Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are current standard of care for patients with EGFR mutation and metastatic non-small-cell lung carcinoma (NSCLC), but most patients using EGFR TKIs acquire resistance later. So, overcoming resistance of EGFR TKIs has become an important issue in the treatment of NSCLC. Previously, therapeutics targeting Bruton’s tyrosine kinase (BTK) have been successful in treating several hematologic malignancies. However, the role of BTK in NSCLC is still unknown. In this study, by examining surgical specimens from 80 NSCLC patients and their clinicopathologic parameters, we found significant correlation between high BTK expression and tumor differentiation, p-stage, lymph node metastatic status, maximum tumor size, and poor prognosis of patients. Using two NSCLC cell lines A540 and PC9, we demonstrated that BTK^pos cells exhibited more stemness (OCT4, SOX2) and EMT (E-Cadherin, Slug) markers than BTK^neg cells. Knockdown of BTK sensitized the NSCLC cells to Gefitinib. Meanwhile, the second-generation BTK inhibitor Acalabrutinib effectively suppressed SOX2, STAT3/JAK2/Akt axis and potentiated the anti-proliferative effect of Gefitinib and Osimertinib in NSCLC cells, including the T790M H1975 cells. Furthermore, Acalabrutinib and Osimertinib combination exhibited significant tumor growth inhibition of H1975-derived tumors in vivo. Our findings suggested that BTK mediates stemness and EMT properties, and inhibition of BTK potentiates the effect of Gefitinib and Osimertinib in NSCLC cells resistant to TKI. This implies a new approach to treat the NSCLC patients with resistance to previous TKI treatment.

表皮生长因子受体 (EGFR) 酪氨酸激酶抑制剂 (TKIs) 是目前治疗 EGFR 突变和转移性非小细胞肺癌 (NSCLC) 患者的标准治疗方法，但大多数使用 EGFR TKI 的患者后来获得耐药性。因此，克服EGFR TKIs耐药已成为NSCLC治疗的重要课题。此前，针对布鲁顿酪氨酸激酶 (BTK) 的疗法已成功治疗多种血液系统恶性肿瘤。然而，BTK在NSCLC中的作用尚不清楚。在这项研究中，通过检查来自 80 名 NSCLC 患者的手术标本及其临床病理参数，我们发现高 BTK 表达与肿瘤分化、p 分期、淋巴结转移状态、最大肿瘤大小和患者预后不良之间存在显着相关性。使用两个 NSCLC 细胞系 A540 和 PC9，^pos细胞比 BTK ^neg细胞表现出更多的干细胞（OCT4、SOX2）和 EMT（E-Cadherin、Slug）标志物。BTK 的敲除使 NSCLC 细胞对吉非替尼敏感。同时，第二代BTK抑制剂Acalabrutinib有效抑制了SOX2、STAT3/JAK2/Akt轴，并增强了吉非替尼和奥希替尼在包括T790M H1975细胞在内的NSCLC细胞中的抗增殖作用。此外，Acalabrutinib 和 Osimertinib 组合在体内表现出对 H1975 衍生肿瘤的显着肿瘤生长抑制。我们的研究结果表明，BTK 介导干性和 EMT 特性，并且抑制 BTK 会增强吉非替尼和奥希替尼对 TKI 耐药的 NSCLC 细胞的作用。这意味着一种治疗对先前 TKI 治疗耐药的 NSCLC 患者的新方法。