Vitamin B2 metabolism promotes FSP1 stability to prevent ferroptosis

Abstract Ferroptosis, a regulated form of cell death driven by excessive lipid peroxidation, has emerged as a promising therapeutic target in cancer. Ferroptosis suppressor protein 1 (FSP1) is a critical regulator of ferroptosis resistance, yet the mechanisms controlling its expression and stability remain mostly unexplored. To uncover regulators of FSP1 abundance, we conducted CRISPR–Cas9 screens using a genome-edited, dual-fluorescent FSP1 reporter cell line, identifying both transcriptional and post-translational mechanisms that determine FSP1 levels. Notably, we identified riboflavin kinase and flavin adenine dinucleotide (FAD) synthase, enzymes that are essential for synthesizing FAD from vitamin B2, as key contributors to FSP1 stability. Biochemical and cellular analyses revealed that FAD binding is critical for both FSP1 activity and stability. FAD deficiency and mutations blocking FSP1–FAD binding triggered FSP1 degradation through a ubiquitin–proteasome pathway involving the E3 ligase RNF8. Unlike other vitamins that inhibit ferroptosis by scavenging radicals, vitamin B2 supports ferroptosis resistance through FAD cofactor binding, ensuring proper FSP1 stability and function. This study provides a rich resource detailing mechanisms that regulate FSP1 abundance and highlights a novel connection between vitamin B2 metabolism and ferroptosis resistance, with implications for therapeutic strategies targeting FSP1 in cancer.