Glutaminase Activity Research Articles

L-Asparaginase from Erwinia carotovora: insights about its stability and activity.

Enzymatic prospection indicated that L-asparaginase from Erwinia carotovora (ECAR-LANS) posses low glutaminase activity and much effort has been made to produce therapeutic ECAR-LANS. However, its low stability precludes its use in therapy. Herein, biochemical and biophysical assays provided data highlighting the influence of solubilization and storage into ECAR-LANS structure, stability, and activity. Moreover, innovations in recombinant expression and purification guaranteed the purification of functional tetramers. According to solubilization condition, the L-asparaginase activity and temperature of melting ranged up to 25-32%, respectively. CD spectra indicate the tendency of ECAR-LANS to instability and the influence of β-structures in activity. These results provide relevant information to guide formulations with prolonged action in the bloodstream.

Pcal_0970: an extremely thermostable L-asparaginase from Pyrobaculum calidifontis with no detectable glutaminase activity.

The genome sequence of Pyrobaculum calidifontis contains two open reading frames, Pcal_0144 and Pcal_0970, exhibiting homology with L-asparaginases. In search of a thermostable L-asparaginase with no glutaminase activity, we have cloned and expressed the gene encoding Pcal_0970 in Escherichia coli. Recombinant Pcal_0970 was produced in insoluble and inactive form which was solubilized and refolded into enzymatically active form. The refolded Pcal_0970 showed the highest activity at or above 100°C. Optimum pH for the enzyme activity was 6.5. Addition of divalent metal cations or EDTA had no significant effect on the activity. The enzyme was capable of hydrolyzing D-asparagine with a 20% activity as compared to 100% with L-asparagine. Pcal_0970 did not show any detectable activity when L-glutamine or D-glutamine was used as substrate. Pcal_0970 exhibited a Km value of 4.5 ± 0.4mmol/L and Vmax of 355 ± 13μmolmin-1mg-1 towards L-asparagine. The activation energy, from the linear Arrhenius plot, was determined as 39.9 ± 0.6kJmol-1. To the best of our knowledge, Pcal_0970 is the most thermostable L-asparaginase with a half-life of more than 150min at 100°C and this is the first report on characterization of an L-asparaginase from phylum Crenarchaeota.

GLS hyperactivity causes glutamate excess, infantile cataract and profound developmental delay.

Loss-of-function mutations in glutaminase (GLS), the enzyme converting glutamine into glutamate, and the counteracting enzyme glutamine synthetase (GS) cause disturbed glutamate homeostasis and severe neonatal encephalopathy. We report a de novo Ser482Cys gain-of-function variant in GLS encoding GLS associated with profound developmental delay and infantile cataract. Functional analysis demonstrated that this variant causes hyperactivity and compensatory downregulation of GLS expression combined with upregulation of the counteracting enzyme GS, supporting pathogenicity. Ser482Cys-GLS likely improves the electrostatic environment of the GLS catalytic site, thereby intrinsically inducing hyperactivity. Alignment of +/-12.000 GLS protein sequences from >1000 genera revealed extreme conservation of Ser482 to the same degree as catalytic residues. Together with the hyperactivity, this indicates that Ser482 is evolutionarily preserved to achieve optimal-but submaximal-GLS activity. In line with GLS hyperactivity, increased glutamate and decreased glutamine concentrations were measured in urine and fibroblasts. In the brain (both grey and white matter), glutamate was also extremely high and glutamine was almost undetectable, demonstrated with magnetic resonance spectroscopic imaging at clinical field strength and subsequently supported at ultra-high field strength. Considering the neurotoxicity of glutamate when present in excess, the strikingly high glutamate concentrations measured in the brain provide an explanation for the developmental delay. Cataract, a known consequence of oxidative stress, was evoked in zebrafish expressing the hypermorphic Ser482Cys-GLS and could be alleviated by inhibition of GLS. The capacity to detoxify reactive oxygen species was reduced upon Ser482Cys-GLS expression, providing an explanation for cataract formation. In conclusion, we describe an inborn error of glutamate metabolism caused by a GLS hyperactivity variant, illustrating the importance of balanced GLS activity.

Highly efficient novel recombinant L-asparaginase with no glutaminase activity from a new halo-thermotolerant Bacillus strain.

Introduction: The bacterial enzyme has gained more attention in therapeutic application because of the higher substrate specificity and longer half-life. L-asparaginase is an important enzyme with known antineoplastic effect against acute lymphoblastic leukemia (ALL). Methods: Novel L-asparaginase genes were identified from a locally isolated halo-thermotolerant Bacillus strain and the recombinant enzymes were overexpressed in modified E. coli strains, OrigamiTM B and BL21. In addition, the biochemical properties of the purified enzymes were characterized, and the enzyme activity was evaluated at different temperatures, pH, and substrate concentrations. Results: The concentration of pure soluble enzyme obtained from Origami strain was ~30 mg/L of bacterial culture, which indicates the significant improvement compared to L-asparaginase produced by E. coli BL21 strain. The catalytic activity assay on the identified L-asparaginases (ansA1 and ansA3 genes) from Bacillus sp. SL-1 demonstrated that only ansA1 gene codes an active and stable homologue (ASPase A1) with high substrate affinity toward L-asparagine. The Kcat and Km values for the purified ASPase A1 enzyme were 23.96s-1 and 10.66 µM, respectively. In addition, the recombinant ASPase A1 enzyme from Bacillus sp. SL-1 possessed higher specificity to L-asparagine than L-glutamine. The ASPase A1 enzyme was highly thermostable and resistant to the wide range of pH 4.5–10. Conclusion: The biochemical properties of the novel ASPase A1 derived from Bacillus sp. SL-l indicated a great potential for the identified enzyme in pharmaceutical and industrial applications.

