组蛋白去甲基化酶LSD1的抑制剂通过调节转录因子治疗MLL白血病的研究

Epigenetic drugs has great potential for new drugs in leukemia therapy, such as inhibitors of histone modification enzymes. However, the mechanism associated with epigenetic drugs in leukemia therapy is still unclear, which influences the improvement of the development and therapeutic efficacy for epigenetic drugs. Histone demethylase LSD1 inhibitors specifically upregulate histone H3 lysine 4 dimethylation (H3K4me2) and treat MLL leukemia using an unknown molecular mechanism. Based on the regulatory association of H3K4me2 with recruitment and transcriptional activity of key transcription factors (such as PU.1), we will apply our preliminarily established models of LSD1 inhibitors-induced MLL leukemia cell differentiation, to investigate critical changes in genome-wide distribution of H3K4me2 and transcriptome upon using LSD1 inhibitors. We will also explore the effect of H3K4me2 changes on the recruitment and transcriptional activity of PU.1. Then, we will further find other key transcription factors important in this process using sequence analysis of differential H3K4me2 enrichment regions, and confirm the regulatory relationship between these transcription factors and H3K4me2 using a ChIP-seq assay. Finally, we will perform functional experiments including gene knockdown functional assays, to validate that functional effects induced by LSD1 inhibitors depend on these key transcription factors, and determine transcriptional regulation of these LSD1 inhibitors and transcription factors on their downstream genes. These studies will reveal the mechanism by which LSD1 inhibitors treat MLL leukemia via targeting key transcriptional factors, and lay the foundation for developing more efficacious epigenetic drugs.

表观遗传药物如组蛋白修饰酶抑制剂是白血病治疗中有极大潜力的新型药物。然而相关的作用机理不清影响了这一类药物的发展和疗效提高。组蛋白去甲基化酶LSD1抑制剂能特异性升高组蛋白H3赖氨酸4的二甲基化（H3K4me2），通过未知的分子机制治疗MLL白血病。基于H3K4me2与关键转录因子（如PU.1）募集和转录活性的功能调节关系，我们将用前期已初步建立的LSD1抑制剂诱导MLL白血病细胞的分化模型，研究诱导后H3K4me2的基因组分布和转录组的关键改变，确定H3K4me2改变与PU.1的募集和转录活性的关联。进一步通过序列信息分析寻找其他关键转录因子，并用ChIP测序确定其与H3K4me2间的调节关系。最后，用基因敲低等实验验证LSD1抑制剂的功能效应依赖于转录因子，并确定对下游基因的转录调节。本研究将揭示LSD1抑制剂靶向关键转录因子治疗MLL白血病的机制，为开发更有效的表观遗传药物奠定基础。

表观遗传变异引起的异常转录调控在白血病的起始、发生和发展中发挥关键作用。通过靶向表观调控因子调节异常的表观调控，有望达到治疗白血病的效果。然而，现仅发现少数有效靶点能够治疗白血病。此外，已有的靶向小分子/药物的作用机制不明，限制了表观靶向治疗在白血病中的应用。研究白血病中参与产生和维持异常表观修饰的调控因子具有重要的科学意义和临床价值。.LSD1（KDM1A）是组蛋白去甲基化酶，能特异性去除组蛋白H3K4或H3K9的一/二甲基化。近年发现 LSD1是治疗急性髓系白血病的酶分子靶点，其抑制剂已用于临床试验研究（如TCP），但抑制LSD1治疗白血病的分子机制仍不清。我们利用LSD1抑制剂TCP诱导小鼠白血病细胞分化的模型，揭示了LSD1抑制剂通过介导H3K4me2修饰增加调节关键转录因子PU.1的靶向募集，进而激活PU.1转录程序促进MLL白血病细胞分化，发挥治疗MLL白血病的作用。该研究为开发更有效的表观遗传药物奠定基础。.Polycomb蛋白家族成员ASXL1/2突变高频出现于多种髓系恶性肿瘤，与预后不良紧密相关，但其在正常造血和肿瘤中的作用机制不明。我们利用基因敲除小鼠模型结合组学研究，功能上证明了ASXL1/2缺失单独/协同NF1基因缺失促进髓系肿瘤的发生，机制上揭示了造血发育和白血病发生相关转录程序的改变，而且这些变化与转录激活相关组蛋白修饰异常相关。更重要的是，异常激活通路的体内联合抑制有效阻碍白血病的起始和进展。这些研究既提供了潜在治疗策略，也为ASXL1突变作为克隆性造血突变增加正常人群白血病发病风险的现象提供了认识。.综上，我们发现了靶向LSD1的药物发挥疗效的分子机理，为改进药物疗效提供重要参考；揭示了ASXL1/2突变单独/协同介导发病的分子机制，为靶向携带这些基因突变的患者治疗提供了新靶点和潜在策略。