组蛋白H3K9三甲基化修饰在MLL白血病中的作用及机制研究

Mixed lineage leukemia (MLL) fusion AML is characterized by poor-prognosis and high rate of relapse, probably due to the existence of leukemia stem cells (LSCs). Growing evidences have revealed an important role of epigenetic regulation in the development of MLL leukemia and the correlation between H3K9me3 modification and AML development. However, the specific function of H3K9me3 in the regulation of AML and LSCs is still largely unknown. Previously, by establishing two AML murine models induced by classic fusion MLL-AF9 and infrequent fusion MLL-NRIP3 respectively, we obtained two AML cell groups with one enriched with LSCs (LSC-high) and the remaining leukemic cells (LSC-low). ChIP-qPCR analysis revealed that the distribution of H3K9me3 on the promoter of LSC genes in LSC-high cells was lower than in LSC-low groups. This suggests a negative correlation between H3K9me3 levels and AML development and LSC maintenance. Here in the proposed study, we will analyze the genome-wide distribution of H3K9me3 in AML LSCs. In addition, we will observe the phenotype of AML mice induced by changing levels of H3K9me3 in LSCs. We will further delineate the underlying mechanism with series of molecular/biological experiments and validate the result in patient AML samples. We expect to reveal the role and mechanism of H3K9me3 modifications in AML development and LSC maintenance, and to provide the biological basis for new clinically treatment of MLL-r AML.

伴MLL融合的白血病以预后差、易复发为主要特点，较为明确的因素是白血病干细胞（LSC）的存在。研究表明，表观遗传调控在MLL白血病发生发展过程中发挥重要作用，而转录抑制性修饰H3K9me3也与白血病发生发展相关，但其具体作用和分子机制未知。我们前期通过建立经典融合突变MLL-AF9及罕见融合突变MLL-NRIP3白血病小鼠模型，分离富含LSC和去除LSC的白血病细胞群体，结合ChIP-qPCR分析发现LSC中H3K9me3在干性维持基因启动子区域低水平分布。因此，我们推测H3K9me3修饰的分布水平与白血病发展和LSC功能维持负相关。在本研究中，我们将通过分析白血病小鼠LSC中H3K9me3修饰在全基因组水平的分布，观察人为改变H3K9me3水平后的表型，并结合分子生物学手段和临床样本验证，揭示H3K9me3修饰调控白血病发展和LSC功能的分子机制，以期为临床治疗MLL白血病提供依据。

伴MLL基因重排的急性髓系白血病以预后差、易复发为主要特点，较为明确的因素是白血病干细胞（leukemic stem cells，LSC）的存在。研究表明，表观遗传调控在MLL白血病发生发展过程中发挥重要作用，而转录抑制性修饰H3K9me3也与白血病发生发展相关，但其具体作用和分子机制未知。SUV39H1是造血系统中占主导地位的H3K9甲基转移酶，其表达随年龄增长而下降。然而，SUV39H1通过其介导的抑制性修饰H3K9me3在白血病发生/白血病进展中的作用仍有待探索。与正常人相比，我们发现SUV39H1在包括MLL-r急性髓系白血病在内的多种白血病中下调。与造血干/祖细胞（HSPC）相比，MLL-r诱导的AML小鼠模型的LSCs中Suv39h1的表达降低，同时观察到基因组和TSS附近H3K9me3的分布水平降低。在MLL-r急性髓系白血病小鼠模型中，Suv39h1的过表达增加了白血病的潜伏期，降低了有功能的LSCs数目，而Suv39h1的敲降则加速了疾病的进展并伴随LSCs数量的增加。机制上，Suv39h1过表达导致Hoxb13和Six1失活，以及Hoxa9/Meis1下游靶基因的表达发生逆转，从而减缓了白血病的进展。有趣的是，在MLL-AF9诱导的AML细胞中，Hoxb13的表达上调，而在MLL-AF9白血病细胞中，Hoxb13的敲除显著延长了白血病小鼠的存活时间并降低了LSC数目。我们的数据显示SUV39H1在MLL-AF9诱导的AML进展中起抑制作用，其调控白血病干细胞数目的分子机制可以为临床治疗MLL白血病提供理论依据。