ENO1介导的靛玉红衍生物PHII-7通过AMPK/mTOR信号途径对AML抗白血病作用机制的研究

The incidence of acute myeloid leukemia (AML) is high and the clinical efficacy is limited. Relapse and resistance are the main causes of the failure of AML treatment. The abnormal patterns of energy metabolism and AMPK/mTOR pathway in AML cells are closely related to cell resistance and leukemia stem cells (LSC). ENO1 is a key enzyme in energy metabolism, which has been found in AML patients, but there haven't any report about the study of ENO1 function in AML yet. PHII-7 is a compound that has been screened by our research group and has desired activity in overcoming multidrug resistance and low toxicity to normal tissues. It is found that ENO1 is the target protein of PHII-7 by chemical biologic method so that PHII-7 is an ideal tool for studying ENO1. Our previous results showed that the tumorigenicity of LSC could be inhibited by down regulation of ENO1. This project will explore the mechanism of PHII-7 anti-leukemia efficacy meidated by ENO1 through AMPK/mTOR pathway. We will clarify the role of ENO1 in PHII-7 anti-leukemia activity, get advantage of clinical specimens, leukemia cell models and NODSCID AML animal model. Our study may provide new idear for the AML therapy in the future.

急性髓系白血病（AML）发病率高且临床治疗有效率有限。复发和耐药是导致AML治疗失败的主因。AML细胞能量代谢模式及AMPK/mTOR信号通路异常与耐药、白血病干细胞（LSC）干性的维持密切相关。ENO1是能量代谢的关键酶，已在AML患者中发现高表达，但目前尚未报道ENO1在AML中的功能。我们之前已发现下调ENO1可显著抑制LSC的致瘤能力。PHII-7是我们课题组筛选得到的具有较好抗耐药白血病活性的靛玉红衍生物，且已鉴定获知ENO1是PHII-7的靶蛋白。以上述结果及理论为基础，我们提出假说：ENO1可能参与介导了PHII-7所致的AML细胞能量代谢抑制并因此激活了AMPK/mTOR信号途径。本研究拟从临床研究、细胞模型以及动物水平等多角度探明ENO1介导的PHII-7通过AMPK/mTOR信号途径对AML及LSC细胞的抗白血病作用及机制，有望为今后AML的治疗提供新的靶点与思路。

FLT3-ITD突变是AML不良预后标记之一，治疗手段有限且生存期短。烯醇化酶ENO1是我们以抗肿瘤化合物靛玉红衍生物PHII-7为靶标、通过化学生物学、蛋白质组学鉴定、分子对接及药理学研究发现的髓系白血病潜在致病基因。我们通过对多个公共数据库队列研究数据进行挖掘分析发现ENO1高表达与FLT3-ITD(+)AML的不良预后显著相关，这一现象在随后的本中心生物样本库的AML中得到验证。为进一步研究ENO1的功能，我们以FLT3-ITD(+)AML细胞系Molm-13为模型，构建了ENO1稳定敲减的细胞株Molm-13.shENO1，体内外研究发现ENO1抑制后可增强AML细胞对FLT3抑制剂索拉非尼、吉瑞替尼的敏感性，减慢细胞增殖及AML细胞的致瘤能力。转录组学研究发现抑制ENO1可下调NADPH氧化酶表达，且ENO1表达与GPX4表达呈正相关，以PHII-7或ENO1敲减抑制ENO1的表达都将导致细胞内氧化还原的失衡、氧化应激产生，进而促进药物引起的细胞死亡。此外，我们还通过体内外药动学研究发现PHII-7可通过ENO1激活AMPK/PXR/CYP3A5信号通路，提升CYP3A5底物药物他克莫司在体内的药物浓度，发挥减毒、增效的作用。上述结论为研发AML治疗的新靶点与新策略奠定了理论基础。