Selective reduction in glutaminase activity of l‑Asparaginase by asparagine 248 to serine mutation: A combined computational and experimental effort in blood cancer treatment

Type II l‑asparaginase (l‑ASNase) is an FDA approved enzyme drug with extensive applications for treatment of certain blood cancers. However, the therapeutic efficiency of this enzyme is hampered by its undesirable glutaminase activity. Given the pivotal role of this enzyme against cancer, designing engineered mutants with diminished glutaminase activity would be of great therapeutic interest.To this end, N248S mutation was selected as the potential mutation with beneficial effects. Various in silico analyses including MD simulation, molecular docking and QMMM studies were performed to assess the effects of N248S mutation on the activity of the enzyme. Thereafter, this mutation along with N248A, N248V and N248T mutations as controls were exerted in l‑ASNase gene.The results from in silico analyses and experimental efforts indicated that N248S mutation is associated with the suitable l‑ASNase activity, while the glutaminase activity is disturbed due to impaired interactions. It has been shown that glutamine turnover was affected much more strongly than asparagine hydrolysis.The approach of exploiting in silico tools to design mutated enzymes lead to staggering time and cost reduction. Following this strategy, we have designed a mutant l‑ASNase with diminished glutaminase activity, which could be of interest for improved biomedical applications.

Overview of the Development of Glutaminase Inhibitors: Achievements and Future Directions.

It has been demonstrated that glutamine metabolism has become the main energy and building blocks supply for the growth and viability of a potentially large subset of malignant tumors. The glutamine metabolism often depends upon mitochondrial glutaminase (GLS) activity, which converts glutamine to glutamate and serves as a significant role for bioenergetic processes. Thus, recently, the GLS has become a key target for small molecule therapeutic intervention. Numerous medicinal chemistry studies are currently aimed at the design of novel and potent inhibitors for GLS, however, to date, only one compound (named CB-839) have entered clinical trials for the treatment of advanced solid tumors and hematological malignancies. The perspective summarizes the progress in the discovery and development of GLS inhibitors, including the potential binding site, biochemical techniques for inhibitor identification, and approaches for identifying small-molecule inhibitors, as well as future therapeutic perspectives in glutamine metabolism are also put forward in order to provide reference and rational for the drug discovery of novel and potent glutamine metabolism modulators.

Abstract B003: NRH:quinone oxidoreductase 2 (NQO2) and glutaminase (GLS) both play a role in large extracellular vesicles (LEV) formation in preclinical LNCaP-C4-2B prostate cancer model of progressive metastasis

