剪接因子DHX15通过调控RUNX1-ETO9a/RUNX1-ETO比例参与急性髓细胞白血病（AML）发生发展的机制研究

Patients with t(8;21) /RUNX1-ETO (RE)-positive AML are generally classed into favorable outcome group, but enormous differences exist among these patients. Therefore, it’s of great significance to study the underlining mechanism of this type of AML. We previously found a high mutation rate and expression level of DHX15 in RE-positive AML patients, and knock-down of DHX15 inhibited the proliferation of RE-positive cells. Besides, we proved the importance of DHX15 in the regulation of proliferation, apoptosis, and differentiation of leukemic cells. Based on the fact that DHX15 is a splicing factor, and that the alterative spliced isoform of transcript, RE9a, is implicated in leukemogenesis in mice, we aimed at proving the hypothesis that DHX15 and its mutant are implicated in the pathogenesis and development of acute myeloid leukemia (AML) through their role in regulation of RUNX1-ETO9a/RUNX1-ETO ratio. And our project consists of the following dimensions: ① the analysis of relevance between the expression level or mutant of DHX15 and ratio of RUNX1-ETO9a/RUNX1-ETO on patient samples; ② the knock-down and restoration of expression of DHX15 with wide-type or mutant DHX15 on RE-positive cells from leukemia cell lines or leukemia mice transplanted with the combination of RE and RE9a, and the evaluation of effects of DHX15 on proliferation and apoptosis of RE-positive cells in vivo and in vitro; ③ the establishment of a splicing assay, and the evaluation of effects of down-regulation or mutant of DHX15 on RE alterative splicing. This project would help to reveal the mechanism underlining the pathogenesis of RE-positive AML and to improve the treatment outcome of those patients.

t(8;21)/RUNX1-ETO（RE）阳性AML被归为预后良好组，但其疗效差异大，发病机制有重要研究价值。我们发现：①RE阳性AML患者中DHX15突变频率高且DHX15表达升高，在RE阳性细胞中下调DHX15可抑制细胞增殖等；②DHX15参与白血病细胞增殖、凋亡与分化调控。因DHX15是剪接因子，RE异常剪接体RE9a可致小鼠白血病，本项目拟从如下方面验证DHX15及突变通过调控RE9a/RE比例参与RE阳性AML发生发展的假说：①临床上分析DHX15表达量及突变与RE9a/RE比例的相关性；②分别在RE阳性细胞、RE9a/RE联合导入后发病的小鼠骨髓细胞中进行DHX15下调及突变、野生型DHX15回复实验，观测对体内外RE阳性细胞增殖、凋亡等的影响；③分子水平上，建立剪接实验体系，观察DHX15下调或突变对RE可变剪接的影响。此研究有助于阐释RE阳性AML发病机制，改善其治疗效果。

DHX15是ATP依赖的RNA解旋酶，前期研究发现DHX15在AML中高表达且预后不良，在AML中检测到DHX15 R222G重现性突变，研究DHX15及其突变在白血病中的功能具有重要的临床意义。本项目揭示了DHX15 R222G突变可能不影响患者预后，且与野生型具有相似的生物学功能。进一步研究发现，从临床及细胞水平证实DHX15的表达量与AE9a/AE成负相关关系，分子水平揭示DHX15的蛋白与AE的RNA相互作用，蛋白水平明确DHX15的RecA1结构域与AE蛋白的RHD和NHR4结构域相互作用，提示DHX15参与AE9a的形成与调控作用。 通过生信、ChIP、EMSA等实验证明ETS1和SP1调控DHX15的转录并具有叠加效应。在dhx15敲除的斑马鱼模型中，揭示dhx15为斑马鱼生长发育必须基因，通过激活 UPR 信号通路导致斑马鱼CHT部位的次级造血缺陷。DHX15在Kasumi-1细胞中敲除致死，且敲低DHX15影响白血病细胞株增殖、周期、凋亡等生物学功能，通过RNAseq及WB检测其分子机制，发现DHX15通过ATM/AKT-p53-p21通路调控细胞周期、通过MTP-Caspase级联途径引起Raji细胞凋亡。上述研究阐释了DHX15在白血病发生发展中的作用及其可能机制，为DHX15的开发应用奠定了理论基础。