背景:FMS样酪氨酸激酶3内部串联重复(FLT3-ITD)是急性髓性白血病(AML)中常见的突变类型,通常与患者预后不良有关。随着分子诊断的进步和酪氨酸激酶抑制剂(TKI)的发展,FLT3-ITD突变的AML患者的总生存期(OS)在一定程度上延长,但是复发和耐药性仍然是巨大的挑战。宁替尼是一种新型的TKI,可抵抗与肿瘤发病机理有关的各种激酶,目前正在进行肺癌的临床试验。在这项研究中,我们在体内和体外研究了宁替尼对FLT3突变的AML的抗肿瘤活性.
方法:在AML细胞系和表达各种FLT3突变的Ba/F3细胞中进行细胞增殖测定,以验证宁吉替尼的体外抗白血病活性。免疫印迹试验用于验证宁替尼对FLT3蛋白和下游途径的影响。分子对接和CETSA用于验证宁替尼与靶蛋白的相互作用。在Ba/F3-FLT3-ITD-,MOLM13,Ba/F3-FLT3-ITD-F691L-,MOLM13-FLT3-ITD-F691L诱导的白血病小鼠模型。我们还使用患者来源的原代细胞来确定宁替尼的疗效。
结果:宁替尼抑制细胞增殖,阻断了细胞周期,诱导细胞凋亡并结合FLT3抑制其下游信号通路,包括STAT5、AKT和ERK通路,在FLT3-ITDAML细胞系中。在FLT3-ITD和FLT3-ITD-F691L突变的小鼠模型中,宁替尼表现出优于现有临床药物gilteritinib和quizartinib的抗白血病活性,显著延长小鼠的生存期。此外,宁替尼对患者来源的携带FLT3-ITD突变的原代细胞表现出活性.
结论:总体而言,我们的研究证实了宁替尼在具有FLT3-ITD突变的AML中的治疗作用,为临床耐药患者提供了一个潜在的新选择。
BACKGROUND: FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is a common mutation type in acute myeloid leukemia (AML) and is usually associated with poor patient prognosis. With advancements in molecular diagnostics and the development of tyrosine kinase inhibitors (TKI), the overall survival (OS) of AML patients with FLT3-ITD mutations has been prolonged to some extent, but relapse and drug resistance are still substantial challenges. Ningetinib is a novel TKI against various kinases in relation to tumour pathogenesis and is undergoing clinical trials of lung cancer. In this study, we explored the antitumor activity of ningetinib against AML with FLT3 mutations both in vivo and in vitro.
METHODS: Cell proliferation assays were performed in AML cell lines and Ba/F3 cells expressing various FLT3 mutations to validate the antileukemic activity of ningetinib in vitro. Immunoblot assays were used to verify the effect of ningetinib on the FLT3 protein and downstream pathways. Molecular docking and CETSA were used to validate the interaction of ningetinib with target proteins. The survival benefit of ningetinib in vivo was assessed in Ba/F3-FLT3-ITD-, MOLM13, Ba/F3-FLT3-ITD-F691L-, MOLM13-FLT3-ITD-F691L-induced leukemia mouse models. We also used patient-derived primary cells to determine the efficacy of ningetinib.
RESULTS: Ningetinib inhibited cell proliferation, blocked the cell cycle, induced apoptosis and bound FLT3 to inhibit its downstream signaling pathways, including the STAT5, AKT and ERK pathways, in FLT3-ITD AML cell lines. In the mouse models with FLT3-ITD and FLT3-ITD-F691L mutation, ningetinib showed superior anti-leukemia activity to existing clinical drugs gilteritinib and quizartinib, significantly prolongating the survival of mice. In addition, ningetinib exhibited activity against patient-derived primary cells harboring FLT3-ITD mutations.
CONCLUSIONS: Overall, our study confirmed the therapeutic role of ningetinib in AML with FLT3-ITD mutations, providing a potential new option for clinically resistant patients.