CLEC11A的表观遗传学沉默影响急性髓系白血病预后的机制研究

Acute myeloid leukemia (AML) is a common type of leukemia with a clonal disorder of myeloid hematopoiesis and a predominantly fatal hematopoietic malignancy with high heterogeneity. Clinical and genetic features incompletely predict the outcome in AML, so it is difficult to achieve precise diagnosis and treatment. DNA methylation is the core and most widely studied epigenetic modification in numerous cancers and often correlates with prognosis. Aberrant DNA methylation and tumor suppressor gene silencing is a common theme and a hallmark in the leukemogenesis of AML with potential prognostic significance. In our previous study based on genome-CpGs-scale methylC-capture sequencing (MCC-Seq), the DNA methylation level of AML patients was detected and a panel of hyper-differentially methylated genes with inferior outcome was identified. CLEC11A is the most valuable prognostic marker. It can make further stratification for intermediate-risk AML and guide demethylation chemotherapy by its DNA methylation level. Based on the previous study, we will further explore the mechanism of epigenetic silencing of CLEC11A, the effect of CLEC11A involved in signaling pathways, and explore the role of hypomethylation agent on CLEC11A. This will clarify the mechanism of CLEC11A on the prognosis of AML, and confirm that CLEC11A can be used as a new prognostic marker associated with demethylation treatment. It will provide theoretical basis for precise diagnosis and treatment of AML.

急性髓系白血病（AML）是白血病的主要类型，发病机制复杂、临床异质性大，目前临床及遗传学特征不能完全准确地评估预后，难以实现精准诊疗。DNA甲基化是一种重要的表观遗传学调控方式，异常的DNA甲基化模式和抑癌基因的表达沉默是AML发生发展的重要因素，具有潜在的预后意义。课题组前期利用全基因组CpG位点甲基化捕获测序技术检测AML患者DNA甲基化，筛选出预后不良相关的甲基化调控基因，发现CLEC11A高甲基化是AML患者预后不良因素，并可对中危患者进一步精准分层及指导去甲基化治疗。在此基础上，课题组拟进一步研究CLEC11A甲基化沉默的机制、CLEC11A调节下游信号通路的作用及生物学功能，并探讨去甲基化药物对CLEC11A的作用。这将阐明CLEC11A的表观遗传学沉默影响AML预后的机制，证实CLEC11A可作为AML的新的预后标志物并可指导去甲基化治疗，为AML的精准诊疗提供理论依据。

AML是一种具有高度异质性的疾病，现有的预后因素不足以完全解释AML的异质性，因此需要采用新的技术方法来寻找有效的预后指标。在本课题中我们采用全基因组CpG位点甲基化捕获测序（Genome-CpGs-scale methylC-capture sequencing，MCC-Seq）技术筛选AML患者中的甲基化差异基因。通过MCC-seq技术在初治AML患者中筛选出甲基化差异基因CLEC11A，对人急性髓系白血病细胞系中CLEC11A的表达进行检测，发现THP1中CLEC11A表达水平最高，而在U937中的表达最低。通过检测CLEC11A的甲基化状态和地西他滨对CLEC11A的调控作用发现，CLEC11A的高甲基化导致基因的低表达，地西他滨能够上调CLEC11A表达，发挥促进分化、抑制肿瘤增殖、周期阻滞和克隆形成的功能。在体外实验中证实CLEC11A在AML中发挥抑癌基因的功能，能够促进分化、抑制肿瘤增殖、周期阻滞和抑制克隆形成的功能。并在裸鼠内证明了CLEC11A抑制肿瘤增殖的作用在初治AML中发现CLEC11A高甲基化AML患者CR率低，长期生存差。利用TCGA公开数据库中同时有表达芯片和甲基化测序结果的149例AML患者的信息进行大样本人群中的验证，同样发现CLEC11A的甲基化水平与预后相关，甲基化水平高的AML患者的OS和DFS均差于低甲基化的患者，CLEC11A低表达的患者OS和DFS差于高表达的患者，甲基化和基因表达的结果趋势一致。这些结果证明CLEC11A的甲基化可以作为AML不良预后的一项指标。CLEC11A在AML中发挥抑基因癌作用，而CLEC11A甲基化导致基因沉默，在AML中是一种不良预后因素。地西他滨能够通过去甲基化上调CLEC11A的表达，发挥治疗作用。目前单基因甲基化指导AML预后分层的研究尚无报道，我们的结果有助于建立对基因甲基化检测进行指导预后的体系，CLEC11A是一个具有明确预后价值的甲基化检测基因。