Department of Pathology, Changhai Hospital, Second Military Medical University, Shanghai, China.
Department of Pathology, Yueyang Integrative Medicine Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
J Cancer Res Clin Oncol. 2021 Apr;147(4):1065-1075. doi: 10.1007/s00432-020-03490-6. Epub 2021 Jan 3.
Imatinib, a small-molecule tyrosine kinase inhibitor, has shown good clinical activity by inhibiting adenosine triphosphate (ATP) binding to the receptor. Unfortunately, majority of patients eventually develop drug resistance, which limits the long-term benefits of the tyrosine kinase inhibitors and poses a significant challenge in the clinical management of GIST. The aim of our study was to explore the feasibility of blocking KIT dimerisation upstream of the phosphorylation in imatinib-resistant GIST.
KITMAb was prepared using hybridoma technique. The biological function of KITMAb was examined in KIT-dimer-expressing cells constructed by transfecting with liposomes using enzyme linked immunosorbent assay (ELISA), immunohistochemistry, western blot, MTT, Annexin V/FITC, and flow cytometry assay, respectively.
KIT-dimer was expressed in 293 cells transfected with c-kit mutated-type pcDNA3.1. Treatment of KIT-dimer-expressing cells with the KITMAb significantly decreased the expression of both KIT-dimer and other phosphorylated proteins of KIT downstream signalling pathway. Furthermore, KITMAb slowed down cell growth and reduced the proportion of cells in the proliferative phase (S + G2-M). Finally, we also found that KITMAb treatment accelerated cell apoptosis. These results indicate that KITMAb strongly inhibits KIT receptor dimerisation-mediated signalling pathway and cell growth responses in vitro.
We demonstrate c-kit mutation-driven KIT auto-dimerisation prior to tyrosine kinase phosphorylation as same as the procedure in ligand-dependent signalling pathway and describe a monoclonal antibody, KITMAb, with strong affinity to the dimerisation domain of KIT that blocks the important step in both the KIT signalling pathways. Further, the results suggest that treatment with KITMAb may be potentially therapeutic in imatinib-resistant GIST.
伊马替尼是一种小分子酪氨酸激酶抑制剂，通过抑制三磷酸腺苷（ATP）与受体结合，显示出良好的临床活性。不幸的是，大多数患者最终会产生耐药性，这限制了酪氨酸激酶抑制剂的长期获益，并对 GIST 的临床管理构成重大挑战。我们的研究目的是探索在伊马替尼耐药 GIST 中阻断 KIT 二聚化以阻止磷酸化的可行性。
采用杂交瘤技术制备 KITMAb。通过脂质体转染构建 KIT 二聚体表达细胞，用酶联免疫吸附试验（ELISA）、免疫组织化学、western blot、MTT、Annexin V/FITC 和流式细胞术分别检测 KITMAb 的生物学功能。
用突变型 pcDNA3.1-c-kit 转染 293 细胞后表达 KIT 二聚体。用 KITMAb 处理 KIT 二聚体表达细胞，明显降低 KIT 二聚体和 KIT 下游信号通路其他磷酸化蛋白的表达。此外，KITMAb 减缓细胞生长，减少增殖期（S+G2-M）细胞比例。最后，我们还发现 KITMAb 治疗可加速细胞凋亡。这些结果表明，KITMAb 可强烈抑制 KIT 受体二聚化介导的信号通路和细胞生长反应。
我们证明了 c-kit 驱动的 KIT 自身二聚化发生在酪氨酸激酶磷酸化之前，与配体依赖性信号通路相同，并描述了一种单克隆抗体 KITMAb，它与 KIT 的二聚化结构域具有很强的亲和力，可阻断 KIT 信号通路中的重要步骤。此外，结果表明，用 KITMAb 治疗可能对伊马替尼耐药 GIST 具有潜在的治疗作用。
