三羧酸循环改变在PMF转白的机制研究

Tumor cells requires a large amount of energy to support the rapid proliferation, which leads to three carboxylic acid cycle disorder.This metabolic disorder had been regarded as one of hallmarks of rapid turnover of tumor cells, which is associated with poor prognosis in several types of tumors. Primary myelofibrosis (PMF) is a subgroup of myeloproliferative neoplasms. Currently, there is no effective strategy to prevent the initiation and development of this disease. PMF patients have genes mutations including JAK2V617F, CALR, MPL and IDH1/2 in a small part of patients. IDH1/2 are the key genes of three carboxylic acid cycle,and these genes mutations will lead to 2-HG accumulation and in turn 2-HG promotes the conversion of PMF to acute myeloid leukemia (AML) by aberrant epigenetic regulation. We conduct this study to explore the mechanism of initiation of PMF and development to AML by analyses of metabolomics and DNA methylation patterns in PMF patients, and therefore identify the prognostic significance of metabolic markers. In order to confirm the significance of small metabolites on initiation of PMF and development to leukemia, we treat JAK2V617F induced cell lines and mice model with sodium chloroacetate to regulate the abnormality of three carboxylic acid cycle, and perform metabolomics and epigenetic profiling analyses. This study will provide reliable biomarkers and treatment targets to predict the development of leukemia from PMF.

肿瘤细胞增殖需要利用大量的能量，可导致三羧酸循环紊乱，三羧酸循环紊乱是肿瘤增殖旺盛的特性之一，和许多肿瘤发生与预后相关。原发性骨髓纤维化（PMF）是骨髓增殖性疾病的一种，目前尚无有效防治其发生、发展的方法。PMF患者伴有JAK2V617F、CALR、MPL等基因突变，部分伴有IDH1/2突变，后者是三羧酸循环中的关键基因，该基因突变导致2-HG产生增多，加剧表观遗传学紊乱，可能是促使PMF向急性髓细胞白血病（AML）转化的机制。本研究拟检测PMF及向AML转化患者的代谢组、表观遗传学变化，明确小分子代谢物及表观遗传学相关基因在PMF预后判断中的意义。构建JAK2V617F突变细胞株及小鼠模型，检测小分子代谢物及表观遗传学相关基因，并采用调节三羧酸循环的小分子物质二氯乙酸钠处理细胞株及小鼠，证实这些代谢物与PMF发生和转白的相关性，为临床预测PMF转化为白血病提供可靠的标志物。

原发性骨髓纤维化疾病原因未明，最终多数进展为骨髓衰竭或向急性髓细胞白血病转化，目前没有有效的治疗方法。研究其发病机制，以及探索如何有效地防止PMF的形成及向AML转化是本课题的研究目标。我们收集了142例骨髓增殖性疾病患者的骨髓细胞和血清，对其中30例骨髓纤维化及30例健康人的血清样本进行代谢组学检测。代谢组学分析证实三羧酸循环在原发性骨髓纤维化患者中上调，提示该通路可作为诊断标志物和治疗的靶点。对30例骨髓纤维化患者进行追踪随访，结果发现JAK2阳性率为50%，MPL阳性率为7%，CALR阳性率为23%，三阴率25%。同时追踪随访并总结11例骨髓纤维化转白患者的临床特点，检测JAK2/MPL/CALR/IDH1/IDH2/ASXL1基因突变，并对分子学特点及生存进行分析。发现原发性骨髓纤维化转白发生率是所有骨髓增殖性疾病中最高的，占比90%。转白患者基因检测发现，IDH1、IDH2及ASXL1突变发生率高，也证实这些基因与转白密切相关。转白后患者的预后极差，对治疗反应不佳，耐受性差，中位生存期仅10个月。我们成功构建JAK2-V617F质粒，验证HEL和SET-2均存在JAK2-V617F突变：1849G>T突变。同时将ASXL1转染至HEL细胞系中，并筛选出ASXL1稳定细胞株，进而在体内外实验中，进行了细胞株的一系列分子生物学的功能试验。随后，我们采用JAK抑制剂卢可替尼联合PLK1抑制剂Volasertib处理HEL及SET2细胞系，达到非常明显的协同抑制作用，进一步采用RNA-seq方法发现两药作用的共同基因为myc基因，采用western blot方法验证myc蛋白的下调为联合作用的共同靶蛋白，进而发挥联合抑制疗效。本项目为骨髓纤维化转白的机制提供理论基础，同时为骨髓增殖性疾病尤其是原发性骨髓纤维化的治疗提供新的方法，同时有望延缓白血病转化的发生。

内容获取失败，请点击重试