Metabolic circuitry of NADH buffering reveals a vulnerability in redox-adaptive cancer cells.

Mitochondrial dysfunction elevates cellular NADH/NAD+ ratios, inducing reductive stress that impairs biosynthesis and cell viability. However, the mechanisms by which cells buffer excess NADH to maintain redox homeostasis remain unclear. In this study, we identify a mitochondrial-cytosolic metabolic circuitry involving pyruvate carboxylase (PC), malate dehydrogenases (MDH1/2), and malic enzyme 1 (ME1) that mitigates NADH overload by coupling anaplerotic flux with NADH oxidation. Under reductive stress induced by electron transport chain (ETC) dysfunction, oxaloacetate derived from PC is converted to malate by MDH1/2, which is then oxidized by ME1, transforming both cytosolic and mitochondrial NADH into cytosolic NADPH. Although cellular NADPH is typically associated with antioxidant defense and biosynthesis, our experiments show that TPNOX, which can oxidase NADPH to NADP+, restores proliferation under ETC inhibition, and this rescue is entirely dependent on the mitochondrial-cytosolic metabolic circuitry. This finding highlights that the primary function of the circuit is to maintain NADH homeostasis rather than to generate NADPH. Notably, glucose-6-phosphate dehydrogenase (G6PD) enhances ME1 activity independently of its catalytic function by acting as a scaffold, thereby preventing net NADPH production while facilitating the conversion of NADH to NADPH. Disruption of the PC-MDH1/2-ME1 pathway through PC inhibition sensitizes cells to complex I inhibitors and/or glutaminase blockade, synergistically suppressing tumor growth both in vitro and in vivo. These findings uncover a redox-buffering strategy that redirects NADH into NADPH production, revealing a metabolic vulnerability in redox-adapted cancer cells.