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

Abstract

Extracellular hydrogen peroxide can induce oxidative stress, which can cause cell death if unresolved. However, the cellular mediators of H2O2-induced cell death are unknown. We determined that H2O2-induced cytotoxicity is an iron-dependent process in HAP1 cells and conducted a CRISPR/Cas9-based survival screen that identified four genes that mediate H2O2-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 H2O2 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 H2O2-induced cell death and that riboflavin depletion protected HAP1 cells from H2O2 toxicity through a specific process that could not be rescued by other flavin compounds. Interestingly, riboflavin mediated cell death specifically by regulating H2O2 entry into HAP1 cells, likely through an aquaporin channel. Our study results reveal the general and specific effectors of iron-dependent H2O2-induced cell death and also show for the first time that a vitamin can regulate membrane transport. IMPORTANCE Using a genetic screen, we discovered that riboflavin controls the entry of hydrogen peroxide into a white blood cell line. To our knowledge, this is the first report of a vitamin playing a role in controlling transport of a small molecule across the cell membrane.

Hydrogen Peroxide-induced Cell Death in Mammalian Cells

“…This factor not only affects the cell death modality employed by the cells, but it also affects the nature of the identified mediators of H 2 O 2 -induced cell death. Interestingly, the mediators have included genes involved in redox biology, riboflavin metabolism, iron homeostasis, vesicle trafficking, and the peroxisomal import pathway [ 17 , 18 ]. Here, we briefly discuss some of the mediators that are involved in redox biology, namely cytochrome P450 reductase (POR), retinol saturase (RETSAT), and KEAP1 [ 17 , 18 ].…”

“…Interestingly, the mediators have included genes involved in redox biology, riboflavin metabolism, iron homeostasis, vesicle trafficking, and the peroxisomal import pathway [ 17 , 18 ]. Here, we briefly discuss some of the mediators that are involved in redox biology, namely cytochrome P450 reductase (POR), retinol saturase (RETSAT), and KEAP1 [ 17 , 18 ]. The mediators involved in riboflavin metabolism, namely riboflavin kinase (RFK), flavin adenine dinucleotide synthetase 1 (FAD synthase), and SLC52A2 which is a riboflavin transporter with the highest affinity for riboflavin, are discussed in more depth [ 17 , 18 ].…”

“…Here, we briefly discuss some of the mediators that are involved in redox biology, namely cytochrome P450 reductase (POR), retinol saturase (RETSAT), and KEAP1 [ 17 , 18 ]. The mediators involved in riboflavin metabolism, namely riboflavin kinase (RFK), flavin adenine dinucleotide synthetase 1 (FAD synthase), and SLC52A2 which is a riboflavin transporter with the highest affinity for riboflavin, are discussed in more depth [ 17 , 18 ]. The mechanism of cell death employed by POR, RETSAT, KEAP1 and SLC52A2 in HAP1 cells is iron-dependent, suggesting that these proteins mediate H 2 O 2 -induced cell death through ferroptosis in HAP1 cells [ 18 ].…”

“…The mediators involved in riboflavin metabolism, namely riboflavin kinase (RFK), flavin adenine dinucleotide synthetase 1 (FAD synthase), and SLC52A2 which is a riboflavin transporter with the highest affinity for riboflavin, are discussed in more depth [ 17 , 18 ]. The mechanism of cell death employed by POR, RETSAT, KEAP1 and SLC52A2 in HAP1 cells is iron-dependent, suggesting that these proteins mediate H 2 O 2 -induced cell death through ferroptosis in HAP1 cells [ 18 ]. It is important to note that these same proteins may mediate H 2 O 2 -induced cell death in other cell types through other mechanisms of cell death such as apoptosis.…”

“…CRISPR/Cas9 genome wide screens in human cell lines, K562 cells, HeLa cells, and HAP1 cells, confirmed the importance of RETSAT as an essential mediator of H 2 O 2 -induced cell death in human cell lines [ 17 ]. As an inhibitor of Nrf2 antioxidant activity, it is not surprising that KEAP1 was identified in CRISPR/Cas9 screens in K562, HeLa, and HAP1 cells [ 17 , 18 ].…”