Project Summary/Abstract Glioblastoma (GBM) is an incurable brain tumor for which improved therapies are badly needed. There are roughly 10,000 cases diagnosed in the US each year, and median survival is ~14 months. Evidence has been accumulating over recent years linking cytomegalovirus (CMV) to GBM and other cancers. However, the role of CMV in GBM remains poorly defined. CMV is a prevalent virus in humans, where it resides lifelong in a latent state but can be reactivated under certain conditions. Several clinical trials targeting CMV using diverse approaches including immunotherapies and the anti-viral drug CMV have shown very promising responses in GBM patients, but the underlying mechanisms are not clear. To improve our understanding of this area we have developed an immunocompetent murine model of intracranial GBM grown in the context of a systemic latent CMV infection. This consistently leads to significantly shortened survival compared with mock infected controls in multiple distinct murine GBM models. These effects can be reversed by treatment with anti-viral drugs. Our new preliminary human data shows reduced progression free survival in CMV seropositive humans and similarities in patterns of differentially expressed genes supporting the relevance of the mouse model. Our model features increased blood vessel formation, with accumulation of perivascular pericytes, increased CD8+ T cell infiltrates and resistance to chemotherapy. We discovered that platelet-derived growth factor-δ (PDGFD) is upregulated by CMV infection of tumor cells, and that CRISPR-mediated knockdown of PDGFD prevents pericyte recruitment and severely impairs tumor growth. Therefore in Specific Aim 1 we will further investigate PDGFD as a potential GBM therapeutic target. Also, we have preliminary data with a replication- defective viral mutant suggesting that CMV replication is critical to this phenotype, and we will use this mutant to explore the mechanisms by which CMV enhances tumor progression. We also have shown that the tumor microenvironment is significantly impacted by CMV, therefore in Specific Aim 2 we will examine the therapeutic implications of CMV on tumor/immune interactions, and the importance of viral replication in these effects. In Specific Aim 3 we will use our model to investigate the interactions of CMV with standard of care chemoradiation, in terms of viral reactivation and sensitization to therapies by anti-viral drugs. Results from the proposed experiments will bring us to a deeper understanding of the impact that CMV has on GBM progression, providing badly needed targets and rational therapeutic combinations for future clinical trials in this devastating disease.