Glutaminase Inhibitor Research Articles

Urban airborne PM2.5-activated microglia mediate neurotoxicity through glutaminase-containing extracellular vesicles in olfactory bulb

Emerging evidence has showed that exposure to airborne particulate matter (PM) with an aerodynamic diameter less than 2.5 μm (PM2.5) is associated with neurodegeneration. Our previous studies in vitro found that PM2.5 exposure causes primary neurons damage through activating microglia. However, the molecular mechanism of microglia-mediated neurotoxicity remains to elucidate. In this study, five groups (N = 13 or 10) of six-week-old male C57BL/6 mice were daily exposed to PM2.5 (0.1 or 1 mg/kg/day body weight), Chelex-treated PM2.5 (1 mg/kg/day body weight), PM2.5 (1 mg/kg/day body weight) plus CB-839 (glutaminase inhibitor), or deionized water by intranasal instillation for 28 days, respectively. Compared with the control groups, We found that PM2.5 triggered reactive oxygen species (ROS) generation and microglia activation evidenced by significant increase of ionized calcium binding adaptor molecule-1 (IBa-1) staining in the mouse olfactory bulbs (OB). Data from transmission electron microscope (TEM) images and Western blot analysis showed that PM2.5 significantly increased extracellular vesicles (EVs) release from OB or murine microglial line BV2 cells, and glutaminase C (GAC) expression and glutamate generation in isolated OB and BV2 cells. However, treatment with N-acetylcysteine (NAC) or CB-839 significantly diminished the number of EVs and the expression of GAC and abolished PM2.5-induced neurotoxicity. These findings provide new insights that PM2.5 induces oxidative stress and microglia activation through its metal contents and glutaminase-containing EVs in OBs, which may serve as a potential pathway/mechanism of excessive glutamate generation in PM2.5-induced neurotoxicity.

Molecular modeling and LC-MS-based metabolomics of a glutamine-valproic acid (Gln-VPA) derivative on HeLa cells.

Glutaminase plays an important role in carcinogenesis and cancer cell growth. This biological target is interesting against cancer cells. Therefore, in this work, in silico [docking and molecular dynamics (MD) simulations] and in vitro methods (antiproliferative and LC-MS metabolomics) were employed to assay a hybrid compound derived from glutamine and valproic acid (Gln-VPA), which was compared with 6-diazo-5-oxo-L-norleucine (DON, a glutaminase inhibitor) and VPA (contained in Gln-VPA structure). Docking results from some snapshots retrieved from MD simulations show that glutaminase recognized Gln-VPA and DON. Additionally, Gln-VPA showed antiproliferative effects in HeLa cells and inhibited glutaminase activity. Finally, the LC-MS-based metabolomics studies on HeLa cells treated with either Gln-VPA (IC60 = 8mM) or DON (IC50 = 3.5mM) show different metabolomics behaviors, suggesting that they modulate different biological targets of the cell death mechanism. In conclusion, Gln-VPA is capable of interfering with more than one pharmacological target of cancer, making it an interesting drug that can be used to avoid multitherapy of classic anticancer drugs.

A comparative pharmaco-metabolomic study of glutaminase inhibitors in glioma stem-like cells confirms biological effectiveness but reveals differences in target-specificity

Cancer cells upregulate anabolic processes to maintain high rates of cellular turnover. Limiting the supply of macromolecular precursors by targeting enzymes involved in biosynthesis is a promising strategy in cancer therapy. Several tumors excessively metabolize glutamine to generate precursors for nonessential amino acids, nucleotides, and lipids, in a process called glutaminolysis. Here we show that pharmacological inhibition of glutaminase (GLS) eradicates glioblastoma stem-like cells (GSCs), a small cell subpopulation in glioblastoma (GBM) responsible for therapy resistance and tumor recurrence. Treatment with small molecule inhibitors compound 968 and CB839 effectively diminished cell growth and in vitro clonogenicity of GSC neurosphere cultures. However, our pharmaco-metabolic studies revealed that only CB839 inhibited GLS enzymatic activity thereby limiting the influx of glutamine derivates into the TCA cycle. Nevertheless, the effects of both inhibitors were highly GLS specific, since treatment sensitivity markedly correlated with GLS protein expression. Strikingly, we found GLS overexpressed in in vitro GSC models as compared with neural stem cells (NSC). Moreover, our study demonstrates the usefulness of in vitro pharmaco-metabolomics to score target specificity of compounds thereby refining drug development and risk assessment.

Undermining Glutaminolysis Bolsters Chemotherapy While NRF2 Promotes Chemoresistance in KRAS-Driven Pancreatic Cancers.