Abstract Advanced-stage prostate cancer is characterized by osseous metastases whose establishment involves the dynamic interplay of factors and exchange of cellular contents by constituents of the tumor bony microenvironment. Among the factors that transport cellular cargoes and facilitate the transmission of signaling complexes for establishment of the bone metastatic lesions are large extracellular vesicles (LEVs), also known as large oncosomes. Information on the contents of LEVs and the mechanisms by which LEVs are formed and regulated is incomplete. In the course of studies aimed at elucidating the mechanisms of bony metastases using the LNCaP-C4-2B prostate cancer progression model, we show that the expression and cellular distribution of nicotinamide riboside: quinone oxidoreductase 2 (NQO2) and glutaminase (GLS, kidney type) both participate in the induction of membrane-localized oxidative stress and the formation of LEVs. The salient features of our findings are the following: The LNCaP model system exhibits gradually increasing oxidative stress levels as it progresses to the bone metastatic derivative C4-2B, while at the same time showing a progressive decrease in the expression level of NQO2. Reverse transcription PCR studies showed that NQO2 mRNA levels were unchanged between LNCaP and its metastatic derivatives C4, C4-2 and C4-2B. As a control, the expression level of the analogous enzyme NADP(H):quinone oxidoreductase 1 (NQO1) was also unchanged. These results suggested the possibility that the NQO2 enzyme in the bone metastatic C4-2B derivative could be localized elsewhere, possibly at an extracellular location. Further studies showed that a fraction of the NQO2 enzyme was associated with the detergent resistant membrane fraction of the C4-2B prostate cancer cells. Molecular modeling studies showed that the NQO2 enzyme had two caveolin-1 and NQO1 enzyme had one caveolin-1 binding sites. While the caveolin-1 binding site of NQO1 is buried inside the molecule and hence inaccessible to caveolin-1, one of the two binding sites available in NQO2 was external and accessible to caveolin-1. We have validated the specific interaction between NQO2 and caveolin-1 using deletion constructs of caveolin-1 fused with glutathione S-transferase (GST). Mitochondrial preparations of LNCaP, C4, C4-2, and C4-2B cells showed increasing level of expression of the GLS enzyme, suggesting a correlation between increased oxidative stress, glutaminase activity, and metastatic potential. Moreover, we found that the metastatic C4-2B derivative extruded LEVs in which the enzyme NQO2 and caveolae specific caveolin-1 could be visualized as cargo using double immunofluorescence studies. The extrusion of these LEVs could be inhibited by the GLS specific inhibitor BP-TES. Characteristically, curcumin was also found to inhibit the extrusion of these LEVs. This naturally occurring plant-based compound was shown earlier to inhibit prostate cancer bone metastasis in vivo using the C4-2B model. These results indicate a role for the NQO2 enzyme at the membrane level, possibly increasing the oxidative stress in a localized manner by lipid peroxidation. These results also suggest that a glutamine/GLS-mediated metabolic reprogramming in the LNCaP model system as it progresses toward the bone metastatic C4-2B is an integral component of the force driving the metastatic process. Based on the results of our studies, a combinatorial strategy using antimetastatic therapies such as nitrogen-containing bisphosphonates (NBPs) and anti-GLS therapy or, alternatively, a naturally occurring plant compound-based anti-NQO2 and anti-GLS therapy, could be considered for treating advanced prostate cancer patients with bone metastatic complications. Citation Format: Thambi Dorai, Ankeeta B. Shah, Faith Summers, Rajamma Mathew, Jing Huang, Tze-chen Hsieh, Joseph M. Wu. NRH:quinone oxidoreductase 2 (NQO2) and glutaminase (GLS) both play a role in large extracellular vesicles (LEV) formation in preclinical LNCaP-C4-2B prostate cancer model of progressive metastasis [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B003.

JHU-083 selectively blocks glutaminase activity in brain CD11b+ cells and prevents depression-associated behaviors induced by chronic social defeat stress.

There are a number of clinically effective treatments for stress-associated psychiatric diseases, including major depressive disorder (MDD). Nonetheless, many patients exhibit resistance to first-line interventions calling for novel interventions based on pathological mechanisms. Accumulating evidence implicates altered glutamate signaling in MDD pathophysiology, suggesting that modulation of glutamate signaling cascades may offer novel therapeutic potential. Here we report that JHU-083, our recently developed prodrug of the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON) ameliorates social avoidance and anhedonia-like behaviors in mice subjected to chronic social defeat stress (CSDS). JHU-083 normalized CSDS-induced increases in glutaminase activity specifically in microglia-enriched CD11b+ cells isolated from the prefrontal cortex and hippocampus. JHU-083 treatment also reverses the CSDS-induced inflammatory activation of CD11b+ cells. These results support the importance of altered glutamate signaling in the behavioral abnormalities observed in the CSDS model, and identify glutaminase in microglia-enriched CD11b+ cells as a pharmacotherapeutic target implicated in the pathophysiology of stress-associated psychiatric conditions such as MDD.

“Pinching” the ammonia tunnel of CTP synthase unveils coordinated catalytic and allosteric-dependent control of ammonia passage

Molecular gates within enzymes often play important roles in synchronizing catalytic events. We explored the role of a gate in cytidine-5′-triphosphate synthase (CTPS) from Escherichia coli. This glutamine amidotransferase catalyzes the biosynthesis of CTP from UTP using either l-glutamine or exogenous NH3 as a substrate. Glutamine is hydrolyzed in the glutaminase domain, with GTP acting as a positive allosteric effector, and the nascent NH3 passes through a gate located at the end of a ~25-Å tunnel before entering the synthase domain where CTP is generated. Substitution of the gate residue Val 60 by Ala, Cys, Asp, Trp, or Phe using site-directed mutagenesis and subsequent kinetic analyses revealed that V60-substitution impacts glutaminase activity, nucleotide binding, salt-dependent inhibition, and inter-domain NH3 transport. Surprisingly, the increase in steric bulk present in V60F perturbed the local structure consistent with “pinching” the tunnel, thereby revealing processes that synchronize the transfer of NH3 from the glutaminase domain to the synthase domain. V60F had a slightly reduced coupling efficiency at maximal glutaminase activity that was ameliorated by slowing down the glutamine hydrolysis reaction, consistent with a “bottleneck” effect. The inability of V60F to use exogenous NH3 was overcome in the presence of GTP, and more so if CTPS was covalently modified by 6-diazo-5-oxo-l-norleucine. Use of NH2OH by V60F as an alternative bulkier substrate occurred most efficiently when it was concomitant with the glutaminase reaction. Thus, the glutaminase activity and GTP-dependent activation act in concert to open the NH3 gate of CTPS to mediate inter-domain NH3 transport.

