Epstein-Barr virus (EBV) was the first oncogenic virus to be discovered in humans, and it infects over 95% of adults worldwide. The virus establishes latency in a subset of host memory B cells (MBCs) and typically persists as a benign but lifelong infection. However, EBV is associated with ~2% of all cancers including many B cell lymphomas. Specifically, EBV is etiologic in endemic Burkitt Lymphoma (eBL), has frequent association with Diffuse Large B Cell Lymphoma (DLBCL), and causes many HIV-related Non-Hodgkin Lymphomas (HIV-NHLs) and post-transplant lymphoproliferative disease (PTLD). The frequency of virus-positive HIV-NHLs (~30-40%) and PTLD cases (~80-90%) highlights the risk for people living with suppressed immune systems to develop EBV-driven cancers. Such lymphomas arise due to viral oncoproteins driven proliferation of EBV+ MBCs when EBV-specific T cell function is depleted. Similar lymphoproliferation can be modeled in vitro with lymphoblastoid cell lines (LCLs), which grow out from B cells immortalized by EBV. Despite many studies of EBV infection in LCLs and other models, mechanisms by which EBV oncoproteins control complex cell responses in malignancy are incompletely understood. Filling this knowledge gap is essential to develop effective therapies for EBV+ lymphomas. My research applies single-cell techniques to define and dissect host-virus interactions that lead to diverse outcomes of EBV-infected B cells. The ultimate goal of this research is to develop comprehensive high- resolution insights on vulnerabilities in viral oncogenesis that can guide effective treatments. This proposal examines how EBV oncoproteins EBNA3A and EBNA3C coordinate with cellular epigenetic regulator EZH2 to promote pathogenic lymphoproliferation and host-EBV responses to EZH2 inhibition. Notably, EZH2 is essential for germinal center (GC) B cell proliferation and maturation and provides a promising selective target for B cell lymphoma therapy. The central hypothesis is that inhibiting EBNA3A/3C-EZH2 limits EBV+ lymphoproliferation by disrupting virus-induced GC dynamics and promoting differentiation into non-proliferative plasma cells (PC). This hypothesis is supported by preliminary single-cell data showing that EZH2 and constitutive GC-like B cell responses are induced by EBV and maintained in latently infected LCLs. Further, EZH2 and other mediators of GC responses and PC differentiation were identified as EBNA3A/3C-regualted target genes. Finally, preliminary EZH2 inhibition screens confirmed reduced growth of EBV+ LCLs and induction of PC biomarkers. The proposal’s rationale is that it will yield invaluable mechanistic insight on oncogenic viral epigenetic regulation of host cell fate to explore a therapeutic vulnerability. I will test the central hypothesis in three aims: I) Resolve mechanisms by which the EBNA3A/3C-EZH2 axis controls EBV+ B cell fate; II) Evaluate terminal fate induction by targeting EBNA3A/3C-EZH2 in EBV+ lymphoma in vivo; and III) Resolve genome-wide EBNA3A/3C mechanisms mediating cell proliferation and differentiation. Completing these aims will enhance knowledge of EBV-host dysregulation and lay a solid foundation for clinically relevant studies to address prevalent EBV+ lymphomas.

PROJECT NARRATIVE Epstein-Barr virus contributes to B cell lymphomas, especially in people with suppressed immune systems. These studies will define viral mechanisms that promote host malignancy at single-cell resolution and test their disruption to inform therapies for EBV-positive tumors.