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Competing interests C.J.D. is on the Scientific Advisory Board of Mirati Therapeutics. A.C.K. has financial interests in Vescor Therapeutics, LLC. A.C.K. is an inventor on patents pertaining to KRAS-regulated metabolic pathways, redox control pathways in pancreatic cancer, targeting GOT1 as a therapeutic approach, and the autophagic control of iron metabolism. A.C.K. is on the Scientific Advisory Board of Cornerstone/Rafael Pharmaceuticals.

The mitochondrial enzyme glutaminase (GLS) is frequently up-regulated during tumorigenesis and is being evaluated as a target for cancer therapy. GLS catalyzes the hydrolysis of glutamine to glutamate, which then supplies diverse metabolic pathways with carbon and/or nitrogen. Here, we report that SIRT5, a mitochondrial NAD+-dependent lysine deacylase, plays a key role in stabilizing GLS. In transformed cells, SIRT5 regulates glutamine metabolism by desuccinylating GLS and thereby protecting it from ubiquitin-mediated degradation. Moreover, we show that SIRT5 is up-regulated during cellular transformation and supports proliferation and tumorigenesis. Elevated SIRT5 expression in human breast tumors correlates with poor patient prognosis. These findings reveal a mechanism for increasing GLS expression in cancer cells and establish a role for SIRT5 in metabolic reprogramming and mammary tumorigenesis.

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 ...

Altered glycolytic flux in cancer cells (the "Warburg effect") causes their proliferation to rely upon elevated glutamine metabolism ("glutamine addiction"). This requirement is met by the overexpression of glutaminase C (GAC), which catalyzes the first step in glutamine metabolism and therefore represents a potential therapeutic target. The small molecule CB-839 was reported to be more potent than other allosteric GAC inhibitors, including the parent compound BPTES, and is in clinical trials. Recently, we described the synthesis of BPTES analogs having distinct saturated heterocyclic cores as a replacement for the flexible chain moiety, with improved microsomal stability relative to CB-839 and BPTES. Here, we show that one of these new compounds, UPGL00004, like CB-839, more potently inhibits the enzymatic activity of GAC, compared to BPTES. We also compare the abilities of UPGL00004, CB-839, and BPTES to directly bind to recombinant GAC, and demonstrate that UPGL00004 has a similar binding affinity as CB-839 for GAC. We go on to show that UPGL00004 potently inhibits the growth of triplenegative breast cancer cells, as well as tumor growth when combined with the anti-VEGF antibody bevacizumab. Finally, we compare the Xray crystal structures for UPGL00004 and CB-839 bound to GAC, verifying that UPGL00004 occupies the same binding site as CB-839 or BPTES, and that all three inhibitors regulate the enzymatic activity of GAC via a similar allosteric mechanism.These results provide insights regarding the potency of these inhibitors that will be useful in designing novel small-molecules that target a key enzyme in cancer cell metabolism.The Warburg effect in cancer cells refers to the significant alteration of the glycolytic pathway that results in the increased generation of lactate, decreased mitochondrial metabolism of pyruvate, and an accompanying reduction in oxidative phosphorylation (1-3). This altered glucose flux is thought to be advantageous for rapidly proliferating cells, such as cancer cells, by providing the building blocks for biosynthetic processes, at the expense of ATP synthesis. A significant consequence of the Warburg effect is that cancer cells need to develop alternative mechanisms that provide inputs into the citric acid cycle. One of the most common of these mechanisms results in an addiction to glutamine, an amino acid that is abundant in the bloodstream and can enter the citric acid cycle through an anaplerotic pathway initiated by the catalytic activity of the enzyme glutaminase (4,5).Two isozymes of mammalian glutaminase have been identified: kidney-type glutaminase encoded by the GLS gene, and liver-type glutaminase encoded by GLS2. Each gene expresses two major A new potent glutaminase allosteric inhibitor 2 splice variants, with the GLS gene expressing the KGA (kidney-type glutaminase) and the C-terminal truncated splice variant GAC (glutaminase C) isoforms, while the GLS2 gene also expresses one longer and one shorter isoform, collectively referred to here as GLS2 (6,7). Of the...

