Sustained biomass accumulation requires cancer cells to undergo metabolic reprogramming. Many tumors exhibit a highly elevated uptake of glutamine, which can be a critical carbon source for the tricarboxylic acid (TCA) cycle via hydrolysis to glutamate and subsequent deamination to α-ketoglutarate. CB-839, which selectively inhibits the mitochondrial glutaminase isozyme GLS, is undergoing clinical trials for treatment of triple-negative breast cancer (TNBC), but has yielded few objective responses as a monotherapy. We have found that ectopic overexpression of the glutaminase isozyme GLS2 in TNBC cells renders them resistant to CB-839 class inhibitors. Here, we propose studies to determine whether up-regulation of GLS2 expression occurs spontaneously in TNBC as a mechanism for CB-839 resistance, and to identify other potential resistance mechanisms. We also propose to test whether combined inhibition of GLS and GLS2 by a structurally distinct class of inhibitors is a more effective treatment strategy than selective inhibition of GLS. Finally, we will use metabolic tracing to address the roles of GLS and GLS2, a topic of both biological and clinical interest to the breast cancer research community, and more broadly relevant to other malignant diseases.