hours. Cell viability assay and the immunoblot assay were independently repeated three times. EGFR-KDD, epidermal growth factor receptor kinase domain duplication; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig. 3

Characteristics of the EGFR-KDD^T790M Ba/F3 cell lines. (A) Cell viability assays in EGFR-KDD^WT and EGFR-KDD^T790M Ba/F3 cell lines. Cells were exposed to EGFR TKIs for 72 hours. Graphs represent mean±SD values. (B) Immunoblot assays of EGFR-KDD^WT and EGFR-KDD^T790M Ba/F3 cell lines. Cells were exposed to erlotinib, afatinib, or osimertinib for 4 hours. Cell viability assays and immunoblot assays were independently repeated three times. EGFR-KDD, epidermal growth factor receptor kinase domain duplication; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig. 4

ENU mutagenesis screening to identify the potential resistance mechanism underlying the EGFR-KDD^BDT mutation. (A) Graphical scheme of ENU mutagenesis screening. EGFR-KDD^BDT Ba/F3 cells were exposed to 50 μg/mL ENU and selected with 2 μM osimertinib. (B) Sanger sequencing of osimertinib-resistant EGFR-KDD^BDT Ba/F3 cells. EGFR C797S mutation in kinase domain 2. EGFR-KDD, epidermal growth factor receptor kinase domain duplication; ENU, N-ethyl-N-nitrosourea.

Fig. 5

Characteristics of the EGFR-KDD^T/T+C Ba/F3 cell line. (A) Cell viability assay of the EGFR-KDD^T/T+C Ba/F3 cell line. Cells were exposed to EGFR TKIs for 72 hours. (B) Immunoblot assays of the EGFR-KDD^T/T+C Ba/F3 cell line. Cells were exposed to EGFR TKIs for 4 hours. (C) Cell viability assay in the EGFR-KDD^T/T+C Ba/F3 cell line. Cells were exposed to EGFR TKIs and cetuximab for 72 hours. For combination treatment, 10 μg/mL cetuximab was added. (D) Growth curve of the EGFR-KDD Ba/F3 cell lines. Cells were grown in interleukin-3 free media and the Ba/F3 parental cells were used as controls. EGFR-KDD, epidermal growth factor receptor kinase domain duplication; TKI, tyrosine kinase inhibitor.