Cancer Research Institute, Seoul National University, Seoul, Korea.
Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea.
Cancer Res Treat. 2022 Jan;54(1):140-149. doi: 10.4143/crt.2021.385. Epub 2021 May 3.
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.
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.
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.
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.
表皮生长因子受体激酶结构域重复（EGFR-KDD）是一种罕见且尚未被充分了解的非小细胞肺癌（NSCLC）致癌突变。本研究旨在探究 EGFR-KDD 对 EGFR-TKIs 获得性耐药的机制。
我们从一名患有 IV 期肺腺癌的患者的肿瘤组织中鉴定出 EGFR-KDD，并建立了患者来源的细胞系 SNU-4784。我们还建立了几种 EGFR-KDD Ba/F3 细胞系：EGFR-KDD 野生型（EGFR-KDDWT）、EGFR-KDD 结构域 1 T790M（EGFR-KDDD1T）、EGFR-KDD 结构域 2 T790M（EGFR-KDDD2T）和 EGFR-KDD 双结构域 T790M（EGFR-KDDBDT）。我们用 EGFR 酪氨酸激酶抑制剂（TKIs）处理这些细胞，并进行细胞活力测定、免疫印迹分析和 ENU（N-乙基-N-亚硝脲）诱变筛选。
在细胞活力测定中，SNU-4784 细胞和 EGFR-KDDWT Ba/F3 细胞对 2 代和 3 代 EGFR TKIs 敏感。相比之下，T790M 阳性的 EGFR-KDD Ba/F3 细胞系（EGFR-KDDT790M）仅对 3 代 EGFR TKIs 敏感。在 ENU 诱变筛选中，我们在 EGFR-KDDBDT Ba/F3 细胞中鉴定出 EGFR-KDD 激酶结构域 2 中的 C797S 突变。基于这一发现，我们建立了一个 EGFR-KDD 结构域 1 T790M/结构域 2 顺式 T790M+C797S（EGFR-KDDT/T+C）Ba/F3 模型，该模型对 EGFR TKIs 和抗 EGFR 单克隆抗体联合 EGFR TKIs 耐药。
本研究表明，EGFR-KDD 中的 T790M 突变赋予了对 1 代和 2 代 EGFR TKIs 的耐药性，但对 3 代 EGFR TKIs 敏感。此外，我们发现 EGFR-KDDT790M 中激酶结构域 2 的 C797S 突变介导了对 3 代 EGFR TKIs 的耐药机制。
