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Abstract

Glioblastoma is the most common central nervous system malignancy in adults with a very poor outcome due to its invasiveness, intratumoral heterogeneity, poorly differentiated features, and immunosuppressive microenvironment. Previous work suggested that Branched-chain amino acid transaminase 1 (BCAT1) is often highly expressed in glioblastoma and multiple modes of action for its oncogenic potential have been proposed. In this thesis, I focus on investigating a novel role of BCAT1 in maintaining mitotic fidelity, and how it impacts the cellular plasticity of glioblastoma cells and the tumor immune microenvironment. We have found that BCAT1 localizes to the key mitotic structures during cell division as well as in the nucleus during interphase. Using co-immunoprecipitation mass spectrometry, I showed that BCAT1 associates with many components of the mitotic spindle and the kinetochore during mitosis. Through proteomic and phosphoproteomic analysis I showed that the central kinases of the spindle assembly checkpoint, TTK and AURKB, showed significantly decreased activity during mitosis upon BCAT1-KO. By analyzing the expression patterns of human and mouse glioblastoma cells and tumor samples of the TCGA-GBM cohort, I found that BCAT1 expression is strongly correlated with the cellular state of glioblastoma, with high expression being indicative of a mesenchymal phenotype and low or no expression with a neuronal cellular state. I further confirmed these observations through a series of differentiation experiments of murine glioblastoma stem cells where the Bcat1-KO showed a much higher tendency towards differentiation and lacked the plasticity of the control cells. Consistently, in vivo findings corroborated these results with a complete lack of tumor outgrowth of the Bcat1-KO cells in immunocompetent mice, and a significant growth delay in immunodeficient mice. Lastly, I explored the impact of tumor BCAT1 expression on the immune microenvironment. I found that low BCAT1 expression was associated with a higher immune infiltration of both myeloid and T-cells in human tumor samples. These findings were additionally confirmed in in vivo experiments in immunocompetent mice. Furthermore, Bcat1-KO tumors did develop in the immunodeficient NSG and Rag2KO mouse models, highlighting the importance of the immune compartment in completely abrogating their growth. In conclusion, the data presented here confirm the novel role of BCAT1 in maintaining mitotic fidelity of glioblastoma cells. Furthermore, it shows that BCAT1 expression is necessary for maintaining the plasticity of glioblastoma cells and an immunosuppressive tumor microenvironment.