Pyridoxal Reprograms Peroxisomal Hydrogen Peroxide Metabolism to Induce Lethal Oxidative Stress in Ovarian Cancer

ABSTRACT While pyridoxal 5′‐phosphate (PLP) is considered the bioactive form of vitamin B6 with context‐dependent anticancer roles, we redefine its non‐phosphorylated precursor, pyridoxal (PL), as a direct and potent cytotoxic agent against ovarian cancer. Using validated patient‐derived organoids encompassing diverse histological subtypes and orthotopic in vivo models, we demonstrate that unmetabolized PL triggers lethal oxidative stress independent of PLP conversion. Mechanistically, PL induces PEX11A‐dependent peroxisome fission and disrupts PEX5‐dependent catalase import, fundamentally switching peroxisomal function from a protective hydrogen peroxide (H 2 O 2 ) scavenger to a dominant H 2 O 2 generator through accelerated fatty acid β‐oxidation. This peroxisome‐driven H 2 O 2 surge propagates severe oxidative damage, causing mitochondrial dysfunction, DNA fragmentation, and intrinsic apoptosis. The cytotoxic cascade is significantly rescued by antioxidants, confirming redox imbalance as the primary death mechanism. Our findings reveal an unprecedented mechanism wherein PL exploits peroxisomal metabolism to create a lethal H 2 O 2 overload, effectively repurposing a cellular detoxification organelle into a vulnerability. This work establishes unmodified PL as a novel cancer therapeutic agent by reprogramming redox homeostasis and positions peroxisomal H 2 O 2 flux as a druggable target in ovarian cancer. Inhibiting PL phosphorylation enhances efficacy, offering a promising strategy for targeting therapy‐resistant disease.