小剂量阿糖胞苷依赖Beclin1介导的mTOR通路靶向自噬促进AML细胞分化的机制研究

Low-dose Ara-C (LD-Ara) exhibits potent antileukemic effects in old and relapsed/refractory AML patients with low toxic side effects, but the precise mechanism by which it generates such response is not well understood. We have previously found that treatment with LD-AraC induced AML cells differentiation without apoptosis. Meanwhile,autophagy was confirmed by transmission electron microscopy and accumulated LC3-II protein during this period.We also provided evidence that PI3K was activated and AKT-mTOR pathway was inhibited while induction of autophagy. To illustrate the mechanism in depth, this study will: 1) To establish that autophagic cell death is critical for the growth inhibition of LD-AraC on AML cells and the exact role of autophagy in the AML cell differentiation; 2) To confirm that autophagy and differentiation effects are regulated through Beclin1-mediated PI3K-AKT-mTOR pathway by gene transfection and RNA interference; 3) To further verify the autophagy effect in murine model and patients primary cells. Raise the potential to enhance the antileukemic properties of LD-AraC by combination with autophagy inducer. It is assumed that pharmacological targeting of autophagy provides promise for the management of AML therapy and combination of autophagy inducers might be attractive for the treatment of AML.

小剂量阿糖胞苷（LD-AraC）为主的化疗方案对复发难治及老年急性髓细胞白血病（AML）获得了明确疗效和低毒副作用，但作用机制尚有争论。我们前期研究发现LD-AraC未诱导AML细胞凋亡，但发生自噬及分化作用，且LD-AraC可激活PI3K并抑制AKT-mTOR诱导自噬。作为前期研究的延续和深入，本项目拟：1）明确LD-AraC通过诱导AML细胞自噬调节分化，并成为其抗白血病的主要机制。2）利用基因转染、RNA干扰等手段确定LD-AraC是否主要通过Beclin1介导的PI3K-AKT-mTOR通路，正向调控自噬及分化过程。3）在患者原代细胞和小鼠模型中进一步验证自噬与LD-AraC疗效关系，初步探讨联用自噬诱导剂能否促进细胞分化，增强LD-AraC敏感性。我们希望进一步阐明LD-AraC的作用机制，论证自噬作为白血病治疗靶点和联合自噬诱导剂增敏的可行性，探索AML靶向治疗新思路。

阿糖胞苷（Ara-c）用于急性髓系白血病（AML）的治疗已经有超过40年的历史。小剂量Ara-c虽在临床广泛应用，但其治疗机制至今仍不明确。此课题探究了小剂量Ara-c通过自噬产生白血病治疗作用的可能性。.采用不同浓度Ara-c对白血病细胞株U937及HEL进行24/48小时的处理，结果发现其治疗效果呈浓度及时间依赖性。采用50nM浓度的阿糖胞苷治疗24小时后，在U937及HEL细胞株中均观察到了LC3、Beclin1表达的升高，p62下调及自噬现象的发生；同时伴有Akt-mTOR通路的下调。观察发现，在自噬发生后，U937细胞表面CD11b表达升高并且在细胞形态上向成熟分化。进一步干扰Beclin1表达或加入自噬抑制剂3-MA及CQ共培养后，自噬及分化均被逆转，小剂量Ara-c的治疗作用被显著弱化。.综上所述，我们认为阿糖胞苷在急性髓系白血病细胞中能诱导自噬并且由此产生治疗作用，自噬或将称为AML治疗新的靶点。