PROJECT 3 – PROJECT SUMMARY Epstein-Barr virus (EBV) is associated with multiple epithelial cell diseases, including gastric cancer and nasopharyngeal carcinoma. Despite its B-cell tropism, more than half of the 200,000 EBV-associated cancers that occur annually are gastric and nasopharyngeal carcinomas. The Cancer Genome Atlas project identified EBV-infected gastric cancer as one of the four biologically distinct subtypes. Extreme tumor genome CpG island methylator phenotype (CIMP) and gain-of-function PI3K mutations are salient features, suggesting interconnected driver roles in EBV+ GC. In fact, EBV+ gastric cancer has the highest level of DNA methylation of any human cancer. Hypermethylation and elevated PI3K activity are also characteristic of EBV+ NPC and Burkitt lymphoma, further suggesting a close pathogenetic relationship of these oncogenic properties with highly restricted forms of EBV latency. Yet, much remains to be learned about how latent EBV mutation together with tumor driver mutations result in epithelial cancers, and how these can be targeted by precision approaches. We therefore used EBV+ epithelial tumor cell RNAseq and CRISPR/Cas9 screens to identify host factors whose knockout is synthetic lethal with reversal of CIMP by the hypomethylating agent decitabine or upon blockade of PI3K hyperactivity by the highly selective antagonist alpelisib. Our central hypothesis is that EBV+ epithelial cancers rely on cross-talk between latent viral genomes, and hyperactive PI3K and CIMP, disruption of which can be targeted in synthetic lethal approaches. Our Aims are to (1) Define the role of latent EBV in driving CIMP; (2) Define the role of hyperactive PI3K signaling in EBV+ epithelial cancers in support of CIMP; (3) Define key synthetic lethal vulnerabilities upon EBV+ epithelial cancer CIMP reversal. Collectively, these studies are expected to identify how EBV-infected epithelial cancers subvert host methionine and PI3K metabolism pathways to support CIMP, and how in turn CIMP not only silences tumor suppressors, but also plays key roles in guarding against DNA damage. Our studies may therefore support strategies to develop rational therapeutic approaches for EBV-associated epithelial cancers.