Lysyl oxidase inhibition disrupts mitochondrial homeostasis to create vulnerability to ferroptosis in TNBC.
High metabolic heterogeneity and plasticity of triple-negative breast cancer (TNBC) contribute to therapy resistance, necessitating identification of therapeutic vulnerabilities. Here, we identify non-canonical functions of the extracellular matrix (ECM) remodeler, lysyl oxidase (LOX), in regulating glucose metabolism and mitochondrial homeostasis and show that inhibiting LOX generates targetable vulnerability to ferroptosis. Mechanistically, LOX interacts with PARKIN and its upstream kinase PINK1, which we identified as a substrate of LOX. LOX-mediated PINK1 oxidation suppresses PARKIN phosphorylation, stabilizing hypoxia-inducible factor 1-alpha (HIF-1α) and increasing glycolysis. Concomitantly, LOX inhibits PARKIN-mediated mitophagy and maintains mitochondria-ER contacts through VDAC1 stabilization, while the LOX-HSP90 complex promotes mitochondrial Ca2+ transport and ATP production. Inhibiting LOX suppresses glycolysis, disrupts mitochondrial dynamics, reduces OXPHOS and GPX4/FSP1, and induces compensatory DHODH activity. Our "one-two punch" approach combining LOX inhibition with clinical DHODH inhibitor suppresses tumor growth in vivo in chemo-free setting. Notably, LOX protein correlates with HIF-1α/GLUT1/GPX4 in TNBC patient tumors, supporting its clinical relevance.