PO.MCB07.02 · 分子与细胞生物学
AMPK在染色质上与PRC1.1复合物相互作用,以响应急性淋巴细胞白血病中的代谢应激调控NADK表达
AMPK interacts with PRC1.1 complex on chromatin to regulate NADK expression in response to metabolic stress in acute lymphoblastic leukemia
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摘要 Abstract
中文摘要
急性淋巴细胞白血病(ALL)仍是儿童癌症相关死亡的主要原因。在AMP激活蛋白激酶(AMPK,细胞能量稳态的主调控因子)激活后,ALL细胞对代谢应激特别易感。我们和他人已经证明,AMPK与染色质相关蛋白相互作用,以响应能量应激调节基因表达。使用TurboID邻近标记蛋白质组学进行的全面AMPK相互作用组分析,确定非经典多梳抑制复合物1.1(PRC1.1)为AMPK相关复合物。共免疫沉淀证实在KASUMI-2(Bp-ALL)和KE-37(T-ALL)细胞中AMPK与PRC1.1组分(PCGF1、RING1、KDM2B、BCOR、BCORL1、USP7、SKP1和RYBP)之间的相互作用,这些相互作用在使用变构AMPK激活剂(PF-06409577、991)处理后增强。为评估AMPK激活对PRC1.1活性的功能后果,我们测量了代谢应激下的H2AK119ub水平,观察到显著增加。相比之下,用AMPK激活剂处理的AMPKalpha1/alpha2双敲除(DKO)HEK293T细胞未表现出变化,证实了AMPK依赖性。在KASUMI-2和KE-37细胞中的ChIP-seq分析确定烟酰胺腺嘌呤二核苷酸激酶(NADK)为AMPK相关的染色质靶标。ChIP-qPCR证明在代谢应激下AMPK、BCOR和KDM2B在NADK启动子处的积累,与H2AK119ub增加和RNA聚合酶II占据减少相关,提示转录抑制。RT-qPCR和免疫印迹分析证实暴露于代谢应激的ALL细胞中NADK下调,而AMPK(BAY-3827)或RING1(PRT4165)的药理学抑制则上调NADK表达。同样,在HEK293T AMPKalpha1/alpha2缺陷细胞中NADK抑制被消除,而USP7的药理学抑制(CDDO-Me和Eupalinolode B)增加了ALL细胞中的NADK表达。NADK催化NAD⁺磷酸化为NADP⁺,支持合成代谢和氧化还原稳态所必需的NADPH生成。NADK抑制耗竭NADPH,诱导氧化应激,并抑制增殖。我们提出,AMPK介导的NADK转录抑制代表了一种在能量应激下强制维持代谢稳态的适应性机制。与此一致,AMPK激活剂ASP4132与NADK抑制剂硫代烟酰胺联合处理在ALL细胞中诱导协同细胞毒性,提示靶向AMPK-PRC1.1-NADK信号通路的潜在治疗策略。
查看英文原文 English abstract
Acute lymphoblastic leukemia (ALL) remains a leading cause of pediatric cancer-related mortality. ALL cells are particularly vulnerable to metabolic stress following activation of AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis. We and others have demonstrated that AMPK interacts with chromatin-associated proteins to modulate gene expression in response to energy stress. A comprehensive AMPK interactome analysis using TurboID proximity labeling proteomics identified the non-canonical Polycomb Repressive Complex 1.1 (PRC1.1) as an AMPK-associated complex. Co-immunoprecipitation confirmed interactions between AMPK and PRC1.1 components (PCGF1, RING1, KDM2B, BCOR, BCORL1, USP7, SKP1, and RYBP) in KASUMI-2 (Bp-ALL) and KE-37 (T-ALL) cells, which were enhanced following treatment with allosteric AMPK activators (PF-06409577, 991). To evaluate the functional consequence of AMPK activation on PRC1.1 activity, we measured H2AK119ub levels under metabolic stress and observed a marked increase. In contrast, AMPKalpha1/alpha2 double knockout (DKO) HEK293T cells treated with AMPK activators exhibited no change, confirming AMPK dependency. ChIP-seq analysis in KASUMI-2 and KE-37 cells identified Nicotinamide adenine dinucleotide kinase ( NADK ) as an AMPK-associated chromatin target. ChIP-qPCR demonstrated accumulation of AMPK, BCOR, and KDM2B at the NADK promoter under metabolic stress, correlating with increased H2AK119ub and reduced RNA polymerase II occupancy, indicative of transcriptional repression. RT-qPCR and immunoblot analyses confirmed NADK downregulation in ALL cells exposed to metabolic stress, while pharmacological inhibition of AMPK (BAY-3827) or RING1 (PRT4165) upregulated NADK expression. Similarly, NADK repression was abrogated in HEK293T AMPKalpha1/alpha2-deficient cells, and pharmacological inhibition of USP7 (CDDO-Me and Eupalinolode B) increased NADK expression in ALL cells. NADK catalyzes phosphorylation of NAD⁺ to NADP⁺, supporting NADPH generation essential for anabolic metabolism and redox homeostasis. NADK inhibition depletes NADPH, induces oxidative stress, and suppresses proliferation. We propose that AMPK-mediated transcriptional repression of NADK represents an adaptive mechanism to enforce metabolic homeostasis under energy stress. Consistently, combined treatment with the AMPK activator ASP4132 and the NADK inhibitor thionicotinamide induced synergistic cytotoxicity in ALL cells, suggesting a potential therapeutic strategy targeting AMPK-PRC1.1-NADK signaling.
利益披露 Disclosure
A. Shvab, None..
G. J. Leclerc, None.