Mitochondrial DNA (mtDNA) G-quadruplexes (G4s) have important regulatory roles in energy metabolism, yet their specific functions and underlying regulatory mechanisms have not been delineated. Using a chemical-genetic screening strategy, we demonstrated that the JAK/STAT3 pathway is the primary regulatory mechanism governing mtDNA G4 dynamics in hypoxic cancer cells. Further proteomic analysis showed that activation of the JAK/STAT3 pathway facilitates the translocation of RelA, a member of the NF-κB family, to the mitochondria, where RelA binds to mtDNA G4s and promotes their folding, resulting in increased mtDNA instability, inhibited mtDNA transcription, and subsequent mitochondrial dysfunction. This binding event disrupts the equilibrium of energy metabolism, catalyzing a metabolic shift favoring glycolysis. Collectively, the results provide insights into a strategy employed by cancer cells to adapt to hypoxia through metabolic reprogramming.

线粒体DNA（mtDNA）G四链体（G4s）在能量代谢中具有重要的调节作用，但其具体功能和潜在的调节机制尚未阐明。使用化学遗传筛选策略，我们证明了 JAK/STAT3 通路是控制缺氧癌细胞中 mtDNA G4 动态的主要调节机制。进一步的蛋白质组学分析表明，JAK/STAT3通路的激活促进NF-κB家族成员RelA易位至线粒体，其中RelA与mtDNA G4结合并促进其折叠，导致mtDNA不稳定性增加，抑制mtDNA转录以及随后的线粒体功能障碍。这种结合事件破坏了能量代谢的平衡，催化有利于糖酵解的代谢转变。总的来说，这些结果提供了对癌细胞通过代谢重编程来适应缺氧的策略的见解。