组蛋白去甲基化酶KDM3B对白血病干细胞增殖与分化的表观遗传学调控

Accumulating evidence has shown that histone modifying enzymes play an importantrole in regulating all aspects of hematopoiesis，and many of them have been shownto be abnormally regulated in leukemia. The epigenetic landscape is clearlyaltered in acute leukemia, due to a variety of acquired lesions in chromatinmodifier genes, or changes in their level of expression. In acute myeloid leukemia(AML), chromosome 5q31 deletion was found in about 20% of the cases, suggesting atumor suppressor gene may exist in the deletion region. In our effort to identifytumor suppressor candidates in the deleted 5q31.1 region, we cloned a candidategene, named 5q nuclear co-activator (5qNCA), and investigated its function andeffect on leukemia cell proliferation and differentiation. It was lately renamed as KDM3B. Recent studies have shown that KDM3B is a H3K9 histone demethylase,participating histone modification and chromatin remodeling. However, the role itplays in leukemogenesis remains unclear. Understanding the role of KDM3B may play in contribution to leukemogenesis could provide the rationale for exploring how these abnormalities can be targeted by new therapeutic approaches. In this proposal, we will investigate the function of the KDM3B and its abnormality in contributing to leukemogenesis. The study includes four specific aims: 1. Using chromatin immuneprecipitation(ChIP) and microarray analysis (ChIP on chip), we will identify KDM3B targeting transcription factors and partners that function as chromatin modifying enzymes, and further characterizing their transcription regulatory role in leukemia. 2. Using viral vector mediated KDM3B gene overexpression in 5q31.1 deleted leukemia cell lines MUTZ-8 and KG-1, and shRNA knock-down KDM3B in HL-60 and OCI-AML-5, we intend to learn the effects of KDM3B on leukemia stem cell self-renew and differentiation, and to understand the mechanism that KDM3B interacts with transcription factors in regulation of leukemia cells proliferation and differentiation. 3. To further investigate the effect of KDM3B in leukemia, we will perform mutation and deletion analysis in large samples of AML patients using high-through-put DNA sequencing. This study may provide evidence for determining the relationship between KDM3B abnormality and clinical outcome of AML patients. 4. In order to develop drugs for specific epigenetic therapies to revise aberrant gene expression in leukemia, we have screened the TimTec Natural Products Library (NPL, 480 compounds) by cell-based high-throughput assay to identify and develop for non-cytosine analogue small molecule compounds that (1) induce cell-cycle exit of leukemia stem cells by late differentiation gene activation, (2) do not cause apoptosis, and (3) do not terminate self-renewal of normal stem cells. The approach may discover small molecular compounds targeting various properties of the epigenetic machinery which is often deregulated in leukemia stem cells.

组蛋白修饰酶异常所致的白血病干细胞（LSC）表观遗传紊乱是导致白血病发生和白血病耐药的重要原因之一。组蛋白去甲基化酶KDM3B基因位于染色体5q31。约20%的急性髓系白血病存在5q31的缺失，提示KDM3B和LSC表观遗传的联系。本项目旨在研究KDM3B对LSC增殖与分化的表观遗传学调控机制。1.探索KDM3B在基因组范围内的特征结合区以及相互作用的造血细胞转录因子，进而了解KDM3B和哪些转录因子相互作用，靶定哪些关键基因，从而对LSC的增殖与分化进行表观遗传学调控；2.揭示KDM3B对白血病干细胞“干”性维持、自我更新与分化等细胞生物学行为的影响；3.弄清KDM3B在白血病患者中基因突变或缺失的情况，以及与其他组蛋白修饰酶和转录因子协调失衡与白血病发病和预后的关系；4.研究从天然小分子化合物文库中筛选出的靶定组蛋白修饰酶，促进LSC分化，抑制其增殖，且对正常造血干细胞无损伤的化合物。

本项目旨在研究KDM3B对LSCs增殖与分化的表观遗传学调控机制。具体研究内容包括：1.了解KDM3B和哪些转录因子相互作用，靶定哪些关键基因，从而对LSCs的增殖与分化进行表观遗传调控；2.揭示KDM3B对白血病干细胞干性维持、自我更新与分化等细胞生物学行为的影响；3.弄清KDM3B在白血病患者中突变或缺失的情况，以及与其它因子协调失衡与白血病发病和预后的关系；4.研究从天然小分子化合物文库中筛选出的靶定组蛋白修饰酶、促进LSCs分化、抑制其增殖、且对正常造血干细胞无损伤的化合物。.我们发现：1.急性髓系白血病（AML）中KDM3B能结合到造血系统关键的HOXA家族基因启动子之上并调控HOXA家族基因如HOXA1、HOXB4和HOXC11等；KDM3B潜在的相互作用蛋白包括RARα等。2.KDM3B抑制AML的发生，是一个潜在的AML抑制基因。3.KDM3B是在AML中表达含量最低的基因之一，并且同p53突变关系密切，同5q33定位基因RBM22、5q32定位基因CSNK1A1的低表达相关、同KDM6A互斥表达，表明这些基因异常可能协同贡献AML的发生；KDM3B也在特定细胞遗传学异常如MLL-rearranged AML和PML-RARA 融合基因AML中低表达，表明KDM3B的低表达可能在MLL-rearranged AML和PML-RARA 融合基因AML的发生中发挥重要作用；我们还在HOXA家族基因调控区上发现了一个同AML相关的可能被KDM3B结合分非编码体细胞突变，提示KDM3B调控AML的新机制。4. 我们通过虚拟筛选，针对KDM3B的酶活性结构域，从天然产物和中药组分库中筛选出KDM3B家族的抑制剂和激动剂，并能分别特异性杀伤MLL-rearranged AML和白血病干细胞（LSCs）。5.KDM3B家族参与调控自然杀伤（NK）细胞，而KDM3B调节小分子能调节NK活性。6.KDM3B参与调控铁死亡。总之，我们按照项目标书的实施方案，鉴定出了KDM3B靶基因和相互作用蛋白，揭示了KDM3B负调控AML，弄清了KDM3B在AML中突变的情况，更重要的是我们筛选到了有抑制AML作用的KDM3B抑制剂和激活剂。不仅如此，我们还探索了KDM3B在免疫调节和铁死亡中的作用。我们的研究对于实现基于KDM3B突变AML的靶向精准治疗具有重要的临床转化潜力。