IDH突变肿瘤代谢物二羟基戊二酸致MDS向AML转化的机制研究

Somatic mutation in the isocitrate dehydrogenase (IDH) genes plays a vital role in myeloid neoplasms. Mutant genes encode neomorphic proteins that produce the presumed oncometabolite 2-hydroxyglutarate (2-HG), which is considered as the key effector of regulatory mechanism for contributing to tumorigenesis. Myelodysplastic syndromes (MDS) are a diverse group of clonal diseases derived from hematopoietic stem cells characterized by substantial risk for progression into acute myeloid leukemia (AML).In our previous studies,IDH mutations occurred at higher frequency in patients with AML which transform from MDS compared to those in low-risk MDS and the pathway of hypoxia-inducible factor 1 (HIF-1) was abnormally activated in high-risk MDS. We will use a series of new methods including magnetic activated cell sorting, gene sequencing technique, western blot, gas chromatography time of flight mass spectrometry, lentiviral transfection technique in this project,to assess the prevalence of IDH gene mutaions and the level of serum 2-HG in MDS. In the further research, to explore how 2-HG induces the disorganized state of deoxyribonucleic acid (DNA) methylated profile and the inhibition of histone demethylation according to repressing the function of ten-eleven translocation 2 (TET2) and histone lysine demethylases subfamily (KDMs), which are the α-ketoglutarate (α-KG) dependent dioxygenases.In addition, we will investigate the underlying principle whether 2-HG interrupt another α-KG dependent dioxygenase,prolylhydroxylases (PHD),to bring about the disorder of hypoxia-inducible factor 1 pathway and the accumulation of HIF-1 α in MDS.Identifying the potential molecular mechanism of IDH gene mutations and its oncometabolite 2-HG triggering MDS clone transforming to AML clone will be significant for providing the theoretical basis of a new and clinical promsing target in the treatment of MDS.

异柠檬酸脱氢酶（IDH）突变是髓细胞肿瘤的一个重要分子事件，IDH突变致肿瘤代谢产物二羟基戊二酸（2-HG）大量合成，以2-HG为核心的调控机制是IDH突变参与肿瘤发生的主要机制。骨髓增生异常综合征（MDS）具有向急性髓细胞白血病（AML）转化的特征，前期研究发现，MDS转化的AML中IDH突变较低危组MDS更为普遍，且存在与AML转化相关的低氧信号通路异常激活。本项目利用磁珠分选、Westernblot、基因测序、气相色谱质谱联用仪、慢病毒转染等技术，通过对大样本MDS骨髓标本IDH突变的检测及血清中2-HG含量的分析，并研究MDS细胞中2-HG如何通过影响α-KG依赖双加氧酶TET2、KDM及PHD对基因组DNA、组蛋白的甲基化以及低氧信号通路的调控作用，以此阐明IDH突变及其产生的肿瘤代谢产物2-HG参与MDS向AML转化的机制，为以IDH突变和2-HG为靶点的靶向治疗提供重要依据。

本研究检测了281例MDS患者突变情况，18例（6.4%）MDS患者检测到IDH1/IDH2突变。用液相色谱质谱法检测患者血清2-HG水平，结果显示，在MDS患者中，血清2-HG水平升高与MDS患者总体生存（OS）率及无白血病生存（LFS）率较低有关。较高的血清2-HG水平与IDH突变相关。IDH2突变往往伴随DNMT3A和SRSF2突变。IDH2突变患者白血病转化的风险较高。IPSS较低危组中，IDH2突变与患者较短的OS和LFS显著相关。以上研究表明IDH突变和2-HG提示MDS预后不良。.进一步研究了2-HG对MDS和AML细胞的作用和机制。结果显示，100-300uM的(2R)-Octyl-2-HG可抑制细胞增殖活性，引起细胞凋亡与G0/G1期阻滞，而对健康外周血单个核细胞的抑制作用较弱。体外诱导IDH2 R140Q突变亦引起细胞增殖抑制。(2R)-Octyl-2-HG作用后，程序性坏死（necroptosis）相关基因RIPK1表达增高。通过FAN法（检测细胞膜通透性）和免疫共沉淀等方法，发现(2R)-Octyl-2-HG作用细胞早期（10小时内），细胞膜通透性和RIPK1-Caspase8复合物的形成明显增加。通过RIPK1特异性抑制剂necrostain-1（NEC-1）和shRIPK1慢病毒抑制RIPK1的功能或表达，可减轻(2R)-Octyl-2-HG对细胞抑制作用。在SKM-1小鼠模型中，(2R)-Octyl-2-HG降低骨髓肿瘤细胞比例，延长生存时间。构建RIPK1敲降小鼠模型，结果表明抑制RIPK1的表达可减轻2-HG的肿瘤抑制作用。.检测50例MDS患者的骨髓单个核细胞RIPK1 mRNA水平，结果显示，RIPK1基因高表达患者的生存时间较长。分析TCGA和GEO公共数据库，显示AML患者骨髓单个核细胞RIPK1表达低于健康人，RIPK1高表达预示AML患者较长生存时间。.以上研究表明，2-HG可早期通过诱导细胞RIPK1基因表达增强，引起RIPK1依赖性程序性坏死，这种程序性坏死不依赖细胞凋亡与细胞周期阻滞，且可能早于细胞凋亡。RIPK1低表达水平提示MDS和AML患者预后不良。这些研究提示RIPK1是潜在的抗髓系肿瘤靶点，2-HG是针对RIPK1的潜在靶向药物，值得进一步研究。