Activation of p53 by costunolide blocks glutaminolysis and inhibits proliferation in human colorectal cancer cells

Colorectal cancer is a leading cause of cancer-related death. Glutaminolysis has been suggested as a therapeutic target for cancer. Costunolide is a natural sesquiterpene lactone showing potent antitumor activity. Our studies were aimed at evaluating how costunolide affected glutaminolysis leading to proliferation inhibition in human colorectal cancer cells. Costunolide suppressed viability and proliferation of HCT116 cells concentration-dependently, but did not apparently affect human intestinal epithelial cells. Costunolide at 20 μM reduced viability and proliferation of HCT116 cells time-dependently. Costunolide also repressed phosphorylation of mTOR and its downstream kinases p70S6K and 4E-BP1. Examinations of glutaminolysis metabolites showed that costunolide increased intracellular glutamine levels, but decreased intracellular levels of glutamate, α-ketoglutarate (α-KG), and ATP in HCT116 cells, suggesting costunolide blockade of glutaminolysis. Furthermore, costunolide inhibited promoter activity of glutaminase 1 (GLS1), the first rate-limiting enzyme in glutaminolysis, and reduced mRNA and protein expression of GLS1 in HCT116 cells, The GLS1 inhibitor BPTES, similar to costunolide, significantly reduced intracellular levels of α-KG and ATP and inhibited proliferation in HCT116 cells. Finally, costunolide increased phosphorylation and nuclear translocation of p53 in HCT116 cells. Both p53 inhibitor pifithrin-α and p53 siRNA significantly rescued costunolide suppression of GLS1 promoter activity and expression in HCT116 cells. These data in aggregate suggested that activation of p53 was required for costunolide inhibition of GLS1 resulting in blockade of glutaminolysis and inhibition of proliferation in colorectal cancer cells, which was a novel mechanism underlying the antitumor activity of costunolide against colorectal cancer.

N-terminal phosphorylation of glutaminase C decreases its enzymatic activity and cancer cell migration

The mitochondrial phosphate-activated glutaminase C (GAC) is produced by the alternative splicing of the GLS gene. Compared to the other GLS isoform, the kidney-type glutaminase (KGA), GAC is more enzymatically efficient and of particular importance for cancer cell growth. Although its catalytic mechanism is well understood, little is known about how post-translational modifications can impact GAC function. Here, we identified by mass spectrometry a phosphorylated serine at the GLS N-terminal domain (at position 95) and investigated its role on regulating GAC activity. The ectopic expression of the phosphomimetic mutant (GAC.S95D) in breast cancer cells, compared to wild-type GAC (GAC.WT), led to decreased glutaminase activity, glutamine uptake, glutamate release and intracellular glutamate levels, without changing GAC sub-cellular localization. Interestingly, cells expressing the GAC.S95D mutant, compared to GAC.WT, presented decreased migration and vimentin level, an epithelial-to-mesenchymal transition marker. These results reveal that GAC is post-translationally regulated by phosphorylation, which affects cellular glutamine metabolism and glutaminase-related cell phenotype.

Abstract 5482: L-2HG/ L2HGDH axis as therapeutic target for kidney cancer

Abstract Background: The D-enantiomer of 2-hydroxygultarate (D-2HG), along with fumarate and succinate, are considered oncometabolites that promote tumorigenesis. Our lab has reported elevated levels of the L-enantiomer of 2-hydroxyglutare (L-2HG) in clear cell renal cell carcinoma (ccRCC), in part due to reduced expression of the enzyme L-2HG dehydrogenase (L2HGDH). Objectives: Here we evaluated the underlying biochemical mechanisms of L-2HG accumulation and characterized the contribution of the L-2HG/L2HGDH axis to tumorigenesis. Experimental strategy: We assessed the role of raised L-2HG in renal carcinogenesis via both in vitro and in vivo means. Mutagenesis and knock down approaches were applied to study involvement of loss of L2HGDH in accumulation of high L-2HG levels and associated tumor phenotypes. Metabolomics coupled with 13C tracer labeling studies were utilized to dissect the biochemical axis that promotes L-2HG accumulation in RCC cells. Results: Translational based studies demonstrate that loss of L2HGDH expression is associated with both cancer progression and worsened outcomes. In connection with our previous findings, we demonstrate that raising cellular L-2HG levels by treatment with cell permeable octyl ester of L-2HG as well as by shRNA-mediated knock down of L2HGDH in renal epithelial cells (HK-2) promotes in vitro tumor phenotypes. Concurrently, the epigenetic mark 5hmc was significantly decreased under high L-2HG levels. Further investigation with restoration of L2HGDH in RCC cells (RXF393 and A498) shows decreased L-2HG levels and suppression of in vivo tumor growth in nude mice (NU/NU). Interestingly, expression of loss-of-function mutant of L2HGDH was unable to decrease L-2HG levels and failed to suppress in vitro and in vivo tumor phenotypes. In addition, high L-2HG levels were found to upregulate epithelial-mesenchymal-transition (EMT) marker SNAIL1 and correspondingly downregulate E-cadherin. Biochemical studies demonstrate that the predominant carbon source for L-2HG in RCC is glutamine through the activity of glutaminase and malate dehydrogenase (MDH). Pharmacological inhibition of the glutamine/MDH axis by treatment of glutaminase inhibitor (CB-839) and MDH inhibitor (4k) in RCC cells reduced L-2HG levels and mitigates in vitro tumor phenotypes. Furthermore, suppression of in vitro phenotypes by CB-839 and shRNA-mediated MDH2 knockdown was rescued by concurrent treatment with octyl ester of L-2HG. Finally, restoration of L2HGDH promoted the expression of genes targeted by the polycomb repressor complex 2 (PRC2), whereas inhibition of PRC2 in high L-2HG cells suppressed tumor phenotypes. Conclusion: Collectively, our data demonstrate the biologic relevance of high L-2HG to renal carcinogenesis and reveal novel therapeutic opportunities for L-2HG driven kidney tumors. Citation Format: Sandeep Balu Shelar, Eun-hee Shim, Garrett Brinkley, Anirban Kundu, Hyeyoung Nam, Francesca Carobbio, Tyler Poston, Jubilee Tan, Daniel Benson, Dinesh Rakheja, Richard Kirkman, Yusuke Sato, Seishi Ogawa, Shilpa Dutta, Sadanandan E. Velu, David Crossman, Anja Becker, Conrad Kunick, Sunil Sudarshan. L-2HG/ L2HGDH axis as therapeutic target for kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5482.

