转录因子RUNX1功能缺陷对造血干细胞生长分化的影响以及与白血病发病的关系

Leukemogenesis is a multi-hit process; multiple genetic and epigenetic abnormalities accumulate to create leukemia initiating cells (LIC). Clinical data suggests that the cell in which the first hit occurs (the cell of origin, CO) is frequently a stem cell. However,it is not resolved whether additional hits that create LIC occur in the stem cell compartment or in lineage-committed daughter cells. Potentially,lineage-committed daughter cells of the CO provide the largest candidate cell pool for additional hits and neoplastic evolution. Runx1 haploinsufficiency (+/-) is a known 'first hit' that occurs in stem cells. Mutations in RUNX1 that produce functional RUNX1 haploinsufficiency (RUNX1 +/-) are the most common identified cause of Myelodysplastic Syndrome(MDS) and AML. In this study, murine Runx1 +/- hematopoietic stem cells were used to examine if an AML associated first-hit, produces potentially important phenotypic consequences in lineage-committed daughter cells. Using lineage-negative cells (Lin-) isolated from Runx1 +/- and WT control bone marrow and cultured the Lin-/CD117+ HSC cells in media with self-renewal promoting cytokines (stem cell factor[SCF], thrombopoietin) or differentiation promoting cytokines (SCF, IL6 and G-CSF), we will focus on the following studies : 1). The effects of RUNX1 haploinsufficiency on the self-renew and differentiation of hematopoietic stem cells(HSC); 2).The potential mechanisms of impaired RUNX1 interaction with other hematopoietic transcription factor CEBPA and CEBPE in regulation of HSC differentiation and in leukemogenesis. 3). Using Lentiviral vector to construct CEBPA specific shRNA expression vector pLenti6-shCEBPA. By transfection of pLenti6-shCEBPA into RUNX1 deficient HSC，a HSC model with RUNX1 and CEBPA double deficiency can be established. Using this HSC model, effect of RUNX1 and CEBPA double deficiency on the HSC self-renew and differentiation will be studied. 4). Determination of RUNX1,CEBPA mutations and CEBPE promoter methylation in a large panel of AML samples and investigation of the possible role of RUNX1 deficiency and reduced CEBPE expression in leukemogenesis. The findings from this study will bring insight into understanding the pathophysiological mechanisms of RUNX1 deficiency in leukemogenesis, and provide experimental data for guiding clinical leukemia therapy.

由基因突变或染色体t（8；21）易位而致RUNX1功能缺陷是引发急性髓系白血病（AML）和MDS发生的重要原因之一。本项目拟用RNUX1 Knock-Out杂合子小鼠模型，研究转录因子RUNX1基因功能缺陷对骨髓造血干细胞（HSC）生长和分化的影响，及与AML发病的关系。拟解决以下科学问题：1. RUNX1功能缺陷对HSC生长与分化功能的影响；2. RUNX1 与其它转录因子的协同作用，在调节HSC生长与分化中的作用机制及与白血病发生的关系；3. RUNX1和CEBPA双重缺陷对HSC生长分化的影响及与白血病发生的关系；4.AML病人RUNX1、CEBPA基因突变及CEBPE启动子甲基化程度。以验证RUNX1功能缺陷可使其与下游转录因子CEBPA, CEBPE的结合活化能力异常，致使HSC过度增生，细胞分化功能减弱，以致HSC在遭受基因突变的二次打击下，易引发白血病产生这一科学假设。

本课题研究应用基因敲除RUNX1+/- 缺陷型小鼠为模型，研究转录因子RUNX1功能缺陷对造血干细胞生长分化的影响以及与白血病发病的关系。结果表明，RUNX1+/- 缺陷型小鼠的LIN-/CD117+ 细胞，在体外培养G-CSF 诱导下与野生型对比具有明显的生长优势。 CEBPE 的表达量在RUNX1+/-小鼠Lin-/CD117+ HSC 细胞中显著减小。且RUNX1+/- Lin-/CD117+ 细胞在诱导分化后表达表面细胞分化标记Gly6G 和CD11b显著减底， 细胞克隆形成能力却明显增加。用Pyro-Sequencing 测定野生型和RUNX1+/- Lin-/CD117+ HSC CEBPE 启动子 CpG 甲基化状态，表明CEBPE 启动子 中3个CpG 位点在RUNX1+/- Lin-/CD117+ HSC 细胞中的甲基化程度明显高于野生型。通过检测33例急性髓系白血病（AML）患者和100例正常对照人群RUNX1的P1启动子、P2启动子、+23增强子、内含子5.2增强子、PU.1启动子、CEBPA启动子、CEBPE启动子。我们发现RUNX1及其靶基因启动子上存在多个单核苷酸多态性位点。遗传学分析表明这些遗传位点中，位于RUNX1内含子5.2上增强子处的rs2249650和rs2268276与AML发病相关。表明RUNX1内含子5.2上存在着新的AML易感性基因座。并发现rs2249650和rs2268276等位基因的不同组合导致不同的RUNX1内含子5.2增强子活性。而RUNX1下游重要的转录因子PU.1能结合于rs2249650和rs2268276位点。该实验结果揭示了RUNX1及其靶基因协同调控白血病发病以及易感性的潜在分子机制。对于白血病的早期检测、易感性的揭示，有重要意义。.根据本课题研究结果，我们已递交发表SCI收录论文1篇， 稿件在审稿中（Leukemia，IF 9.1分）。EI收录论文1篇（印刷中）。与国外科研机构合作发表与本课题相关的SCI收录论文2篇。培养硕士生3名及实验人员2名。组织召开了一次由国内外知名血液学专家参加的学术会议“泰山学术论坛研-白血病与淋巴瘤专题”。每位项目组成参加国内学术会议至少1次。选派了一名项目组成员去美国休斯顿MD Anderson Cancer Center进修学习6个月。