靶向AMPK依赖的自噬克服FLT3-ITD 突变AML对Sorafenib的耐药及分子机制的研究

Acute Myeloid leukemia (AML) with FLT3-ITD mutation is a type of high-risk AML. The kinase inhibitor Sorafenib is a new agent for treating this type of AML by targeting FLT3-ITD mutation. However, resistance to Sorafenib remains a big problem. Previously, we found that Sorafenib could induce autophagy in AML cell line with FLT3-ITD mutation, which may be related to the activation of AMPK pathway. Furthermore, cellular apoptosis was increased when the autophagy was inhibited by a specific autophagy inhibitor CQ. On the basis of these findings, this work aims to investigate that Sorafenib can induce protective autophagy in AML cells with FLT3-ITD mutation and inhibition of autophagy may reverse the drug-resistance. We will further investigate whether Sorafenib can mediates intracellular accumulation of free calcium by stimulating the endoplasmic reticulum (ER) stress response, which in turn actives the CAMKKβ-AMPK-dependent autophagy. Also we will try to find the other up-and down-stream pathways for regulating it. Furthermore, we will validate that Sorafenib together with autophagy inhibitor can reverse resistance of Sorafenib to AML with FLT3-ITD mutation in mice model. Our study can provide innovation achievements in the field of reversal of resistance to Sorafenib in the treatment of AML with FLT3-ITD mutation.

伴有FLT3-ITD突变的急性髓系白血病（AML）属于高危AML，靶向FLT3-ITD的激酶抑制剂Sorafenib是治疗此类型AML的新希望，但存在继发耐药问题。我们前期研究发现Sorafenib可诱导FLT3-ITD突变AML细胞发生自噬，与AMPK激活相关。用特异性自噬抑制剂能显著增加细胞凋亡。在此基础上，本课题验证Sorafenib可诱导FLT3-ITD突变AML细胞发生保护性自噬，抑制自噬可逆转耐药；进一步研究Sorafenib是否诱导内质网应激反应，促进胞浆内游离钙堆积,进而诱发CAMKKβ-AMPK 途径依赖性自噬，阐明参与其调控的上下游分子机制；进而在小鼠动物实验中验证Sorafenib联合自噬抑制剂能否逆转伴FLT3-ITD突变的AML细胞耐药。本研究将在克服Sorafenib耐药和FLT3-ITD突变AML治疗领域获得创新性成果，具有重要的理论意义和应用前景。

伴有FLT3-ITD突变的急性髓系白血病（AML）属于高危AML，靶向FLT3-ITD的激酶抑制剂Sorafenib是治疗此类型AML的新希望，但存在继发耐药问题，使患者预后不佳, 急需解决方法。本课题主要研究自噬在Sorafenib对于伴FLT3-ITD突变的AML细胞耐药中的作用及其相关机制。实验结果表明，Sorafenib可诱导FLT3-ITD突变AML细胞发生自噬。氯喹、Compound C (AMPK inhibitor）能抑制自噬，促进细胞凋亡，提示Sorafenib诱导细胞发生AMPK依赖的保护性自噬，抑制自噬可逆转耐药；Sorafenib可通过AMPK/mTOR /ULK1/Beclin1信号通路促进FLT3-ITD突变的AML细胞发生自噬；小鼠动物实验表明 Sorafenib诱导细胞AMPK激活和细胞自噬水平升高，应用Compound C抑制AMPK活性，抑制细胞自噬水平可显著增强细胞对Sorafenib的敏感性, 小鼠模型中肿瘤明显缩小，肿瘤细胞凋亡增加。以上实验提示靶向AMPK依赖的自噬可克服FLT3-ITD 突变AML对Sorafenib 的耐药，其途径AMPK/mTOR /ULK1/Beclin1信号通路。另一方面，我们进行了靶向FLT3-ITD蛋白的研究。我们发现天然产物新藤黄酸（NGA）通过降解Flt3-ITD蛋白，从而抑制FLT3-ITD突变的AML细胞的增殖。本项目研究成果在Oncology Reports等SCI期刊发表论文5篇，已投稿并俢回SCI论著1篇，待投稿论文1篇。协助培养毕业硕士生1名。本研究在克服Sorafenib耐药和FLT3-ITD突变AML治疗领域获得创新性成果，具有重要的理论意义和应用前景。