Abstract 374: Direct measurements of cellular ATP levels in tumor cell lines using real-time, quantitative live-cell analysis

Abstract Metabolic reprogramming is a hallmark of tumor cells, owing to the increased energetic and biosynthetic demands associated with maintaining high rates of proliferation. Therapeutic strategies targeting unique, critical metabolic pathways of tumor cells are under investigation and in clinical trials. Standard approaches to monitoring drug induced metabolic perturbations are limited to endpoint assays that provide population-based measurements and limited kinetic information. We developed a genetically encoded ATP sensor to enable direct, automated analysis of cellular ATP levels using IncuCyte® S3. In this study, our live cell imaging approach was utilized to evaluate the effect of cancer therapeutics on ATP levels in tumor cell lines, focusing primarily on compounds that target metabolic vulnerabilities. Cell lines stably expressing a genetically encoded, fluorescent ATP sensor or a control (non-ATP binding) sensor were generated. ATP levels were monitored and analyzed using an IncuCyte® S3 equipped with a specialized filter set and data acquisition module. Cellular ATP levels were measured over the course of hours to days following compound treatment. Reductions in ATP levels could be observed within an hour of administration, highlighting the sensitivity and temporal resolution of the IncuCyte® S3 ATP sensor assay. Transient reductions in ATP followed by recovery, which would have been missed by typical endpoint assays, were noted using our live cell analysis approach. Sustained decreases in ATP were associated with enhanced antiproliferative efficacy compared to conditions under which recovery of ATP levels were observed. For example, triple-negative breast cancer (TNBC) cell lines have been shown to be more dependent on activity of glutaminase 1 (GLS1), which catalyzes the first step in utilization of glutamine to fuel mitochondrial metabolism, than their receptor-positive counterparts. Using our live cell analysis approach, we observed a rapid drop in ATP upon glutamine deprivation or inhibition of GLS1 by CB-839 in the TNBC cell line MDA-MB-231. ATP levels remained below that of vehicle-treated cells for the duration of the three-day time course. In contrast, the estrogen receptor-positive cell line MCF-7 displayed a more modest decrease in ATP and full recovery within 48 hours. Quantification of phase confluence confirmed that CB-839 had a stronger antiproliferative effect in MDA-MB-231 cells compared to MCF-7 cells. Visualization of morphology in tandem with automated ATP sensor analysis provide additional insight into the heterogeneity of cellular responses to compound addition. Overall, these data highlight the ability of IncuCyte® S3 to provide direct, kinetic measurement of ATP by live-cell analysis. Citation Format: Cicely L. Schramm, Grigory S. Filonov, Michael L. Bowe, Yong X. Chen, Laura A. Skerlos, Dyke P. McEwen, Daniel M. Appledorn. Direct measurements of cellular ATP levels in tumor cell lines using real-time, quantitative live-cell analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 374.

Abstract 1452: The receptor tyrosine kinase EphA2 promotes glutamine metabolism in tumors by activating the transcriptional coactivators YAP and TAZ

