PROJECT 2 – PROJECT SUMMARY EBV(+) epithelial cancers represent 75% of all the EBV(+) malignancies. Despite the presence of virus infection, these EBV(+) tumors receive the same treatment as EBV(-) cancers. The long-term goal of this Program Project is to identify specific targetable mechanisms of EBV-mediated oncogenesis in epithelial cells. We previously showed that EBV latency and oncogenicity are regulated by factors that link epigenetics with metabolism, such Poly-ADP-ribose Polymerases (PARPs). Drugs that target PARPs have the potential to be effective therapeutic options for EBV(+) tumors. However, our incomplete understanding of the epigenetic and metabolic mechanisms regulating EBV latency in epithelial cells limits the application of such drug options to treat EBV(+) epithelial malignancies. In this project (Project 2), we investigated the role of PARP1 in regulating EBV genome maintenance, gene expression, and metabolic sensing in epithelial cells. We found that PARP1 forms a complex with CTCF on the EBV genome. We now show that this complex includes UHRF1, an epigenetic reader protein involved in DNA methylation pattern propagation. We found that treatment of EBV(+) cells with PARP1 inhibitors disrupts this complex altering both viral and cellular gene expression and causing global DNA hypomethylation. Consistently, PARP inhibitors synergized with DNA hypomethylating agents to elicit cytotoxicity in EBV+ gastric cancers both in vitro and in vivo. Based on these data, we hypothesize that in EBV+ epithelial cells, PARP1, by interacting with CTCF and UHRF1 and modulating their functions, is a key epigenetic factor that connects 3D chromatin conformation and DNA methylation to enable EBV latency and EBV-driven epithelial cell oncogenesis .We base our hypothesis on our preliminary data showing that EBV+ epithelial cells hyper-activate PARP1 and that this drives changes in the 3D conformation of the EBV genome supporting viral latent gene expression. We observed that multiple mechanisms for hyper-activation of PARP1 exist in EBV+ cells, including regulation of the ERK/MEK pathway. We observed that differences exist in NAD+/NADH ration between EBV+ and EBV- epithelial cells, indicating that EBV+ cells are metabolically equipped to sustain PARP1 activation. This observation is consistent with our observation that EBV+ cells are sensitive to inhibitors of NAD salvage pathways, indicating that NAD metabolism plays a key and underappreciated role in EBV -driven oncogenesis in epithelial cancer cells. Programmatic Interactions. This project is highly integrated with other projects and cores. Collaborations with Project 1 on the effect of PARP1 and NAD metabolism on EBNA1 functions; and with Project 3 on the relevance of the PARP1/CTCF/UHRF1 complex for CIMP reversal and PI3K blockade. We have engaged all three cores: Core B for the development of novel small molecule PARP1 degraders, Core D for analysis of chromosome conformations and gene regula...