Epstein-Barr virus latency programs dynamically sensitize B-cells to ferroptosis

Abstract Epstein-Barr virus (EBV) causes 200,000 cancers annually. Upon B-cell infection, EBV induces lipid metabolism to support B-cell proliferation. Yet, little is known about how latent EBV infection, or human B-cell stimulation more generally, alter sensitivity to ferroptosis, a non-apoptotic form of programmed cell death driven by iron-dependent lipid peroxidation and membrane damage. To gain insights, we analyzed lipid reactive oxygen species (ROS) levels and ferroptosis vulnerability in primary human CD19+ B-cells infected by EBV or stimulated by key B-cell receptors. Prior to the first mitosis, EBV-infected cells were exquisitely sensitive to blockade of glutathione biosynthesis, a phenomenon not observed with B-cell receptor stimulation. Subsequently, EBV-mediated Burkitt-like hyper-proliferation generated elevated levels of lipid ROS, which necessitated SLC7A11-mediated cystine import and glutathione peroxidase 4 (GPX4) activity to prevent ferroptosis. By comparison, B-cells were sensitized to ferroptosis induction by combinatorial CD40-ligand and interleukin-4 stimulation or anti-B-cell receptor and Toll-like receptor 9 stimulation upon GPX4 inhibition, but not with SLC7A11 blockade. EBV transforming B-cells became progressively resistant to ferroptosis induction upon switching to the latency III program and lymphoblastoid physiology. Similarly, latency I Burkitt cells were particularly vulnerable to blockade of SLC7A11 or GPX4 or cystine withdrawal, while latency III Burkitt and lymphoblastoid cells were comparatively resistant. The selenocysteine biosynthesis kinase PSTK was newly implicated as a cellular target for ferroptosis induction including in Burkitt cells, likely due to roles in GPX4 biosynthesis. These results highlight ferroptosis as an intriguing therapeutic target for the prevention or treatment of particular EBV-driven B-cell malignancies. Significance EBV contributes to B-cell Burkitt and post-transplant lymphoproliferative disease (PTLD). EBV transforming programs activate lipid metabolism to convert B-cells into immortalized lymphoblastoid cell lines (LCL), a PTLD model. We found that stages of EBV transformation generate lipid reactive oxygen species (ROS) byproducts to varying degrees, and that a Burkitt-like phase of B-cell outgrowth is dependent on lipid ROS detoxification by glutathione peroxidase 4 and its cofactor glutathione. Perturbation of this redox defense in early stages of B-cell transformation or in Burkitt cells triggered ferroptosis, a programmed cell death pathway. LCLs were less dependent on this defense, a distinction tied to EBV latency programs. This highlights ferroptosis induction as a novel therapeutic approach for prevention or treatment of EBV+ lymphomas.