Hu Jiayuan, Yuan Zihao, Shu Yan, Ren Jun, Yang Jing, Tang Lisha, Wei Xingyu, Liu Yongcan, Jin Fangfang, Xiao Qiaoling, Chen Xinyi, Wu Nan, Zhao Wen, Li Ziwei, Zhang Ling
Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, College of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, China.
Department of Laboratory Medicine, Chongqing University Fuling Hospital, School of Medicine, Chongqing University, Chongqing, China.
Cancer Metab. 2025 Jun 18;13(1):32. doi: 10.1186/s40170-025-00401-6.
Acute myeloid leukemia (AML) with nucleophosmin 1 (NPM1) mutations represents a distinct subtype of leukemia. Emerging evidence suggests that regulation of redox metabolism contributes to tumorigenesis and reveals a metabolic vulnerability in anti-tumor therapies. However, the role of redox homeostasis between reactive oxygen species (ROS) and antioxidant systems plays in NPM1-mutated AML has not been fully elucidated.
First, ROS-related metabolic pathways in NPM1-mutated AML were analyzed using RNA-sequencing data. Intracellular and mitochondrial ROS levels in leukemia cells were detected using flow cytometry (FCM). The expression of nuclear factor (erythroid-derived 2)-like 2 (NRF2) was analyzed in public databases and further validated in AML primary blasts and cell lines by quantitative real-time PCR (qRT-PCR), western blotting, and immunofluorescence. Next, the mechanism underlying NRF2 expression was investigated through the RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation (MeRIP) and rescue experiments. Additionally, the downstream target gene of NRF2 was identified by bioinformatics analysis and chromatin immunoprecipitation (ChIP) assays. Furthermore, RNA interference and the NRF2 inhibitor ML385 were applied to explore the role of NRF2 in leukemia. Finally, the anti-leukemic effects of ML385 alone or in combination with the B-cell lymphoma 2 (BCL-2) inhibitor venetoclax on AML cells were investigated using FCM analysis and western blotting, and further explored in cell line-derived xenograft (CDX) mouse models.
In this study, we identified significant ROS accumulation in leukemia cells with NPM1 mutations. Meanwhile, elevated NRF2 expression and its nuclear localization were observed in NPM1-mutated AML cells. The high NRF2 expression levels were at least partially induced by fat mass and obesity-associated protein (FTO) via mA modification. Functionally, NRF2 exerts its antioxidant effects by transcriptionally upregulating malic enzyme 1 (ME1) expression and enhancing its activity. Targeting NRF2/ME1 axis reduced NADPH/NADP ratio, increased ROS levels, impaired leukemia cell viability, and promoted apoptosis. More importantly, NRF2 inhibitor ML385 in combination with venetoclax showed synergistic anti-leukemic activity in vitro and in vivo.
Overall, our findings provide new insight into the therapeutic potential of targeting NRF2 and guide the development of innovative combination therapies in NPM1-mutated AML.
伴有核磷蛋白1（NPM1）突变的急性髓系白血病（AML）是白血病的一种独特亚型。新出现的证据表明，氧化还原代谢的调节有助于肿瘤发生，并揭示了抗肿瘤治疗中的代谢脆弱性。然而，活性氧（ROS）与抗氧化系统之间的氧化还原稳态在NPM1突变的AML中所起的作用尚未完全阐明。
首先，利用RNA测序数据分析NPM1突变的AML中与ROS相关的代谢途径。采用流式细胞术（FCM）检测白血病细胞内和线粒体中的ROS水平。在公共数据库中分析核因子（红系衍生2）样2（NRF2）的表达，并通过定量实时PCR（qRT-PCR）、蛋白质免疫印迹和免疫荧光在AML原代细胞和细胞系中进一步验证。接下来，通过RNA免疫沉淀（RIP）、甲基化RNA免疫沉淀（MeRIP）和拯救实验研究NRF2表达的潜在机制。此外，通过生物信息学分析和染色质免疫沉淀（ChIP）实验鉴定NRF2的下游靶基因。此外，应用RNA干扰和NRF2抑制剂ML385来探究NRF2在白血病中的作用。最后，采用FCM分析和蛋白质免疫印迹研究ML385单独或与B细胞淋巴瘤2（BCL-2）抑制剂维奈克拉联合对AML细胞的抗白血病作用，并在细胞系来源的异种移植（CDX）小鼠模型中进一步探究。
在本研究中，我们发现NPM1突变的白血病细胞中存在明显的ROS积累。同时，在NPM1突变的AML细胞中观察到NRF2表达升高及其核定位。高脂肪和肥胖相关蛋白（FTO）通过mA修饰至少部分诱导了NRF2的高表达水平。在功能上，NRF2通过转录上调苹果酸酶1（ME1）的表达并增强其活性来发挥抗氧化作用。靶向NRF2/ME1轴降低了NADPH/NADP比值，增加了ROS水平，损害了白血病细胞的活力，并促进了细胞凋亡。更重要的是，NRF2抑制剂ML385与维奈克拉联合在体外和体内均显示出协同抗白血病活性。
总体而言，我们的研究结果为靶向NRF2的治疗潜力提供了新的见解，并为NPM1突变的AML中创新联合治疗的开发提供了指导。
