Vitamin B2 keeps a key anti-ferroptosis enzyme stable, and a riboflavin mimic can switch it off in cancer cells

Ferroptosis is a form of regulated cell death driven by membrane lipid peroxidation and implicated in cancer, neurodegeneration and ischaemia-reperfusion injury. Cells resist it through defenses including GPX4 and ferroptosis suppressor protein 1 (FSP1). FSP1 uses NAD(P)H to regenerate antioxidants such as ubiquinone and vitamin K. Skafar and colleagues investigated factors that support this protective activity, identifying riboflavin metabolism as an important regulator.

The researchers engineered HT1080 cancer cells lacking GPX4 but overexpressing FSP1, making their survival dependent on FSP1. An FSP1 inhibitor killed these cells, while the ferroptosis inhibitor liproxstatin-1 rescued them. Two independent CRISPR–Cas9 screens targeting about 3,000 potentially druggable genes compared conditions with and without liproxstatin-1. The top hits were SCD1 and RFK, which encodes riboflavin kinase. RFK phosphorylates riboflavin to produce FMN, a precursor of FAD, the flavin cofactor used by FSP1.

CRISPR-mediated RFK deletion in A375 cells reduced FSP1 protein and increased sensitivity to the GPX4 inhibitors RSL3 and ML210. RFK loss also impaired viability and induced ferroptosis in the FSP1-dependent HT1080 model. However, unedited cells rapidly outcompeted RFK-deficient cells, limiting further experiments. DepMap analysis likewise indicated that RFK loss was poorly tolerated by most cell types examined.

The researchers therefore disrupted FADS, encoded by FLAD1, which catalyses the final step in FAD synthesis. FADS loss was better tolerated than RFK loss and allowed the establishment of deficient clones and addback controls. FADS-deficient A375 cells showed increased sensitivity to GPX4 inhibition and increased lipid peroxidation, with similar effects reported in the engineered HT1080 model. The excerpt also describes proteomic analysis using five technical replicates, an unpaired two-sided t-test, P < 0.05 and a minimum fold-change threshold of 1.5; it does not provide the detailed protein-level results claimed in the original summary.

The abstract and introduction conclude that riboflavin supports FSP1 stability and activity, enabling antioxidant recycling and limiting phospholipid peroxidation. The abstract additionally reports that roseoflavin, a riboflavin antimetabolite, markedly impairs FSP1 function and sensitizes cancer cells to ferroptosis. The supplied excerpt does not include its dose-response results or detailed mechanism.

These findings suggest a preclinical strategy for weakening FSP1-dependent ferroptosis resistance through riboflavin metabolism. Several immunoblots in the supplied figures were performed once, and the proteomics used technical replicates. Because the text is truncated during the Results section, later experiments and the study's complete limitations cannot be assessed. The excerpt provides no clinical evidence supporting changes to vitamin B2 intake.