Abstract Malignant tumors reprogram cellular metabolism to support cancer cell proliferation and survival. Although most cancers depend on a high rate of aerobic glycolysis, many cancer cells also display addiction to glutamine. Glutamine transporters and glutaminase activity are critical for glutamine metabolism in tumor cells. We found that the receptor tyrosine kinase EphA2 activated the TEAD family transcriptional coactivators YAP and TAZ (YAP/TAZ), likely in a ligand-independent manner, to promote glutamine metabolism in cells and mouse models of HER2-positive breast cancer. Overexpression of EphA2 induced the nuclear accumulation of YAP and TAZ and increased the expression of YAP/TAZ target genes. Inhibition of the GTPase Rho or the kinase ROCK abolished EphA2-dependent YAP/TAZ nuclear localization. Silencing YAP or TAZ substantially reduced the amount of intracellular glutamate through decreased expression of SLC1A5 and GLS, respectively, genes that encode proteins that promote glutamine uptake and metabolism. The regulatory DNA elements of both SLC1A5 and GLS contain TEAD binding sites and were bound by TEAD4 in an EphA2-dependent manner. In patient breast cancer tissues, EphA2 expression positively correlated with that of YAP and TAZ, as well as that of GLS and SLC1A5. Although high expression or EphA2 predicted enhanced metastatic potential and poor patient survival, it also rendered HER2-positive breast cancer cells more sensitive to glutaminase inhibition. The findings define a previously unknown mechanism of EphA2-mediated glutaminolysis through YAP/TAZ activation in HER2-positive breast cancer and identify therapeutic targets in patients. Citation Format: Deanna N. Edwards, Verra M. Ngwa, Shan Wang, Eileen Shiuan, Dana Brantley-Sieders, Laura Kim, Albert B. Reynolds, Jin Chen. The receptor tyrosine kinase EphA2 promotes glutamine metabolism in tumors by activating the transcriptional coactivators YAP and TAZ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1452.

Abstract 5493: Targeted inhibition of glutaminase in GRM1-expressing melanoma cells inhibits cell proliferation by reducing glutamate bioavailability

Abstract Aberrant glutamatergic signaling has been implicated in many cancer types. Our laboratory has previously illustrated the role of metabotropic glutamate receptor 1 (GRM1) in neoplastic transformation of melanocytes in vitro and spontaneous metastatic melanoma in vivo. Glutamate, the natural ligand of GRM1 is also the predominant excitatory neurotransmitter in the central nervous system. We have demonstrated significant upregulation of glutaminase (GLS) expression in GRM1 expressing melanoma cells, resulting in excess glutamate production and the establishment of autocrine loops in vitro. GLS catalyzes the first step in glutamine metabolism–the conversion of glutamine to glutamate. Comparison of glutamate levels in circulating blood plasma between melanoma prone and wild type mice showed elevated glutamate levels in melanoma prone GRM1-transgenic mice, suggesting that aberrant GRM1 expression also promotes an increase in circulating glutamate, to ensure constitutive activation of the GRM1 receptor and its downstream signaling affecters in vivo. Inclusion of CB-839, a potent, selective, and orally bioavailable inhibitor of GLS, resulted in significant inhibition of GRM1+ melanoma cell proliferation compared to GRM1- ones. We also demonstrated that treatment with Riluzole, an inhibitor of glutamatergic signaling by decreasing the available ligand, glutamate, led to significantly reduced melanoma cell proliferation in vitro and tumor progression in vivo. We found that simultaneous targeting of glutamate production (CB-839) and release (Riluzole) in GRM1+ melanoma cells led to synergistic suppression of cell proliferation in vitro. Furthermore, we found that CB-839 altered the function not the expression of GLS in melanoma cells. Additionally, we also discovered that manipulation of GRM1 expression by genetic means led to robust parallel changes in gene expression levels and intracellular glutamate concentrations by mass spectrometry analysis, as well as the endogenous levels of GLS in melanoma cells, suggesting association between GRM1 and GLS. The sensitivity of GRM1+ melanoma cells to modulation of GLS point to the dependency of these cells to glutamate amounts via GLS activity. We are continuing to investigate the mechanisms and regulation of GLS in our experimental system, with the goal of developing a rational approach for the treatment of GRM1 expressing tumors. Citation Format: Raj Shah, Mengying Zhu, Andrew Boreland, Fabian Filipp, Suzie Chen. Targeted inhibition of glutaminase in GRM1-expressing melanoma cells inhibits cell proliferation by reducing glutamate bioavailability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5493.

Abstract 5472: Basal-like breast cancer subtype is characterized by deregulated glutamine metabolism and is sensitive to GLS inhibition