Pancreatic cancer is a disease with limited therapeutic options. Resistance to chemotherapies poses a significant clinical challenge for patients with pancreatic cancer and contributes to a high rate of recurrence. Oncogenic KRAS, a critical driver of pancreatic cancer, promotes metabolic reprogramming and upregulates NRF2, a master regulator of the antioxidant network. Here, we show that NRF2 contributed to chemoresistance and was associated with a poor prognosis in patients with pancreatic cancer. NRF2 activation metabolically rewired and elevated pathways involved in glutamine metabolism. This curbed chemoresistance in KRAS-mutant pancreatic cancers. In addition, manipulating glutamine metabolism restrained the assembly of stress granules, an indicator of chemoresistance. Glutaminase inhibitors sensitized chemoresistant pancreatic cancer cells to gemcitabine, thereby improving the effectiveness of chemotherapy. This therapeutic approach holds promise as a novel therapy for patients with pancreatic cancer harboring KRAS mutation. SIGNIFICANCE: These findings illuminate the mechanistic features of KRAS-mediated chemoresistance and provide a rationale for exploiting metabolic reprogramming in pancreatic cancer cells to confer therapeutic opportunities that could be translated into clinical trials. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/8/1630/F1.large.jpg.

From the Editor’s Desk…

From the Editor’s Desk…

Abstract IA12: Fueling T cells in inflammation and cancer

Abstract Lymphocyte activation leads to rapid proliferation and differentiation, and we have shown that CD4 T-cell subsets are metabolically distinct. These specific metabolic programs may allow new understanding and approaches to manipulate immunity. Using metabolic network analysis of metabolomics and proteomics, we defined several metabolic nodes differentially utilized by CD4 T-cell subsets, including glutamine metabolism. By genetically deleting the glucose transporter Glut1 or the glutaminolysis enzyme, glutaminase (GLS), we have shown that glycolysis and glutaminolysis are both used by activated T cells. All effector T cells require Glut1 and glycolysis. In contrast, we found that Th17 cells preferentially require GLS while Th1 cells are actively impaired by this enzyme. Thus, inhibition of GLS both reduces Th17 responses and promotes differentiation of Th1 cells and can lead to signs of T-cell exhaustion. We show that GLS deficiency can protect against Th17-mediated inflammatory models and also can augment effector function of Th1 CAR-T cells against B-cell targets. Understanding mechanisms that regulate T-cell metabolism may provide new tools to modulate immunity in the balance of T-cell effector populations to both suppress inflammation and promote effector function. Citation Format: Jeffrey C. Rathmell. Fueling T cells in inflammation and cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr IA12.

Regulation of GLS1 Expression by Scleraxis in Cardiac Fibroblasts

Cardiac fibrosis is characterized by the excessive deposition of extracellular matrix, including fibrillar collagens, proteoglycans, and fibronectin. This process is energy‐intensive for cardiac fibroblasts, however it remains unclear how these energy metabolic requirements are met. Liver fibrosis, and in particular collagen synthesis, has been shown to be dependent on glutaminolysis, in which glutamine is converted to glutamate by the enzyme glutaminase (GLS) with further conversion to a‐ketoglutarate to fuel oxidative metabolism via the tricarboxylic acid cycle, but a role for glutaminolysis in cardiac fibrosis remains to be demonstrated. We previously showed that scleraxis (Scx) is a transcription factor that is critically required for the conversion of cardiac fibroblasts to myofibroblasts, but its potential role in glutaminolysis in these cells is unknown. We report here that freshly‐isolated rat cardiac fibroblasts (P0 – unpassaged) passaged twice to become myofibroblasts (P2) show concomitant up‐regulation of Scx by 5‐fold and GLS1 by 4‐fold in P2 versus P0. Scx over‐expression induced a 20‐fold increase in GLS1 expression. To determine whether GLS1 expression is dependent on Scx, we examined GLS1 expression in activated mouse cardiac fibroblasts (P1) isolated from wild type or Scx knockout mice. Scx knockout attenuated GLS1 expression by almost 90%. TGFb is a potent inducer of cardiac fibrosis that both dramatically up‐regulates Scx expression, and requires Scx for many of its downstream effects. TGFb treatment of wild type mouse P1 activated fibroblasts doubled the expression of GLS1, but had no effect in Scx KO cells. In silico analysis revealed four putative Scx binding sites in the GLS1 promoter. While TGFb significantly up‐regulated expression of the myofibroblast marker periostin in cardiac fibroblasts, both scleraxis knockout and the glutaminase inhibitor CB‐839 attenuated this induction, indicating a failure to convert to myofibroblasts. These findings suggest that Scx regulates GLS1 expression to facilitate increased energy metabolism during cardiac fibroblast to myofibroblast conversion.Support or Funding InformationCanadian Institutes of Health Research (PJT‐162422).

