Feng Shuya, Yuan Yigang, Lin Zihan, Li Min, Ye Daijiao, Shi Liuzhi, Li Danyang, Zhao Min, Meng Chen, He Xiaofei, Wu Shanshan, Xiong Fang, Ye Siyu, Yang Junjun, Zhuang Haifeng, Hong Lili, Gao Shenmeng
Medical Research Center, The First Affiliated Hospital of Wenzhou Medical University, 1 Xuefubei Street, Ouhai District, Wenzhou, 325000, Zhejiang Province, China.
Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, 1 Xuefubei Street, Ouhai District, Wenzhou, Zhejiang Province, China.
Exp Hematol Oncol. 2024 Feb 20;13(1):19. doi: 10.1186/s40164-024-00489-4.
Ferroptosis is a new form of nonapoptotic and iron-dependent type of cell death. Glutathione peroxidase-4 (GPX4) plays an essential role in anti-ferroptosis by reducing lipid peroxidation. Although acute myeloid leukemia (AML) cells, especially relapsed and refractory (R/R)-AML, present high GPX4 levels and enzyme activities, pharmacological inhibition of GPX4 alone has limited application in AML. Thus, whether inhibition of GPX4 combined with other therapeutic reagents has effective application in AML is largely unknown.
Lipid reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) assays were used to assess ferroptosis in AML cells treated with the hypomethylating agent (HMA) decitabine (DAC), ferroptosis-inducer (FIN) RAS-selective lethal 3 (RSL3), or their combination. Combination index (CI) analysis was used to assess the synergistic activity of DAC + RSL3 against AML cells. Finally, we evaluated the synergistic activity of DAC + RSL3 in murine AML and a human R/R-AML-xenografted NSG model in vivo.
We first assessed GPX4 expression and found that GPX4 levels were higher in AML cells, especially those with MLL rearrangements, than in NCs. Knockdown of GPX4 by shRNA and indirect inhibition of GPX4 enzyme activity by RSL3 robustly induced ferroptosis in AML cells. To reduce the dose of RSL3 and avoid side effects, low doses of DAC (0.5 µM) and RSL3 (0.05 µM) synergistically facilitate ferroptosis by inhibiting the AMP-activated protein kinase (AMPK)-SLC7A11-GPX4 axis. Knockdown of AMPK by shRNA enhanced ferroptosis, and overexpression of SLC7A11 and GPX4 rescued DAC + RSL3-induced anti-leukemogenesis. Mechanistically, DAC increased the expression of MAGEA6 by reducing MAGEA6 promoter hypermethylation. Overexpression of MAGEA6 induced the degradation of AMPK, suggesting that DAC inhibits the AMPK-SLC7A11-GPX4 axis by increasing MAGEA6 expression. In addition, DAC + RSL3 synergistically reduced leukemic burden and extended overall survival compared with either DAC or RSL3 treatment in the MLL-AF9-transformed murine model. Finally, DAC + RSL3 synergistically reduced viability in untreated and R/R-AML cells and extended overall survival in two R/R-AML-xenografted NSG mouse models.
Our study first identify vulnerability to ferroptosis by regulating MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway. Combined treatment with HMAs and FINs provides a potential therapeutic choice for AML patients, especially for R/R-AML.
铁死亡是一种新的非凋亡性且依赖铁的细胞死亡形式。谷胱甘肽过氧化物酶4（GPX4）通过减少脂质过氧化在抗铁死亡中发挥重要作用。尽管急性髓系白血病（AML）细胞，尤其是复发难治（R/R）-AML细胞，呈现出高GPX4水平和酶活性，但单独对GPX4进行药理学抑制在AML中的应用有限。因此，抑制GPX4与其他治疗试剂联合应用在AML中是否有效应用在很大程度上尚不清楚。
使用脂质活性氧（ROS）、丙二醛（MDA）和谷胱甘肽（GSH）检测来评估用去甲基化剂（HMA）地西他滨（DAC）、铁死亡诱导剂（FIN）RAS选择性致死3（RSL3）或它们的组合处理的AML细胞中的铁死亡。联合指数（CI）分析用于评估DAC + RSL3对AML细胞的协同活性。最后，我们在体内评估了DAC + RSL3在小鼠AML和人R/R-AML异种移植NSG模型中的协同活性。
我们首先评估了GPX4表达，发现AML细胞中GPX4水平更高，尤其是那些具有MLL重排的细胞，高于正常对照细胞（NCs）。通过短发夹RNA（shRNA）敲低GPX4以及通过RSL3间接抑制GPX4酶活性在AML细胞中强烈诱导铁死亡。为了降低RSL3的剂量并避免副作用，低剂量的DAC（0.5 μM）和RSL3（0.05 μM）通过抑制AMP激活的蛋白激酶（AMPK）-溶质载体家族7成员11（SLC7A11）-GPX4轴协同促进铁死亡。通过shRNA敲低AMPK增强了铁死亡，而SLC7A11和GPX4的过表达挽救了DAC + RSL3诱导的抗白血病作用。机制上，DAC通过减少MAGEA6启动子高甲基化增加MAGEA6的表达。MAGEA6的过表达诱导了AMPK的降解，表明DAC通过增加MAGEA6表达抑制AMPK-SLC7A11-GPX4轴。此外，在MLL-AF9转化的小鼠模型中，与单独使用DAC或RSL3治疗相比，DAC + RSL3协同降低了白血病负担并延长了总生存期。最后，在两个R/R-AML异种移植NSG小鼠模型中，DAC + RSL3协同降低了未处理和R/R-AML细胞的活力并延长了总生存期。
我们的研究首次通过调节MAGEA6-AMPK-SLC7A11-GPX4信号通路确定了对铁死亡的易感性。HMA与FIN联合治疗为AML患者，尤其是R/R-AML患者提供了一种潜在的治疗选择。
