Targeting NADH homeostasis through GPD2 inhibition and metabolic stress induces tumor regression in colorectal cancer

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality worldwide and is characterized by marked metabolic reprogramming to sustain tumor growth and survival. In this context, mitochondrial glycerol-3-phosphate dehydrogenase (GPD2), a key enzyme in the glycerophosphate shuttle, emerges as a critical regulator of redox balance. Through analysis of publicly available datasets and tumor samples from mouse models of intestinal tumorigenesis, we demonstrate that GPD2 is consistently upregulated at both the mRNA and protein levels in CRC compared to normal mucosa. To investigate its functional role, we employed lentiviral-mediated knockdown of GPD2, which led to a marked decrease in proliferation and viability across multiple CRC cell lines. By a virtual screening of an in-house compound library, we identified a novel allosteric inhibitor of GPD2, termed G2i. Pharmacological inhibition of GPD2 using G2i significantly impaired tumor cell growth in vitro and in mouse models of CRC. Mechanistically, G2i disrupted mitochondrial NADH oxidation, leading to redox imbalance, accumulation of glycerol-3-phosphate (G3P), decreased ATP levels, and impaired mitochondrial respiration. These effects were reversible by restoring NADH oxidation capacity through LbNOX expression, pyruvate supplementation, or LOX-CAT induction, confirming that NADH accumulation and redox collapse are the primary vulnerabilities induced by GPD2 blockade. Consistent with disrupted electron flow and increased redox imbalance, GPD2 inhibition led to elevated ROS levels and increased lipid remodelling. Notably, G2i promoted ferroptosis through redox-mediated ROS production and modulation of lipid metabolism. Given the role of lactate dehydrogenase (LDH) as a compensatory cytosolic NADH sink, we tested the impact of GPD2 and LDH dual inhibition on CRC tumorigenesis. Co-targeting LDH and GPD2 synergistically enhanced redox stress, reduced NAD⁺ regeneration, and significantly suppressed CRC growth both in vitro and in vivo, potentiating G2i-mediated induction of ferroptosis. To further increase metabolic vulnerability, we implemented intermittent fasting (IF) protocols, which limit glycolytic substrate availability. IF potentiated the anti-tumor effects of G2i by amplifying NADH accumulation and energetic collapse, leading to enhanced tumor suppression through ferroptotic cell death. Collectively, our findings position GPD2 as a druggable node in CRC redox metabolism. Its inhibition induces synthetic redox stress that compromises mitochondrial function and tumor viability. This vulnerability can be exploited through combined targeting of compensatory NADH sinks or nutrient restriction strategies, defining a therapeutic framework based on redox imbalance and opening new avenues for metabolic interventions in CRC.