Highlights d CRISPR/Cas9 screen highlights host epigenetic suppressors of the EBV lytic cycle d EBV senses MYC abundance to maintain B cell latency d MYC depletion alters three-dimensional EBV genomic architecture d FACT is a druggable target for Burkitt B cell EBV latency reversal

Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma, the leading childhood cancer in sub-Saharan Africa. Burkitt cells retain aspects of germinal center B-cell physiology with MYC-driven B-cell hyperproliferation, yet little is presently known about their iron metabolism. CRISPR/Cas9 analysis highlighted the little studied ferrireductase CYB561A3 as critical for Burkitt proliferation, but not for that of closely related EBV-transformed lymphoblastoid cells or nearly all other Cancer Dependency Map cell lines. Burkitt CYB561A3 knockout induced profound iron starvation, despite ferritinophagy and plasma membrane transferrin upregulation. Elevated concentrations of ascorbic acid, a key CYB561 family electron donor or the labile iron source ferrous citrate rescued Burkitt CYB561A3 deficiency. CYB561A3 knockout caused catastrophic lysosomal and mitochondrial damage and impaired mitochondrial respiration. By contrast, lymphoblastoid B-cells with the transforming EBV latency III program were instead dependent on the STEAP3 ferrireductase. These results highlight CYB561A3 it as an attractive therapeutic Burkitt lymphoma target.