Abstract Tumor cells display enhanced requirements for energy and building blocks such as amino acids, nucleic acids and lipids to sustain their proliferation. Metabolic demands of cancer cells are often met in the context of limited access to nutrients and the need to maintain redox balance. Glutamine catabolism is considered critical to cancer cell survival by not only delivering carbon and nitrogen atoms needed for nucleic acid and amino acid precursors but also by serving as the source of antioxidants such as NADPH and glutathione. A vital step in the catabolism of glutamine is its conversion to glutamate by the mitochondrial enzyme glutaminase (GLS).In this study, we confirmed the significance of GLS and enhanced glutamine utilization in the basal subtype of breast cancer as evidenced by higher GLS to GLUL (Glutamine synthetase) ratio in a panel of breast cancers in The Cancer Genome Atlas (TCGA) database. Using inducible shRNA mediated gene knockdown, we discovered that loss of GLS function in triple-negative breast cancer (TNBC) cell lines led to profound tumor growth inhibition in vitro and in vivo. This anti-tumor effect of GLS knockdown was rescued either using shRNA resistant cDNAs encoding both GLS isoforms or by addition of an a-ketoglutarate analog thus confirming the critical role of GLS in TNBC. Along with these observations, we have found that pharmacological inhibition of GLS with the small molecule inhibitor CB-839 reduced cell growth and led to a decrease in mammalian target of rapamycin (mTOR) coupled with an increase in the stress response pathway driven by activating transcription factor 4 (ATF4). In line with the observed impact on mTOR pathway we found that GLS inhibition synergizes with mTOR inhibition. In conclusion, our preliminary investigation revealed that glutaminolysis is deregulated in a majority of TNBC cancer patients. Our target credentialing studies confirmed that GLS is essential for the survival of TNBC cell lines and xenografts tumors. The synergistic activity of GLS and mTOR inhibitors in this specific breast cancer subtype suggests a new therapeutic modality that may bring benefit to TNBC patients with high unmet need. Citation Format: Christophe Henry, Michael Lampa, Timothy He, Beatriz Ospina, Bailin Zhang, Gejing Deng, Claude Barberis, Dietmar Hoffmann, Jack Pollard, Adrian Francisco, Heike Arlt, Jason Reeves, Joshua Murtie, Christopher Winter, Victoria Richon, Hong Cheng, Carlos Garcia-Echeverria, Dmitri Wiederschain, Lakshmi Srinivasan. Basal-like breast cancer subtype is characterized by deregulated glutamine metabolism and is sensitive to GLS inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5472.

Abstract 4875: Addition of RP10107, a novel and potent glutaminase inhibitor, accentuates 5-FU activity in lung cancer cell lines in vitro

Abstract Background: Drug resistance remains a significant limitation to the clinical use of 5-Fluorouracil (5-FU) in lung cancer. With cancer cell metabolism emerging as a critical regulator of tumor progression, combining 5-FU with an inhibitor of the metabolic machinery represents a potential therapeutic strategy to prevent lung cancer progression. Cancer cell metabolism is reprogrammed wherein glutamine utilization is increased via elevation of glutaminase activity thereby generating the necessary substrates required for eventual ATP synthesis and energy production. RP10107 is a novel, potent, and selective glutaminase (GLS-1) inhibitor that demonstrated high potency against mouse (IC50=21.2 nM), rat (IC50=18.2 nM) and human (IC50=26.4 nM) enzymes with selectivity over GLS-2 (>380-fold). The objective of this study was to evaluate the effect of a combination of 5-FU and RP10107 in lung cancer cells. Methods: Glutamate concentrations in lung cancer cell lines (A549, NCI-H460, NCI-H1975, NCI-H441, and NCI-H2170) following treatment with RP10107 was estimated by LC-MS/MS. Synergism with 5-FU was determined using different concentrations of the compounds in a 5 x 5 grid. Synergism, additivity, or antagonism was calculated based on the BLISS score. Apoptosis was determined by Annexin V/7AAD staining using a MUSE® Annexin V and dead cell assay kit (Millipore) while the effect on cell cycle was estimated using Guava Cell Cycle Reagent (Millipore). Expression of caspase 3/7, phosphor-p53 (S15), and Bcl-2 were determined by Western Blotting. Results: Treatment of lung cancer cell lines with RP10107 resulted in an increased ratio of glutamine to glutamate with a doubling noticed between 100-300 nM. The change in glutamine utilization was accompanied by a dose-dependent inhibition in cell growth with half-maximal inhibitions ranging from 1.3 to 9.0 μM. Synergism or additivity between RP10107 and 5-FU for anti-proliferative activity was noticed in all the cell lines tested with effect being most pronounced in NCI-H460 and NCI-H441. Additionally, incubation with the combination (1-5 μM RP10107 + 3-5 μM 5-FU) for 48-72 h caused a G2/M or S-phase arrest with a corresponding increase in the percent of apoptotic cells (1-5 to 2.5-fold) compared to the individual agents. Combination of RP10107 and 5-FU increased expression of phospho-p53 expression in all cell lines tested while it led to 40-75% reduction in caspase 3, caspase 7, and Bcl-2 in NCI-H2170 and NCI-H460. Conclusions: Addition of RP10107, a potent glutaminase inhibitor, potentiated 5-FU activity in lung cancer cell lines. Findings provide a rationale for use of the combination in future clinical trials involving lung cancer patients thereby providing a safer and more effective alternative to currently available therapy. Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Seeta Nyayapathy, Swaroop Vakkalanka. Addition of RP10107, a novel and potent glutaminase inhibitor, accentuates 5-FU activity in lung cancer cell lines in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4875.

