A Genome-Wide CRISPR/Cas9 Screen Reveals that Riboflavin Regulates Hydrogen Peroxide Entry into HAP1 Cells

Extracellular hydrogen peroxide can induce oxidative stress, which can cause cell death if unresolved. However, the cellular mediators of H(2)O(2)-induced cell death are unknown. We determined that H(2)O(2)-induced cytotoxicity is an iron-dependent process in HAP1 cells and conducted a CRISPR/Cas9-based survival screen that identified four genes that mediate H(2)O(2)-induced cell death: POR (encoding cytochrome P450 oxidoreductase), RETSAT (retinol saturase), KEAP1 (Kelch-like ECH-associated protein-1), and SLC52A2 (riboflavin transporter). Among these genes, only POR also mediated methyl viologen dichloride hydrate (paraquat)-induced cell death. Because the identification of SLC52A2 as a mediator of H(2)O(2) was both novel and unexpected, we performed additional experiments to characterize the specificity and mechanism of its effect. These experiments showed that paralogs of SLC52A2 with lower riboflavin affinities could not mediate H(2)O(2)-induced cell death and that riboflavin depletion protected HAP1 cells from H(2)O(2) toxicity through a specific process that could not be rescued by other flavin compounds. Interestingly, riboflavin mediated cell death specifically by regulating H(2)O(2) entry into HAP1 cells, likely through an aquaporin channel. Our study results reveal the general and specific effectors of iron-dependent H(2)O(2)-induced cell death and also show for the first time that a vitamin can regulate membrane transport.