Endoplasmic Reticulum-Mitochondria Contacts are apical hotspots of lipid peroxidation driving ferroptosis

Peroxidation of membrane phospholipids (PLs) is a hallmark of ferroptosis. ER and mitochondria have been implicated in ferroptosis, but whether intracellular PL-peroxidation ensues at their contact sites (EMCSs) is unknown. Using super-resolution live imaging we charted the immediate spatiotemporal events triggered by ferroptosis at the inter-organelle level. EMCSs expand minutes after the formation of localized and activated lipid-peroxides that secondary spread to mitochondria, promoting mitochondrial ROS accumulation and fission. Oxidative lipidomics unravels that EMCSs host distinct pro-ferroptotic polyunsaturated (PUFA)-PLs, including doubly PUFA-acylated PLs, demonstrating the persuasive propensity of EMCS to PL-peroxidation. Genetic disruption of EMCSs blunts PL-peroxidation and ferroptosis, while EMCS stabilization enhances them. Analysis of human triple-negative breast cancer (TNBC) data shows that expression of EMCShigh-gene signature associates with the ferroptosis-susceptible TNBC subtype harboring heightened levels of oxidized-PLs. Our data reveal EMCSs as apical orchestrators of lethal lipid-peroxidation production and suggest that empowering EMCSs can promote ferroptosis in vulnerable cancer cells.