Epigenetic Reprogramming Driven By Metabolic Alterations As A Mechanism Of EGFR – Tyrosine Kinase Inhibitor Resistance In Human Lung Adenocarcinoma

Osimertinib, an EGFR tyrosine kinase inhibitor (TKI), is approved for the treatment of lung adenocarcinoma (LUAD) patients with activating EGFR mutations. However, acquired resistance to osimertinib is common. Resistance results from genomic alterations, but also through perturbations in enzyme activities, and epigenetic modifications. We aimed to identify pathways that mediate drug resistance and target them by identifying potent targeted therapies. Using quantitative proteomics and transcriptomics, we revealed altered metabolic pathways such as oxidative phosphorylation, citric acid cycle, and glutamate pathway in LUAD. Osimertinib‐resistant tumors were dependent on alpha‐ketoglutarate (aKG) pool derived from glutamine but not dependent on citrate, aspartate or fumarate for their survival. Moreover, fumarate, a competitive inhibitor of histone demethylases (HDMs) which use aKG as a cofactor, inhibited the viability of resistant cells. We validated these findings in our clinical trial (NCT02759835). Using RNAseq, we also show that in LUAD patients, oxidative phosphorylation, citric acid cycle, and glutamate pathway genes, including glutaminase (GLS) was significantly over expressed upon resistance when compared to their baseline (pre‐treatment) tumors. Moreover, the osimertinib resistant patient‐derived xenograft tumors were resensitized to osimertinib therapy when treated in combination with telaglenastat, a GLS inhibitor. Given that aKG is essential for HDMs, we investigated the activity of HDMs. Compared to baseline, upon development of resistance patients’ tumors and resistant cells exhibited several over expressed HDMs and decreased histone methylation (H3K27me3), which was restored upon treatment with telaglenastat or transient knock down of GLS, suggesting that reduced intracellular aKG due to the inhibition of GLS could have reduced the activity of HDMs. Osimertinib‐resistant xenograft tumors were susceptible to both HDM inhibitor (GSK J4), and to depletion of KDM6B, an aKG‐dependent demethylase. GSK J4 treatment and depletion of KDM6B increased histone methylation in resistant tumors and decreased their viability, thereby potentially suggesting its role in the maintenance of resistant tumors. We further investigated the role of transcription factors in regulating GLS expression. We found that JUN and RELA were over expressed and activated in resistant tumors. Transient depletion of JUN and RELA reduced GLS expression leading to increased histone methylation and reduced viability of resistant cells. Together, these results suggest that the increased glutaminase expression and activity increase the reliance of cells on aKG dependent HDMs, resulting in the transcriptional reprogramming that may be driving the osimertinib resistance that can be potentially targeted by telaglenastat and GSK‐J4 to overcome resistance.Support or Funding InformationIntramural Research Program, National Institutes of Health. Major Opportunity Grant on Metabolism, Center for Cancer Research, National Cancer Institute.

NRF2 activation promotes the recurrence of dormant tumour cells through regulation of redox and nucleotide metabolism.

The survival and recurrence of dormant tumour cells following therapy is a leading cause of death in cancer patients. The metabolic properties of these cells are likely distinct from those of rapidly growing tumours. Here we show that Her2 down-regulation in breast cancer cells promotes changes in cellular metabolism, culminating in oxidative stress and compensatory upregulation of the antioxidant transcription factor, NRF2. NRF2 is activated during dormancy and in recurrent tumours in animal models and breast cancer patients with poor prognosis. Constitutive activation of NRF2 accelerates recurrence, while suppression of NRF2 impairs it. In recurrent tumours, NRF2 signalling induces a transcriptional metabolic reprogramming to re-establish redox homeostasis and upregulate de novo nucleotide synthesis. The NRF2-driven metabolic state renders recurrent tumour cells sensitive to glutaminase inhibition, which prevents reactivation of dormant tumour cells in vitro, suggesting that NRF2-high dormant and recurrent tumours may be targeted. These data provide evidence that NRF2-driven metabolic reprogramming promotes the recurrence of dormant breast cancer.

Kruppel-like factor 2 disturb non-small cell lung cancer energy metabolism by inhibited glutamine consumption.

