HGF协同GATA2在急性红白血病发生中的作用及机制研究

Bone marrow microenvironment alterations play a vital role in the genesis and development of hematopoietic malignancies. Acute erythroid leukemia (AEL) is characterized by abnormal erythroid expansion, while the leukemogenesis of AEL remained unknown. Our previous results identified high frequencies of GATA2 mutations in AEL patients and proved that typical GATA2 mutations could promote erythroid differentiation of hematopoietic stem cells (HSC). With microarray we further found that the level of hepatocyte growth factor (HGF) expression was downregulated in AEL patients. Notably, HGF expression was significantly downregulated in GATA2 mutated AEL patients. Given the fact that GATA2 mutations in HSC alone were not able to generate AEL and HGF was the key regulator in erythroid development in the bone marrow microenvironment, we speculated that GATA2 mutations combined with lower HGF could favor AEL genesis. In this study, we are going to explore: 1) the molecular mechanisms of GATA2 mutations in modulating erythroid differentiation and downregulating HGF expression, 2) the synergistic role of HGF and GATA2 mutations in the abnormal erythroid expansion and the leukemogenesis of AEL in the severe immunodeficiency mice, 3) whether HGF could serve as the therapeutic target in AEL model. This project might partially elucidate the leukemogenesis of AEL and identify potential therapeutic targets, which could provide experimental basis of precise diagnostic and treatment strategies of AEL patients.

骨髓微环境紊乱在恶性血液病的发生发展中具有重要作用。急性红白血病（AEL）以红系异常增生为主要特征，发病机制尚不明确。我们前期研究发现AEL患者存在高频GATA2基因突变，且GATA2突变促进了造血干细胞向红系分化；通过基因表达谱发现AEL患者骨髓中肝细胞生长因子（HGF）表达水平下调，且在GATA2突变AEL患者中下调更为显著。造血干细胞单独GATA2突变并不能导致AEL，而HGF是骨髓微环境中维持红系发育的重要细胞因子，我们推测HGF下调协同GATA2突变促进了AEL的发生。本项目拟进一步通过体内外研究，阐明GATA2突变促进红系分化和下调HGF表达的机制；在免疫缺陷小鼠中，研究HGF下调协同GATA2突变在红系异常增生及AEL发生中的作用及机制；建立AEL模型，探讨HGF是否可作为AEL治疗靶点。本研究将部分阐明AEL的发生机制，鉴定潜在的治疗靶点，对AEL的精准诊疗具有重要意义。

急性红白血病（AEL）以红系异常增生且分化障碍为主要特征，其分子遗传学特征和发病机制尚不明确，缺乏有效的治疗手段，预后较差。项目组前期研究发现高频GATA2基因突变是AEL患者独特的分子遗传学特征，且AEL患者中维持红系发育的肝细胞生长因子（HGF）表达水平显著下调。本项目拟进一步建立GATA2突变患者的研究队列，明确GATA2突变与红系异常的相关性，通过体内外实验和突变敲入小鼠明确GATA2典型突变的作用和分子机制，探讨HGF信号通路的失活是否能协同GATA2突变产生AEL表型，阐明AEL可能的发病机制，为进一步临床诊疗提供重要实验基础。.本项目在实施期间，根据2016年WHO对AEL的最新诊断标准，总结分析了新标准下AEL的临床、表达谱和分子遗传学特征，明确高频的GATA2氨基端锌指结构（ZF1）突变是AEL的分子遗传学特征。并进一步建立GATA2突变AML患者研究队列，发现GATA2 ZF1突变与红系分化异常密切相关，ZF1突变患者粒/红细胞比值显著减少，血红素代谢相关通路受抑。体内外实验证明GATA2 ZF1突变中R330X突变显著促进红系祖细胞BFU-E增殖，阻滞红细胞成熟。纯合R330X突变敲入小鼠于E10.5天死于重度贫血，杂合小鼠的胎肝和成体红系幼稚细胞显著增加，红系成熟细胞比例减少，造血干祖细胞的数量和自我更新能力减弱。此外，体外红系定向分化实验表明抑制HGF通路能影响红系分化，但其与GATA2 R330X突变协同作用并不显著。通过CHIP-seq等手段，进一步阐明GATA2 R330X突变丢失了大部分DNA结合位点，保留了对红系分化成熟关键转录因子GATA1和KLF1的转录抑制，从而抑制红细胞成熟的作用机制。研究结果为AEL的发病机制探索及治疗提供了重要的理论指导。.本项目在国际重要学术刊物上，发表论文20篇，主编论著4部，牵头制定指南/共识2部，授权发明专利4项。项目培养了20名研究生，包括8名博士研究生，12名硕士研究生。

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