ABSTRACT T-cell acute lymphocytic leukemia (T-ALL) is an aggressive hematological malignancy. Despite successes in curing pediatric T-ALL with intensive chemotherapy, the majority of adult T-ALL patients will relapse and die of their disease. We and others have demonstrated that in T-ALL, oxidative phosphorylation (OxPhos) generates energy and metabolic intermediates necessary to promote growth and support survival, by regulation of mitochondrial Complex I (CI). This unique metabolic and mitochondrial biology makes T-ALL vulnerable to strategies that target OxPhos. We have identified a first-in-class nanomolar-potent inhibitor of OxPhos (OxPhosi), IACS-010759, that inhibits CI of the OxPhos respiratory chain, blocks oxygen consumption, and destabilizes Hypoxia-Inducible Factor 1α (HIF-1α). Our data demonstrated profound growth-inhibitory effects of this agent in T-ALL cell lines and primary ALL cells at low nM concentrations, with minimal toxicity against normal BM cells. OxPhos blockade in vivo was tolerable as a single agent, yet had only a modest therapeutic benefit. However, targeting the OxPhos-driven biology of T-ALL is likely to be effective in the right combinations. We have demonstrated synergy of IACS-010759 with standard chemotherapy agents used in T-ALL, both in vitro and in the in vivo T-ALL PDX models. We will translate these findings in our Aim 3, by a Phase I/II clinical trial of IACS-010759 combined with a modified hyperCVAD/L-asparaginase regimen in relapsed/refractory ALL patients, using the recommended Phase 2 dose of IACS-010759 from the ongoing AML trial. Matching pre-clinical studies on T-ALL PDX models in Aim 1 will develop biomarkers of response in ways not possible in human subjects. We have further identified a synthetically lethal combination in T-ALL cells of OxPhosi with an inhibitor of the lactate transporter MCT1 (AZD3965, now in Phase 2 trials), and in Aim 2 will investigate mechanisms of this synergy. We will also characterize the effect of HIF-1α blockade by IACS-010759 on T-ALL cells using in vivo two-photon imaging of metabolic NADH and oxygen sensing by phosphorescence lifetime microscopy. Finally, we will conduct further screening with a novel high-content metabolomic drug library to identify other combinations with IACS-010759 that are toxic to T-ALL cells but not normal cells, for future therapeutic applications. We believe that the proposed studies will provide mechanistic insights into the newfound vulnerability of T-ALL to OxPhosi; identify candidate predictive biomarkers for the combination of IACS-010759 with chemotherapy or MCT1 inhibitor; and develop combinations for the next generation of OxPhosi trials for T-ALL.