转录因子AML1-ETO与KLF4相互作用对CDKN1C基因的转录调控及对白血病细胞分化、凋亡的影响

The transcription factor AML1-ETO, formed by chromosomal translocation between chromosome 8 and 21, involved in the leukemogenesis through a variety of mechanisms to regulate gene transcription. However, the current understanding of its biological function and its transcriptional mechanism is still not enough to completely explain its pathogenesis in leukemia. In our previous study, a new binding protein of AML1-ETO, the transcription factor KLF4, was found to interact with AML1-ETO by CHIP-seq, immunofluorescence confocal and immunoprecipitation experiment. In this proposal, we will investigate the roles of the interaction between AML1-ETO and KLF4 in the following aspects: 1) to confirm the interaction between KLF4 and AML1-ETO, and to explore the specific binding domain; 2) to clarify whether AML1-ETO would interfere the interaction between AML1 and KLF4; 3) to investigate the effect of interaction between AML1-ETO and KLF4 on the transcription of CDKN1C, the KLF4 target gene; 4) to investigate the effect of KLF4 and its target gene on the differentiation and apoptosis of AML with AML1-ETO fusion protein; 5) to investigate the effects of KLF4 and its target gene on the in vivo leukemogenesis in AML1-ETO leukemia model. Through this proposal, we could clarify the pathogenesis of AML1-ETO in leukemia and find new therapeutic target for this type of leukemia.

转录因子AML1-ETO是由 t(8;21)易位形成的融合蛋白，通过多种机制调控基因转录，参与白血病的发生发展。目前对该转录因子生物学功能和转录调控机制的认识尚不足以完整的解释其致病机理。我们前期通过CHIP-seq、荧光共聚焦和免疫共沉淀等实验方法发现了一个新的与AML1-ETO相互作用的转录因子KLF4。本项目以AML1-ETO与KLF4相互作用为切入点展开研究，旨在：1) 明确KLF4与AML1-ETO结合并确定具体的结合结构域；2) 明确AML1-ETO能否干扰AML1与KLF4的相互作用；3) 探讨两者相互作用对KLF4靶基因CDKN1C转录的影响；4) 探讨KLF4及其靶基因在携带AML1-ETO的AML中的分化、凋亡作用；5) 探讨KLF4及其靶基因对AML1-ETO白血病细胞体内致病性的影响。本项目将有助于进一步揭示AML1-ETO的致病机制，发现该型白血病新的治疗靶点。

本项目研究中，我们对前期发现的融合基因AML1-ETO与KLF4、CDKN1C之间存在相互作用的基础上，研究了KLF4与AML1-ETO相互结合及其具体的结合结构域、AML1-ETO 与KLF4 相互作用对KLF4靶基因CDKN1C (p57)转录调控的影响、KLF4、CDKN1C过表达对携带t(8;21)染色体易位的白血病细胞分化、凋亡的影响，及其对AML1-ETO小鼠白血病细胞体内致病性的影响。结果发现：1）AML1通过Runt同源结构域(RHD)与KLF4结合，AML1-ETO除了通过RHD还通过ETO部分与KLF4结合，AML1-ETO能竞争性地抑制野生型AML1与KLF4的结合；2）AML1以剂量依赖的方式激活KLF4的启动子/增强子报告质粒，而AML1-ETO对KLF4启动子/增强子报告质粒的转录激活作用明显减弱，KLF4对P57启动子/增强子报告质粒亦存在剂量依赖性的激活效应；3）在t(8;21)白血病细胞(Kasumi-1)中过表达AML1、KLF4和P57均能抑制白血病细胞增殖、阻滞细胞周期、促进细胞凋亡；4)在AML1-ETO+白血病小鼠骨髓细胞中分别过表达KLF4和P57，移植受体小鼠，发现过表达KLF4和P57可以降低t(8;21)白血病小鼠的发病率，延长小鼠生存时间。本项目的研究结果，阐明了转录因子AML1与KLF4之间的作用关系以及AML1-ETO融合蛋白对这种作用关系的影响，进一步揭示了AML1-ETO的致病机制，为该型白血病提供新的治疗靶点。