Intrinsic polyunsaturated fatty acid synthesis capacity dictates ferroptosis sensitivity under restricted arachidonic acid availability.

Ferroptosis is an iron-dependent type of regulated cell death driven by lipid peroxidation, in which polyunsaturated fatty acids (PUFAs) in membrane phospholipids serve as key substrates. Here, we identify PUFA biosynthetic capacity as a key determinant of ferroptosis sensitivity under arachidonic acid (AA)-limited conditions. Lipidomic and stable isotope-tracing analyses in human lung adenocarcinoma cell lines revealed that H1299 cells that harbor wild-type KEAP1 and are sensitive to ferroptosis have unexpectedly impaired PUFA biosynthesis and consequently low PUFA levels. Nevertheless, ferroptosis sensitivity in H1299 cells is maintained under normal culture conditions owing to an exogenous supply of AA. Culturing cells in the B27 supplement, which provides only essential fatty acids such as linoleic acid, in the presence of 1% dialyzed FBS reduced AA-containing phospholipids and rendered H1299 cells resistant to ferroptosis. In contrast, A549 cells with a KEAP1 mutation retained ferroptosis sensitivity under B27 conditions, enabled by their endogenous PUFA-synthesizing capacity. Strikingly, pharmacological inhibition of FADS2 or deletion of ELOVL5 in A549 cells phenocopied H1299 cells, conferring resistance under B27-AO conditions that was reversed by AA supplementation. These findings demonstrate that ferroptosis susceptibility is dictated by intrinsic PUFA biosynthetic capacity under AA-limited conditions resembling physiological lipid availability. Therefore, intrinsic PUFA biosynthetic capacity should be considered in ferroptosis-based cancer therapies.