ABSTRACT A key hallmark of human cancer is cancer-specific metabolic rewiring. Notably, this area of research has gained renewed attention in the last decade after several studies demonstrated that key oncogenes in human cancer, such as AKT, KRas, MYC or NOTCH1, have differential and specific effects on primary cellular metabolism, leading to the hypothesis that selective targeting of those routes might be an attractive therapeutic approach. In this context, I recently identified glutaminolysis as a critical pathway in NOTCH1-driven T-lineage acute lymphoblastic leukemia (T-ALL), a hematological malignancy where relapses occur in up to 20% of pediatric and 50% of adult patients, who ultimately succumb to refractory disease. Importantly, inhibition of glutaminolysis genetically, via deletion of glutaminase (Gls), or pharmacologically, using Gls inhibitors, results in anti-leukemic effects and is highly synergistic with anti-NOTCH1 therapies. Notably, Gls selective inhibitors are currently being explored in clinical trials for hematological malignancies, and glutaminolysis has also been proposed as a therapeutic target in a variety of solid tumors. However, the role of glutamine in vivo is still not well understood and Gls-deficient T-ALLs eventually progress, underscoring the need to understand the mechanisms of relapse. My preliminary data suggests that glutamine-derived carbon might feed into the TCA cycle and Gls- deficient T-ALLs might still use glutamine even in the absence of glutaminase. Moreover, my preliminary data points to a role of glutaminase in stemness. Therefore, this research proposal seeks to: 1) dissect the role of glutaminase in T-ALL in vivo and unravel the mechanisms of escape to Gls loss;; 2) address the role of glutaminolysis in stemness;; and 3) identify synthetic lethal pathways/genes with pharmacological inhibition of glutaminase or with genetic loss of glutaminase using CRIPSR/Cas9 screens in vitro and experimental therapeutic experiments in vivo. These studies will reveal as yet undiscovered fundamental mechanisms implicated in the metabolic and epigenetic rewiring of T-ALL, will advance our understanding of the role of Gls and glutaminolysis in cancer, and will help us rationally design combinations of metabolic or epigenetic targeted therapies that will result in stronger therapeutic effects with decreased chances of relapse.