PROJECT SUMMARY Glioblastoma (GBM) is the most common primary malignant brain tumor in adults that is universally fatal despite multimodal treatment. Novel therapies are therefore critically needed. Immunotherapy, such as checkpoint blockade, has thus far failed to demonstrate clinical efficacy. GBM has high intrinsic resistance to antitumor immunity due to, among other factors, low expression of MHC-I and lack of T-cell infiltration. Strategies to promote immunogenicity of GBM to sensitize tumor to checkpoint therapy is needed. Protein phosphatase 2A (PP2A) is a ubiquitous serine/threonine phosphatase comprised of a catalytic (C), regulatory (B) and scaffolding (A) subunit. We have previously reported that pharmacological inhibition of PP2Ac can enhance the efficacy of anti-PD1 blockade in multiple preclinical models, including GBM. However, the mechanism(s) or cell type(s) responsible for the enhanced antitumor immune response is not well understood. Recently, we found that deficiency of PP2Ac, by genetic modification, in glioma cells resulted in enhanced interferon signaling, which is essential to eliciting antitumor immune response. PP2A deficiency in glioma cells enhanced MHC-I expression, tumor T-cell infiltration and sensitivity to checkpoint blockade in vivo. We also demonstrated that PP2Ac deficiency led to enhanced production of cytoplasmic double-stranded DNA (dsDNA), which is known to activate cGAS-STING signaling, a potent simulator of interferon production. Moreover, from unbiased screening of all known regulatory B subunits, we identified PPP2R2C, a specific B subunit of PP2A, to have a similar role as PP2Ac in promoting MHC-I expression. In this project, we will first elucidate the effect of PP2Ac deficiency in glioma cells on the immunological landscape of the tumor microenvironment and will identify the immune cell types responsible for PP2Ac modulated antitumor immunity. We will then dissect the molecular mechanisms that link PP2A deficiency to cGAS-STING activation and promotion of interferon signaling in the glioma microenvironment. We will also identify the role of the specific regulatory B subunit, PPP2R2C, in modulating dsDNA production and cGAS-STING signaling. Finally, we will investigate the ability of PP2Ac deficiency to enhance the therapeutic efficacy of radiation therapy, a major component of current standard-of-care and a known stimulator of cGAS-STING signaling. The immediate goal of this project is to identify the mechanisms of PP2A modulated immunogenicity in GBM, with the long-term goal of developing precise PP2A targeting strategies to increase effectiveness of immunotherapies for GBM. We believe this study fits the mission of NINDS to seek fundamental knowledge of the nervous system and to use that knowledge to reduce the burden of neurological disease such as brain tumor.