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The mitochondrial enzyme glutaminase C (GAC) catalyzes the hydrolysis of glutamine to glutamate plus ammonia, a key step in the metabolism of glutamine by cancer cells. Recently, we discovered a class of allosteric inhibitors of GAC that inhibit cancer cell growth without affecting their normal cellular counterparts, with the lead compound being the bromo-benzophenanthridinone 968. Here, we take advantage of mouse embryonic fibroblasts transformed by oncogenic Dbl, which hyperactivates Rho GTPases, together with 13 Clabeled glutamine and stable-isotope tracing methods, to establish that 968 selectively blocks the enhancement in glutaminolysis necessary for satisfying the glutamine addiction of cancer cells. We then determine how 968 inhibits the catalytic activity of GAC. First, we developed a FRET assay to examine the effects of 968 on the ability of GAC to undergo the dimer-to-tetramer transition necessary for enzyme activation. We next demonstrate how the fluorescence of a reporter group attached to GAC provides a direct read-out of the binding of 968 and related compounds to the enzyme. By combining these fluorescence assays with newly developed GAC mutants trapped in either the monomeric or dimeric state, we show that 968 has the highest affinity for monomeric GAC and that the dose-dependent binding of 968 to GAC monomers directly matches its dose-dependent inhibition of enzyme activity and cellular transformation. Together, these findings highlight the requirement of tetramer formation as the mechanism of GAC activation and shed new light on how a distinct class of allosteric GAC inhibitors impacts the metabolic program of transformed cells.R ecently, the mitochondrial enzyme glutaminase (GLS1) has gained significant attention as a therapeutic target for cancer (1-3). GLS1 catalyzes the hydrolysis of glutamine to glutamate, which is used in the citric acid cycle (TCA) of cancer cells undergoing an aberrant glycolytic flux (i.e., the Warburg effect) as a non-glucose-derived source for anaplerosis. The elevation in glutamine metabolism exhibited by many cancer cells ("glutamine addiction") is critical for sustaining their proliferative capacity, as well as for other aspects of their transformed phenotypes (4-9). Work from our laboratory has shown that a specific GLS1 splice variant, glutaminase C (GAC), plays an essential role in the transformation of NIH 3T3 fibroblasts by Rho GTPases, as well as in the proliferative and invasive activities of various cancer cells (10, 11). Thus, given the importance of GAC expression and activation for oncogenic transformation, the identification of inhibitors that target this metabolic enzyme offers new opportunities for the development of anticancer drugs.Because glutamine is necessary for a range of biochemical reactions, including nucleotide and protein synthesis, glutamine analogs like the GLS1 inhibitor diazo-O-norleucine (DON) (12, 13) are not ideal candidates for cancer drugs (14). However, two classes of allosteric inhibitors of GAC have been identified and offer ...