DNA甲基化在t(8;21)急性髓系白血病发病中的作用与机理研究

Translocation (8;21) (q22;q22) is one of the most common acute myeloid leukemia (AML) cytogenetic abnormalities and t(8;21) produces the chimeric protein AML1/ETO （AE） which is constituted of the N-terminal AML1-derived part and the C-terminal ETO part that contain a DNA-binding domain and a corepressor-binding domain. Wild AML1 is a critical regulator in hematopoiesis and has an essential function in the establishment of definitive hematopoiesis. AE acts as a suppressor of wild AML1 and blocks myeloid differentiation. Although a lot of evidences show AE is critical for causing myeloid leukemia, but the mechanism detail of AE for leukemogenesis is not clear. AE can alter the epigenetic environment of hematopoietic cells dependent on its ability to interact with histone deacetylases and methylases. By compared with AE negative AML by Infinium HumanMethylation450 BeadChip, we found 14 genes with DNA hypermethylation in AE positive AML. Bioinformatics analysis show that 5 genes (DKK1,LRRC49,PAIP2B,SEPT9 and THAP10) of those 14 genes abound with CpG island and also harbor AML1 binding sites. Interestingly, many reseaches reported that DKK1 gene, SEPT9 gene and THAP10 gene are tumor suppressor genes. Those results indicate that AE protein may cause leukemogenesis after AE protein combined with AML1 binding sites in promotor regions and then silenced those genes with DNA hypermethylation. This project is to study which genes are really methylated by AE protein and the corresponding epigenetic mechanisms, and then further to explore the role of DNA hypermethylation of those 5 genes in the leukemogenesis of AE positive leukemia, and to analyse the relationship between DNA methylation of those 5 genes and prognosis of AML with t(8;21). Thus, we can learn the role of those 5 genes with DNA methylation in AML with t(8;21) and preliminarily screen out some genes to predict the prognosis and to monitor the leukemia.

t(8;21)急性髓系白血病（AML）的发病机制与t(8;21)形成AML1/ETO（AE）融合蛋白有关，但具体机制仍不清楚。通过全基因组甲基化芯片筛查我们发现，有14个基因的DNA甲基化在AE（+）AML细胞显著高于AE（-）AML细胞。生物信息学分析证实，14个基因中的DKK1、LRRC49、PAIP2B、SEPT9及THAP10等5个基因启动子区富含CpG岛并存在AML1结合位点，且其中3个为抑癌基因。因此，我们推测AE蛋白可能通过结合这5个基因启动子的AML1位点，引起基因甲基化沉默而参与白血病发生。本课题通过甲基化PCR和CHIP技术研究5个基因中真正被AE蛋白甲基化调节的基因及其调节机制，体外细胞和裸鼠体内实验探讨5个基因对AE（+）细胞发生白血病转化的影响，并通过病例分析基因甲基化状态与t(8;21)白血病预后的关系，从而了解DNA甲基化在t(8;21)白血病发病中的意义。

不管是从生物学角度，或是从临床的角度来说，t(8;21)急性髓系白血病都是异质性较高的一种疾病，这就导致了此种疾病在精准临床分类、预后分层和靶向用药等方面出现很大困难。在t(8;21)急性髓系白血病中，DNA甲基化谱常常发生异常变化，但目前对于到底是哪些基因的甲基化发生了异常变化尚不十分清楚，对于发生了异常甲基化的基因到底是如何被调控的也不十分清楚。t(8;21)急性髓系白血病（AML）的发病机制与t(8;21)形成AML1/ETO（AE）融合蛋白有关。本项目通过全基因组甲基化芯片筛查发现，有14个基因的DNA甲基化在AE（+）AML细胞显著高于AE（-）AML细胞。生物信息学分析证实，14个基因中的DKK1、LRRC49、PAIP2B、SEPT9及THAP10等5个基因启动子区富含CpG岛并存在AML1结合位点，且其中3个为抑癌基因。因此，我们推测AE蛋白可能通过结合这5个基因启动子的AML1位点，引起基因甲基化沉默而参与白血病发生。本课题以THAP10为突破口，通过甲基化PCR和CHIP技术研究了THAP10基因真正被AE蛋白甲基化调节的基因及其调节机制，实验证实：融合蛋白AE可以通过结合在THAP10基因的启动子区上的AML1结合位点，并且募集了DNA甲基化转移酶（DNMT3a和DNMT3b）以及组蛋白去乙酰化酶（HDAC1），从而使THAP10的启动子区发生了去乙酰化和高甲基化，并进一步导致THAP10基因发生沉默。体外细胞和裸鼠体内实验探讨THAP10基因对AE（+）细胞发生白血病转化的影响，通过高表达THAP10基因，可以明显抑制白血病细胞的增殖和诱导分化。并通过病例分析基因甲基化状态与t(8;21)白血病预后的关系，从而了解DNA甲基化在t(8;21)白血病发病中的意义。