Spi2A抑制自噬解除MLL-AF9白血病干细胞化疗耐药与机制研究

Chemotherapy resistance and relapse are still therapeutic puzzles in leukemia treatment. Numerous studies indicate leukemic stem cells(LSCs) contribute to the chemotherapy resistance and relapse of acute myeloid leukemia(AML). And enhanced autophagy is considered to participate in the resistance mechanism of LSCs. Our previous investigations have demonstrated high expression of Spi2A inhibited lysosomal function and autophagy, leading to suppression of autophagic apoptosis in hematopoietic stem cells. Based on obvious low expression of Spi2A in murine MLL-AF9-positive AML LSCs and our initial studies, which showed over-activated autophagy and depressed apoptosis in MLL-AF9 LSCs, we assume aberrant activation of autophagy induced by lose of Spi2A is most likely to result in chemotherapy resistance in MLL-AF9 LSCs. Therefore, we will investigate whether autophagy would be inhibited in MLL-AF9 LSCs with over-expression of Spi2A, thereby improving apoptosis and senescence and alleviating chemotherapy resistance. Those will provide some theoretical clues to seek novel therapeutic strategies to cure patients with AML.

白血病耐药和复发仍然是目前白血病治疗成功的瓶颈。研究表明①白血病干细胞（LSCs）的存在是急性髓系白血病（AML）耐药和复发的根本原因，②自噬增强是LSCs产生耐药的机制之一。我们的前期研究发现造血干细胞中高表达的Spi2A能抑制溶酶体功能和自噬，从而抑制溶酶体功能过强导致的自噬性凋亡。基于MLL-AF9阳性AML小鼠 LSCs中Spi2A显著低表达，结合我们的初步发现MLL-AF9 LSCs存在高水平自噬和低水平凋亡，推测低水平Spi2A极有可能引起自噬异常激活导致MLL-AF9 LSCs耐药。为此本项目拟证实增强MLL-AF9 LSCs中Spi2A表达能否抑制自噬，促进凋亡和衰老，并缓解MLL-AF9 LSCs耐药，为寻找根治AML的新策略提供线索。

本研究按计划构建出MLL-AF9-AML小鼠模型，但构建小鼠Serpina3g表达慢病毒载体后其Spi2A蛋白表达量低，后续蛋白功能实验实施困难，故选择前期研究急性髓系白血病（AML）病人骨髓CD34+CD38-细胞（白血病干细胞，LSCs）中低水平的miR-34c-5p为研究对象，探讨miR-34c-5p在LSCs化疗耐药机制中的作用。研究中，我们证实体外上调 miR-34c-5p 表达水平可诱导 AML CD34+CD38-细胞衰老，同时，过表达 miR-34c-5p的KG-1a 细胞构建的小鼠白血病模型中白血病细胞负荷明显减少，有效抑制白血病的发生发展并促进 AML干祖细胞的清除。进而，我们继续研究 miR-34c-5p在AML LSCs中低表达进而使其抵抗衰老的分子机制，发现miR-34c-5p可以通过P53-P21cip1-CDKs/Cyclins或P53非依赖CDKs/Cyclins途径诱导AML CD34+CD38-细胞衰老，囊泡外排是AML LSCs内miR-34c-5p表达降低的重要机制之一，miR-34c-5p可通过靶向下调RAB27B抑制外泌体外排，进一步促进细胞内miR-34c-5p表达上调，形成miR-34c-5p/RAB27B/Exo-shedding正反馈调控。总之，本研究证明了AML LSCs通过RAB27B增加miR-34c-5p外排进而抵抗衰老是LSCs化疗耐药的机制之一，为进一步研究白血病化疗耐药机制打下了基础。本项目已经培养1名博士研究生和1名硕士研究生。本研究有2篇SCI论文在投。