伴EVI1基因高表达MDS高风险向AML转化的分子致病机制研究

The clinical and biological studies of myelodysplastic syndrome with high EVI1 expression are relatively rare acquired, and the pathogenesis has not been quite clear so far. In the previous study, we retrospectively analyzed a large cohort of MDS patients, and found that patients with high EVI1 expression had more adverse clinical and cytogenetic characteristics, higher risk of disease progression and mortality, which can be considered as an unfavorable prognostic factor independent of IPSS-R. Based on current research, we put forward the following scientific questions: Firstly, high expression of EVI1 gene is associated with disease progression in MDS. Are there any other synergistic factors involved in disease transformation? Secondly, JQ1, a BRD4 inhibitor, regulates the expression of EVI1 mediated by 3q rearrangement. Can it also down-regulate the expression of EVI1 in MDS patients and further interfere with disease progression? Therefore, in this proposed project, we will use NUP98-HOXD13 transgenic mice as MDS model, EVI1 knock-in mice model, to clarify the molecular pathogenic mechanism of EVI1 overexpression leading to disease progression and adverse prognosis in MDS patients from cell, molecular and animal levels, and to provide experimental basis for the development of appropriate precise treatment strategies.

伴EVI1基因高表达MDS患者临床及分子生物学研究较少，其致病机理也尚不明确。本课题组回顾性分析了国际上较大一组MDS患者，发现EVI1高表达患者具有更高危的临床及细胞遗传学特征，较高的疾病进展风险及死亡率，是独立于IPSS-R的不良预后因素。基于此我们提出科学问题：EVI1基因的高表达与MDS患者疾病进展相关，是否有其他协同因素共同参与疾病的转化？BRD4抑制剂JQ1对3q重排引起的EVI1表达具有调控作用，是否也可以干预MDS患者中EVI1基因的表达，作为潜在的治疗靶点，进一步干预疾病的进展？本申请项目拟以NUP98-HOXD13转基因小鼠为MDS模型联合EVI1敲入小鼠，从分子水平、细胞水平、小鼠模型等多方面研究，明确EVI1高表达MDS患者疾病进展或不良预后的分子致病机制，为发展相应的精准治疗策略提供实验基础。

目的：分析不同EVI1表达量的MDS患者的临床、细胞遗传学及分子生物学特征，通过体内外实验揭示EVI1高表达的分子生物学异常，并初步探讨其可能的致病机制。.方法： 1. 1033例成人MDS患者纳入研究，以300copies/10000ablcopies为cut-off将其分为EVI1高表达及低表达组，回顾性分析其临床和预后特征。2.通过NHD13小鼠与EVI1过表达的敲基因小鼠杂交，比较EVI1过表达在MDS小鼠转白及生存过程中的影响。3. 通过不同MDS阶段小鼠的RNA测序，比较其分子生物学异常，并初步探讨其可能的致病机制。4. 根据上述结果选取针对EVI1高表达的MDS的靶向候选药物，在体内外实验中验证药物的疗效和特异性。.结果：1. 1033例MDS患者中EVI1高表达者229例(22.2%)， EVI1高表达的MDS患者更为年轻，且较低表达组有更多的病例进展比率 (12.7% VS 5.6%, P=0.015)，尤其是向急性白血病转化 (8.7% VS 5.4%, P=0.045)。OS及EFS显著差于EVI1低表达组。2. 高通量NGS检测492例MDS患者，可见EVI1高表达患者中74.6% (97/130) 至少具有一种及以上的基因变异，常见变异为：U2AF1 (23.1%)、ASXL1 (16.2%)和TP53 (12.3%)等。3. 伴有EVI1高表达的NHD13小鼠随着年龄的增加逐渐出现外周血象的不同程度的减低，表现为更低的Hb、HCT、RBC及PLT，且更快进展为急性白血病 (中位转白时间259天, P=0.0044)。4. 对不同EVI1表达 MDS小鼠骨髓前体细胞 RNA测序推测Cdkn2a、Foxc1及Rorc可能在NHD13+EVI1high小鼠更快的转白过程中发挥一定的作用。5. 体外实验提示NHD13+EVI1high对JQ1药物治疗高度敏感，显著减少骨髓c-kit+细胞克隆形成能力。NHD13+EVI1high小鼠体内JQ1单药治疗提示其一定程度阻滞EVI1高表达的MDS小鼠转白进程。.结论：1. EVI1高表达在MDS病人及小鼠体内均带来不良预后及更快的白血病转化进程。2. 表观遗传学调控在转白进程中扮演重要作用，BRD4抑制剂JQ1体外显著抑制前体细胞的集落形成能力，体内亦阻滞EVI1高表达MDS转白进程。

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