SN56 neuronal cell death after 24 h and 14 days chlorpyrifos exposure through glutamate transmission dysfunction, increase of GSK-3β enzyme, β-amyloid and tau protein levels

Chlorpyrifos (CPF) is an organophosphate insecticide described to induce cognitive disorders, both after acute and repeated administration. However, the mechanisms through which it induces these effects are unknown. CPF has been reported to produce basal forebrain cholinergic neuronal cell death, involved on learning and memory regulation, which could be the cause of such cognitive disorders. Neuronal cell death was partially mediated by oxidative stress generation, P75NTR and α7-nAChRs gene expression alteration triggered through acetylcholinesterase (AChE) variants disruption, suggesting other mechanisms are involved. In this regard, CPF induces Aβ and tau proteins production and activation of GSK3β enzyme and alters glutamatergic transmission, which have been related with basal forebrain cholinergic neuronal cell death and development of cognitive disorders. According to these data, we hypothesized that CPF induces basal forebrain cholinergic neuronal cell death through induction of Aβ and tau proteins production, activation of GSK-3β enzyme and disruption of glutamatergic transmission. We evaluated this hypothesis in septal SN56 basal forebrain cholinergic neurons, after 24 h and 14 days CPF exposure. This study shows that CPF increases glutamate levels, upregulates GSK-3β gene expression, and increases the production of Aβ and phosphorylated tau proteins and all these effects reduced cell viability. CPF increases glutaminase activity and upregulates the VGLUT1 gene expression, which could mediate the disruption of glutamatergic transmission. Our present results provide new understanding of the mechanisms contributing to the harmful effects of CPF, and its possible relevance in the pathogenesis of neurodegenerative diseases.

IDH1‐mutated gliomas rely on anaplerosis of glutamate and lactate whereas IDH1 wild‐type gliomas rely on glycolysis and acetate anaplerosis

IntroductionHotspot mutations in isocitrate dehydrogenase 1 (IDH1MUT) initiate low grade glioma (LGG) and secondary glioblastoma and induce neomorphic activity that converts α‐ketoglutarate (α‐KG) to the oncometabolite D‐2‐hydroxyglutarate (D‐2HG). This causes metabolic rewiring and stress that is not fully understood. In vitro studies suggest that this stress is relieved via glutaminolysis, providing cells with α‐KG via activities of glutaminase (GLS) and glutamate dehydrogenase (GLUD). We previously postulated that IDH1MUT gliomas directly use glutamate as a shortcut.ResultsTo test this hypothesis, we first showed by in silico analysis of 269 IDH1 wild‐type (IDH1WT) and 408 IDH1MUT gliomas, obtained from the The Cancer Genome Atlas (TCGA) database, that IDH1WT gliomas have high expression levels of genes encoding for enzymes that are involved in glycolysis and acetate anaplerosis. On the other hand, the tricarboxylic acid (TCA) cycle, rather than glycolytic lactate production, is the predominant metabolic pathway in IDH1MUT gliomas and is driven by lactate and glutamate anaplerosis to facilitate production of α‐KG, and ultimately D‐2‐HG. Secondly, we confirmed these findings by a novel technique of quantitative targeted next‐generation sequencing of RNAs encoding for 104 metabolic enzymes, on a cohort of 66 clinical gliomas. Preferential glutamate processing in IDH1MUT gliomas was confirmed by in situ enzyme activity mapping experiments in clinical gliomas of known IDH status. We functionally validated these findings in a set of glioma xenografts with and without the endogenous IDH1R132H mutation, indicating that IDH1MUT gliomas depend on glutamatolysis, rather than glutaminolysis. We show that transcript levels in our xenocraft models are in good agreement with our in silico analysis of the TCGA database. Finally, we confirmed the glutamate dependency of IDH1MUT gliomas by in vivo magnetic resonance spectroscopic imaging, whereas IDH1WT gliomas showed high lactate levels.ConclusionsWe show that IDH1WT gliomas have a typical Warburg phenotype and rely on acetate anaplerosis whereas IDH1MUT gliomas are glutamate and lactate dependent. This metabolic rewiring in IDH1MUT glioma, enables targeting of glutaminolysis rather than direct inhibition of IDH1MUT for therapy. It diminishes the supply of glutamate‐derived α‐KG and directly inhibits the production of D‐2HG and simultaneously worsen the redox status of the glioma cells by inhibiting NAD(P)H production by GLUD. A candidate drug to inhibit GLUD is epigallocatechin‐3‐gallate gallate, a component of green tea that is currently receiving high interest as anticancer agent.Support or Funding InformationThis work was supported by Dutch Cancer Society grant UvA 2014‐6839 (KL, MK) and by Stichting StopHersentumoren (CNAMvdH). RJM was supported by an AMC PhD scholarship. THP was supported by EFRO/GO project Ultrasense NMR.

First insights of peptidoglycan amidation in Gram-positive bacteria - the high-resolution crystal structure of Staphylococcus aureus glutamine amidotransferase GatD

Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.85 Å resolution. A glutamine molecule is found close to the active site funnel, hydrogen-bonded to the conserved R128. In vitro functional studies using 1H-NMR spectroscopy showed that S. aureus MurT-GatD complex has glutaminase activity even in the absence of lipid II, the MurT substrate. In addition, we produced R128A, C94A and H189A mutants, which were totally inactive for glutamine deamidation, revealing their essential role in substrate sequestration and catalytic reaction. GatD from S. aureus and other pathogenic bacteria share high identity to enzymes involved in cobalamin biosynthesis, which can be grouped in a new sub-family of glutamine amidotransferases. Given the ubiquitous presence of GatD, these results provide significant insights into the molecular basis of the so far undisclosed amidation mechanism, contributing to the development of alternative therapeutics to fight infections.