1344 Metabolic Reprogramming Contributes to Immunosuppressive Microenvironment in Glioblastoma

INTRODUCTION: One of the hallmark features of GBM is its immunosuppressive nature, allowing it to proliferate unchecked in the siloed CNS immune environment. GBM has shown a unique indifference to immunotherapeutic approaches that have transformed the treatment landscape of other cancer types. Purinergic signaling is a critical mechanism of immune regulation and has been shown to contribute to GBM immune evasion, with one mechanism being accumulation of purines through upregulation of ectonucleotidases. A novel mechanism of aberrant purinergic signaling is through accumulation of methylthioadenosine (MTA) after homozygous deletion of the Methylthioadenosine Phosphorylase (MTAP) gene, a critical step of the purine salvage pathway, occuring in >50% of GBM patients. MTA accumulation has both cell-autonomous and nonautonomous effects, including extracellular signaling through purinergic receptors. METHODS: In-silico CYBERSORT analysis was performed on TCGA gene expression data. Quantitative PCR (qPCR) and flow cytometry are used to measure T cell and macrophage markers in vitro. CRISPR knockout of MTAP in murine GBM models and flow cytometry analysis of intacranial tumors are used to analyze tumor immune composition. ELISA was utilized to measure cAMP levels. RESULTS: Using in vitro and in vivo studies we demonstrate that MTA promotes macrophage M2 polarization and impairs T cell activation and proliferation. MTA upregulates M2 marker gene expression in RAW264.7 cells, and inhibits T cell activation and proliferation in mouse splenocytes. By ELISA assay we found that T cell suppression in the presence of MTA is associated with cAMP accumulation, implicating aberrant purinergic signaling as a contributing mechanism. CONCLUSIONS: These findings illuminate a role for MTAP deficiency in GBM pathogenesis and immune evasion, and suggest that manipulation of purinergic signaling is a potential therapeutic strategy necessitating further exploration for improving immunotherapeutic efficacy.