BACKGROUND Osimertinib is the current recommended treatment for EGFR T790M-positive NSCLC following EGFR-TKI therapy. However, resistance to osimertinib therapy is inevitably acquired after a period of effective treatment. We had a patient suffering from EGFR L858R/T790M-positive NSCLC who initially responded to osimertinib therapy but eventually developed resistance. Plasma cell-free DNA analysis revealed the occurrence of exon-16-skipping HER2, which may resulted in the HER2 splice variant, HER2D16. HER2D16 has never been reported in lung cancer, and HER2D16-driven signaling was known to be regulated by Src-kinase in breast cancer. We investigated the role of HER2D16 as an osimertinib-resistant mechanism. METHODS We constructed and established H1975 cells stably expressing HER2D16. The dimeric formation of HER2D16 was tested using non-reducing polyacrylamide gel electrophoresis (PAGE). The effects of drug on signaling transduction were examined using Western blot. The synergistic effect was assessed using Chou-Talalay method. RESULTS We found HER2D16 can form homo-dimer in NSCLC cells. HER2D16-expressing H1975 cells were resistant to osimertinib treatment. We also found mutant EGFR and HER2D16 cooperated to activate downstream signaling for osimertinib-resistance. Besides, co-treatment with osimertinib and Src-kinase inhibitor failed to reverse resistance, indicating that HER2D16-driven signaling in NSCLC was not through canonical pathway. Finally, we revealed that the combination of osimertinib with the pan-HER small molecular inhibitor, afatinib, could synergistically repress cell growth and signaling in H1975-HER2D16 cells. CONCLUSION HER2D16 can contribute to osimertinib resistance through Src independent pathway. HER2D16 should be included in the molecular diagnosis panel for lung cancer.

中文翻译：

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Exon-16-skiping HER2 作为 EGFR L858R/T790M 阳性非小细胞肺癌中奥希替尼耐药的新机制

背景 奥希替尼是目前推荐用于 EGFR-TKI 治疗后 EGFR T790M 阳性 NSCLC 的治疗方法。然而，经过一段时间的有效治疗后，不可避免地会出现对奥希替尼治疗的耐药性。我们有一位患有 EGFR L858R/T790M 阳性 NSCLC 的患者，他最初对奥希替尼治疗有反应，但最终出现了耐药性。血浆无细胞 DNA 分析揭示了外显子 16 跳跃 HER2 的发生，这可能导致 HER2 剪接变体 HER2D16。HER2D16 从未在肺癌中报道过，并且已知 HER2D16 驱动的信号传导在乳腺癌中受 Src-激酶调节。我们研究了 HER2D16 作为奥希替尼耐药机制的作用。方法我们构建并建立了稳定表达HER2D16的H1975细胞。使用非还原聚丙烯酰胺凝胶电泳 (PAGE) 测试 HER2D16 的二聚体形成。使用蛋白质印迹检查药物对信号转导的影响。使用 Chou-Talalay 方法评估协同效应。结果我们发现HER2D16可以在NSCLC细胞中形成同源二聚体。表达 HER2D16 的 H1975 细胞对奥希替尼治疗具有抗性。我们还发现突变的 EGFR 和 HER2D16 协同激活奥希替尼耐药性的下游信号。此外，奥希替尼和 Src 激酶抑制剂的联合治疗未能逆转耐药性，表明 NSCLC 中 HER2D16 驱动的信号传导并非通过经典途径。最后，我们揭示了奥希替尼与泛 HER 小分子抑制剂阿法替尼的组合，可以协同抑制 H1975-HER2D16 细胞中的细胞生长和信号传导。结论 HER2D16 可通过 Src 独立途径导致奥希替尼耐药。HER2D16 应包括在肺癌的分子诊断面板中。