AMPK激动剂对人白血病细胞的抑制作用及机制研究

The competition between leukemic and normal hematopoietic cells determines the outcome of leukemia patients. Leukemia cells and hematopoietic stem cells （HSCs）share the ability to self-renew; thus, the genes and pathways associated with maligancies often also regulate HSC function. Much efforts has gone into identifying genes that are differentially expressed in leukemia relative to HSCs. In previous study, we have defined the different expression pattern of AMP-activated protein kinase (AMPK) between leukemia stem cells and normal HSCs. The phosphorylation of AMPK was depressed in HSC isolated from patients with de novo and relaped leukemia, whereas, up-regulated in HSC of patients achieved complete remission. Moreover, its expression pattern is strongly associated with the prognosis of leukemia patients. Hyperglycemia, which is not uncommon during the treatment of acute lymphocytic leukemia (ALL), has been shown to be an independent predictor of adverse outcomes among hospitalized patients with undiagnosed diabetes; it also may have the potential to affect leukemic cell proliferation through altered metabolism. Metformin is an activator of AMPK which inhibits protein synthesis and gluconeogenesis during cellular stress. The biological function of metformin has been linked to the functions of AMPK, which has essential roles in DNA replication, repair and cell cycle control. In the present project,we will further identify the association between phosphorylation of AMPK and prognosis in a expanded case-control study. The specific function of AMPK agonist metformin in leukemic cells will be investigated through gene, protein, cell and on the whole. And the aberrant up-stream signal pathway regulated AMPK in de novo and relapsed leukemia will be investigated through the animal model. The results obtained from cell lines will be verified in humanized leukemia model and clinical samples. The project will specify the function and mechanism of aberrant phosphorylation of AMPK in human leukemia and provide evidences for metformin application in leukemia therapy.

在前期工作中，我们发现伴有糖耐量异常的急性淋巴细胞白血病（ALL）患者无病生存期缩短，同时比对了白血病患者体内白血病干细胞和正常造血干细胞的AMPK表达情况，发现白血病干细胞AMPK磷酸化水平降低。而磷酸化AMPK/AMPK比值在白血病患者初发、复发时均降低，并与疾病的临床结局具有很强的相关性。进一步研究发现，AMPK激动剂二甲双胍不仅能控制血糖，还能提高AMPK磷酸化水平，并且有一定的抗肿瘤作用。结合临床研究结果，本项目拟进一步扩大病例研究，从mRNA表达水平、蛋白表达水平和细胞水平研究二甲双胍对白血病生物学特征的影响，以及AMPK磷酸化和白血病患者预后的关系；利用基因敲减及相应的功能实验，研究AMPK在白血病细胞中的确切功能；通过高通量检测方法、生物信息学和体内实验，明确二甲双胍调控白血病细胞生物学行为的方式，为AMPK激动剂及其作用通路成为白血病治疗靶点奠定实验基础。

本研究主要探讨了急性淋巴细胞白血病细胞AMPK通路受抑的机制。首先在临床研究中 我们发现伴有糖耐量异常的急性淋巴细胞白血病（ALL）患者无病生存期缩短。为此，我们设计了参照儿童急淋方案的CHALL-01，并在方案中加入了AMPK激动剂二甲双胍。在多因素分析中，二甲双胍的加入有利于提高患者的总生存率，降低复发率。比对了白血病患者体内白血病干细胞和正常造血干细胞的AMPK表达情况，发现白血病干细胞AMPK磷酸化水平降低。而磷酸化AMPK/AMPK比值在白血病患者初发、复发时均降低，并与白血病的临床结局具有很强的相关性。AMPK激动剂二甲双胍不仅能控制血糖，还能提高AMPK磷酸化水平，并且有一定的抗肿瘤作用。在体外研究中，二甲双胍对白血病细胞中AMPK的磷酸化水平呈剂量依赖性，且在50mM浓度下对AMPK的磷酸化水平最高。此外，二甲双胍还能促进肿瘤细胞凋亡，抑制细胞周期。在研究过程中我们还发现中药成分Icariside II能和二甲双胍竞争性结合AMPK位点，具有和二甲双胍协同作用。SPR竞争性实验发现ICAII和AMPK的亲和力比二甲双胍更高。ICAII在体外能促进肿瘤细胞凋亡和细胞周期（G2/M)停滞，且对白血病原代细胞具有抑制作用。. 该研究初步明确了二甲双胍对于急性淋巴细胞白血病具有抑制作用及其潜在机制。发现了中药成分IcarisideII能竞争性抑制AMPK位点，且和二甲双胍具有协同作用。