Endoplasmic reticulum-mitochondria contacts are prime hotspots of phospholipid peroxidation driving ferroptosis.

The peroxidation of membrane phospholipids (PLs) is a hallmark of ferroptosis. The endoplasmic reticulum and mitochondria have been implicated in ferroptosis, but whether intracellular PL peroxidation ensues at their contact sites (endoplasmic reticulum-mitochondria contact sites, EMCSs) is unknown. Using super-resolution live imaging, we charted the spatiotemporal events triggered by ferroptosis at the interorganelle level. Here we show that EMCSs expand minutes after localized PL peroxides are formed and secondarily spread to mitochondria, promoting mitochondrial reactive oxygen species and fission. Oxidative lipidomics unravels that EMCSs host distinct proferroptotic polyunsaturated-PLs, including doubly proferroptotic polyunsaturated-acylated PLs, demonstrating their high propensity to undergo PL peroxidation. Endoplasmic reticulum-mitochondria untethering blunts PL peroxidation and ferroptosis, while EMCS stabilization enhances them. Consistently, distancing EMCSs protects the ferroptosis-susceptible triple-negative breast cancer subtype, harbouring high EMCS-related gene expression and basal PL peroxide levels. Conversely, in insensitive triple-negative breast cancer subtypes, bolstering EMCSs sensitizes them to ferroptosis. Our data unveil endoplasmic reticulum-mitochondria appositions as initial hubs of PL peroxide formation and posit that empowering EMCSs endorses ferroptosis in cancer cells.