靶向LDH-A能量代谢对T细胞急性淋巴细胞白血病的抗白血病效应及机制研究

Lactate dehydrogenase A（LDH-A）is a key enzyme of the glycolytic pathway, and is regulated by NOTCH1 and MYC.Our study will investigate NOTCH1 mutant and wild type cell lines（DU528,MOLT4）through treatment with LDHA inhibitor and LDHA knocked down by means of LDHA short hairpin RNAs.We will observe the affections of LDHA suppression on proliferation and apoptosis.And genetically inhibit LDHA to investigate the affections of attenuated LDHA expression increase GSI sensitive.Using MYC-induced T-ALL transgenic zebrafish model, we are going to construct tetracycline（Tet）-On/off system in zebra?sh.The plasmids: I-SceI-Rag2-rtTA-M2-I-SceI, I-SceI-LDHA-TetR-EGFP-I-SceI modulated conveniently by doxycycline（Dox）, co-microinjection into one-cell stage zebrafish embryos, generation Dox induction LDHA high expression in T-ALL transgenic zebrafish.We will investigate the process that LDHA promotes the T-ALL progression upon Tet on and delays the disease upon Tet off； We will also treat on two groups fishes with LDH-A inhibitor, and observe the anti tumor affection of LDHA.By exploring the LDHA as energy metabolism target in T-ALL, the study will provide theoretical proofs for new targeted therapy and the decreasing of T-ALL resistant rate.

LDH－A是糖酵解途径的关键限速酶，在急性T细胞淋巴细胞白血病（T－ALL）患者，血清LDH常显著增高，并为影响ALL预后的独立危险因素。已知LDH－A的表达受NOTCH1、MYC调节。本项目以NOTCH1突变及野生型细胞株（DU528、MOLT4）为研究对象，观察低氧刺激对细胞株LDH活性、细胞增殖及线粒体凋亡的影响和差异，进一步采用LDH-A抑制剂干预细胞株和特异性敲除LDH-A基因，观察白血病细胞增殖活力和凋亡状态等细胞生物学行为，确立LDH-A的促白血病细胞增殖和诱导凋亡耐受活力。同时以MYC诱导的T-ALL转基因斑马鱼模型为研究模型，构建强力霉素调控性I-SceI-Rag2-rtTA-M2-I-SceI，I-SceI-LDH-A-TetR-EGFP-I-SceI载体，联合注入单细胞期受精卵，建立可调控性LDH-A高表达的T-ALL转基因模型，观察Tet on 状态下LDH-A基因

急性T淋巴细胞白血病（T-ALL）往往表现为高白血病细胞负荷，易出现中枢神经系统浸润；血清乳酸脱氢酶（LDH）显著增高，对目前化疗方案易出现耐药，预后常常不佳。LDH－A是糖酵解途径的关键限速酶，并为影响ALL预后的独立危险因素。本研究以T-ALL患者骨髓单个核细胞和T-ALL细胞株Jurkat为研究对象，用LDHA小分子抑制剂oxamate及LDHA基因敲减，采用MTT方法观察其抗增殖效应，采用AnnexinⅤ分析早期凋亡，并进一步采用Western Blot分析其抗凋亡途径；采用集落形成实验分析其抗侵袭效应。通过上述实验，证明靶向LDHA对T-ALL具有抗增殖，抗侵袭，诱导凋亡的效应。采用qPCR方法分析MYC,NOX5,GLUT1等表达，发现靶向LDHA存在MYC表达下调，提示靶向LDHA可能为MYC依赖途径；且NOX5表达下调，ROS下降，提示靶向LDHA可能以NOX5依赖途径下调ROS; 通过Western Blot分析PI3K/AKT信号通路，发现靶向LDHA通过下调PI3K/AKT信号通路P-GSK，P-AKT发挥抗肿瘤效应。我们通过采用CRISPR Cas9技术敲除T-ALL斑马鱼模型（rag2-LDL-c-myc）的LDHA基因以及用小分子抑制剂oxamate干预转基因斑马鱼来验证其对T-ALL的抗白血病效应。并拟采用TUNEL分析体内抗凋亡效应，并采用免疫组化分析PI3K/AKT信号通路，qPCR分析MYC表达。通过以上实验证实靶向LDHA抗T-ALL效应,为T-ALL靶向治疗提供新的策略。