Oncogenic RAS proteins, which are mutated in approximately 24% of all human cancers, have earned a well-deserved reputation as being “undruggable.” However, several studies have challenged that reputation. With the first small molecules that directly target one oncogenic RAS mutant (G12C) undergoing clinical evaluation, there have been substantial advances in finding anti-RAS therapeutic strategies. Furthermore, new insights have come from the growing appreciation that neither all RAS proteins (HRAS, NRAS, and KRAS4A/KRAS4B) nor all oncogenic RAS mutations (such as at residues Gly12, Gly13, and Gln61) have the same impact on RAS signaling and function. The role of the nonmutated, wild-type RAS proteins in the context of mutant RAS is increasingly considered to be targetable, with reports of strategies that directly disrupt either the RAS interaction with activating guanine nucleotide exchange factors (GEFs) or receptor tyrosine kinase–mediated and GEF-dependent RAS activation (such as by targeting the scaffolding phosphatase SHP2). Last, the development of agents that target downstream effectors of RAS signaling has advanced substantially. In this review, we highlight some important trends in the targeting of RAS proteins in cancer.

The first step in glutamine catabolism is catalysis by the mitochondrial enzyme glutaminase, with a specific isoform, glutaminase C (GAC), being highly expressed in cancer cells. GAC activation requires the formation of homotetramers, promoted by anionic allosteric activators such as inorganic phosphate. This leads to the proper orientation of a flexible loop proximal to the dimer-dimer interface that is essential for catalysis ( the "activation loop"). A major class of allosteric inhibitors of GAC, with the prototype being bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and the related molecule CB-839, binds to the activation loop and induces the formation of an inactive tetramer (two inhibitors bound per active tetramer). Here we describe a direct readout for monitoring the dynamics of the activation loop of GAC in response to these allosteric inhibitors, as well as allosteric activators, through the substitution of phenylalanine at position 327 with tryptophan (F327W). The tryptophan fluorescence of the GAC(F327W) mutant undergoes a marked quenching upon the binding of BPTES or CB-839, yielding titration profiles that make it possible to measure the binding affinities of these inhibitors for the enzyme. Allosteric activators like phosphate induce the opposite effect ( fluorescence enhancement). These results describe direct readouts for the binding of the BPTES class of allosteric inhibitors as well as for inorganic phosphate and related activators of GAC, which should facilitate screening for additional modulators of this important metabolic enzyme.

Glutamine-derived carbon becomes available for anabolic biosynthesis in cancer cells via the hydrolysis of glutamine to glutamate, as catalyzed by GAC, a splice variant of kidney-type glutaminase (GLS). Thus, there is significant interest in understanding the regulation of GAC activity, with the suggestion being that higher order oligomerization is required for its activation. We used x-ray crystallography, together with site-directed mutagenesis, to determine the minimal enzymatic unit capable of robust catalytic activity. Mutagenesis of the helical interface between the two pairs of dimers comprising a GAC tetramer yielded a non-active, GAC dimer whose x-ray structure displays a stationary loop ("activation loop") essential for coupling the binding of allosteric activators like inorganic phosphate to catalytic activity. Further mutagenesis that removed constraints on the activation loop yielded a constitutively active dimer, providing clues regarding how the activation loop communicates with the active site, as well as with a peptide segment that serves as a "lid" to close off the active site following substrate binding. Our studies show that the formation of large GAC oligomers is not a pre-requisite for full enzymatic activity. They also offer a mechanism by which the binding of activators like inorganic phosphate enables the activation loop to communicate with the active site to ensure maximal rates of catalysis, and promotes the opening of the lid to achieve optimal product release. Moreover, these findings provide new insights into how other regulatory events might induce GAC activation within cancer cells.