PROJECT SUMMARY/ABSTRACT Glioblastoma (GBM) is the most common primary malignant brain tumor with a dismal prognosis that lacks effective therapeutic strategies. While the advent of immunotherapy has significantly improved patient outcomes in a variety of extracranial cancers, these novel approaches have achieved limited success in GBM. The goal of this study is to investigate mechanisms underlying brain microenvironment-specific suppression of effective anti- tumor immunity mediated by aberrant epidermal growth factor receptor (EGFR) signaling, and furthermore whether targeting this oncogene creates novel vulnerabilities to enhance immune response in this deadly disease. Extensive preliminary data, which leverages a highly innovative EGFR activated immunocompetent murine glioma model (MADR-mEGFRvIII), indicate tumor-intrinsic EGFR signaling impairs intratumoral T cell infiltration and promotes the development of a tumor microenvironment (TME) enriched in microglia, a myeloid cell type unique to the brain. Moreover, preliminary data demonstrate an association between EGFR activation and an immunosuppressive wound healing signature linked with poor prognosis in the myeloid compartment of immune cells isolated from newly diagnosed GBM patient tumors. Specific Aim 1 utilizes a transgenic T cell system engineered to be antigen-specific in conjunction with multiple modalities of in vivo EGFR ablation to determine the precise impact of aberrant EGFR signaling and tumor-programmed myeloid cells on T cell cytotoxic function, clonal proliferation, and exhaustion. Through the incorporation of functional and phenotypic assays designed to isolate the contributions of each factor in the complex brain immune TME, this approach is expected to provide novel insight into the role of EGFR activation in dysregulating effector T cell responses. Specific Aim 2 endeavors to elucidate the axis by which aberrant EGFR signaling promotes a distinct immunosuppressive microglia phenotype characterized by VEGF and IL-1β in the brain TME with an emphasis on EGFR-mediated tumor-microglia crosstalk. Using in vitro co-culture approaches and in vivo knockdown strategies, the precise impact of EGFR activation in coordination with microglia IL-1β signaling on tumor- associated microglia phenotype and overall anti-tumor immune response will be assessed. Specific Aim 3 focuses on assessing rational combinatorial strategies targeting EGFR using ERAS-801, a novel clinical stage small molecule inhibitor, to drive enhanced T cell infiltration followed by immune checkpoint blockade to support prolonged T cell function within the TME. Overall, this project will provide the first comprehensive assessment of the mechanistic underpinnings and functional consequences of aberrant EGFR activation on the myeloid- dominant tumor immune microenvironment and the resultant impact on anti-tumor immune response in GBM. By identifying the dynamics of interplay between oncogenic EGFR signaling and immune...