Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has emerged as a promising therapeutic target for cancer. However, accumulating evidence indicates that tumor cells exhibit insensitivity to classic ferroptosis inducers. In the present study, through high-throughput screening of a metabolite library, we identified cerulenin as a potent ferroptosis sensitizer. Mechanistically, cerulenin dually targets fatty acid synthase (FASN) and hydroxymethylglutaryl-CoA synthase 1 (HMGCS1), thereby inhibiting their enzymatic activity. FASN inhibition unexpectedly promoted ferroptosis via suppressing carnitine O-palmitoyltransferase 1 (CPT1)-mediated lipid β-oxidation, thereby triggering polyunsaturated fatty acid (PUFA) accumulation to drive ferroptosis. Simultaneously, HMGCS1 inhibition disrupted the mevalonate pathway, leading to impaired selenocysteine tRNA maturation and subsequent suppressing the synthesis of the glutathione peroxidase 4 (GPX4) protein, which enhances the sensitivity of tumor cells to ferroptosis inducers. Clinical bioinformatics analysis of TCGA datasets revealed significant co-overexpression of solute carrier family 7 member 11 (SLC7A11), FASN, and HMGCS1 in multiple malignancies, which correlated with poor patient survival rates. In vivo, cerulenin synergized with classic ferroptosis inducer erastin to suppress xenograft tumor growth without observable toxicity. Collectively, this study revealed that cerulenin dual-targets FASN/HMGCS1 and obtained with remarkable pro-ferroptosis properties, providing a mechanistically distinct therapeutic paradigm for precision cancer therapy.
