NT-39GLUTAMINASE-MEDIATED METABOLIC PATHWAY INVOLVES GLIOBLASTOMA RESISTANCE TO mTOR-TARGETED THERAPIES.

BACKGROUND: Although the phosphatidylinositol 3-kinase (PI3K)/ mammalian target of rapamycin (mTOR) pathway is a master regulator of aerobic glycolysis and cellular biosynthesis, the effect of modulating mTOR activity on other metabolic processes, such as glutamine metabolism is not well understood. AIMS: Through an integrated analysis of glioblastoma (GBM) cell lines and clinical samples, we examined the importance of glutamine metabolism during mTOR-targeted treatments. METHODS: To identify metabolic response to mTOR-targeted treatments in GBM cells, we analyzed metabolites using gas chromatography and mass spectroscopy (GC/MS) in mutated EGFR (EGFRvIII)-expressing GBM cells treated with the mTOR inhibitors (rapamycin or PP242) and examined gene expression of key enzymes for glycolysis and glutaminolysis pathways using RT-PCR methods. RESULTS: Principal component analysis (PCA) based on GC/MS studies identified glutamic acid (glutamate), aspartic acid, citric (or isocitric) acid and succinic acid as key differentiating metabolites between control and mTOR-targeted treatment groups. Gene expression analysis identified upregulation of glutaminase (GLS) to generate glutamate from glutamine after mTOR inhibitor treatments, as well as downregulation of glycolytic enzymes. Importantly suppression of GLS activity with RNA interference or an inhibitor with Compound 968 sensitized U87/EGFRvIII cells to mTOR-targeted therapy through alpha-ketoglutarate (α-KG). CONCLUSIONS: The mTOR-targeted treatments affected glutamine utilization and elicited a switch in the pathways used to deliver glutamine carbon to the tricarboxylic acid (TCA) cycle, with increasing GLS expression. These results may suggest the new therapeutic strategies targeting GLS will be a rational approach in combination with mTOR-targeted therapy.