Metabolic reprogramming is well accepted as a hallmark of cancer. This study aimed to explore the role of Kruppel-like factor 2 (KLF2) in aerobic glycolysis and glutamine consumption of energy metabolism in non-small cell lung cancer (NSCLC) cells. Two different NSCLC cells, A549 and NCI-H1299, were used to investigate the role of KLF2 in glycolysis and glutamine consumption by tracer technique and KLF2 transfection. The results showed that overexpression KLF could inhibit the energy metabolism and proliferation of NSCLC cells, but had no significant effect on glycolysis reaction and only affected the glutamine consumption of NSCLC cells. In NSCLC cells exposed to glutamine deprivation, the effect of overexpression of KLF2 on cell proliferation and energy metabolism disappeared. It was found that KLF2 could inhibit the expression of glutaminase (GLS) by metabolite tracing technique and so on. However, when GLS inhibitors were given to overexpressing KLF2 NSCLC cells, the intervention effect of KLF2 disappeared. Kruppel-like factor 2 could decrease the level of glutamine, participate in the consumption of glutamine by cancer cells, and then inhibit the energy metabolism of cancer cells.

Glutaminase-1 (GLS1) inhibition limits metastatic progression in osteosarcoma

BackgroundOsteosarcoma (OS) is a malignant bone tumor that often develops during the period of rapid growth associated with adolescence. Despite successful primary tumor control accompanied by adjuvant chemotherapy, death from pulmonary metastases occurs in approximately 30% of patients within 5 years. As overall survival in patients remains unchanged over the last 30 years, urgent needs for novel therapeutic strategies exist. Cancer metastasis is characterized by complex molecular events which result from alterations in gene and protein expression/function. Recent studies suggest that metabolic adaptations, or “metabolic reprogramming,” may similarly contribute to cancer metastasis. The goal of this study was to specifically interrogate the metabolic vulnerabilities of highly metastatic OS cell lines in a series of in vitro and in vivo experiments, in order to identify a tractable metabolically targeted therapeutic strategy for patients.MethodsNutrient deprivation and drug treatment experiments were performed in MG63.3, 143B, and K7M2 OS cell lines to identify the impact of glutaminase-1 (GLS1) inhibition and metformin treatment on cell proliferation. We functionally validated the impact of drug treatment with extracellular flux analysis, nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry. 13C-glucose and 13C-glutamine tracing was employed to identify specific contributions of these nutrients to the global metabolic profiles generated with GLS1 inhibition and metformin treatment in vivo.ResultsHighly metastatic OS cell lines require glutamine for proliferation, and exposure to CB-839, in combination with metformin, induces both primary tumor growth inhibition and a distinct reduction in metastatic outgrowth in vivo. Further, combination-treated OS cells showed a reduction in cellular mitochondrial respiration, while NMR confirmed the pharmacodynamic effects of glutaminase inhibition in tumor tissues. We observed global decreases in glycolysis and tricarboxylic acid (TCA) cycle functionality, alongside an increase in fatty acid oxidation and pyrimidine catabolism.ConclusionsThis data suggests combination-treated cells cannot compensate for metformin-induced electron transport chain inhibition by upregulating glutaminolysis to generate TCA cycle intermediates required for cell proliferation, translating into significant reductions in tumor growth and metastatic progression. This therapeutic approach could be considered for future clinical development for OS patients presenting with or at high risk of developing metastasis.

Glutaminases as a Novel Target for SDHB-Associated Pheochromocytomas/Paragangliomas.

Pheochromocytoma/paragangliomas (Pheo/PGL) are rare endocrine cancers with strong genetic background. Mutations in the SDHB subunit of succinate dehydrogenase (SDH) predispose patients to malignant disease with limited therapeutic options and poor prognosis. Using a host of cellular and molecular biology techniques in 2D and 3D cell culture formats we show that SDH inhibition had cell line specific biological and biochemical consequences. Based on our studies performed on PC12 (rat chromaffin cell line), Hela (human cervix epithelial cell line), and H295R (human adrenocortical cell line) cells, we demonstrated that chromaffin cells were not affected negatively by the inhibition of SDH either by siRNA directed against SDHB or treatment with SDH inhibitors (itaconate and atpenin A5). Cell viability and intracellular metabolite measurements pointed to the cell line specific consequences of SDH impairment and to the importance of glutamate metabolism in chromaffin cells. A significant increase in glutaminase-1 (GLS-1) expression after SDH impairment was observed in PC12 cells. GLS-1 inhibitor BPTES was capable of significantly decreasing proliferation of SDH impaired PC12 cells. Glutaminase-1 and SDHB expressions were tested in 35 Pheo/PGL tumor tissues. Expression of GLS1 was higher in the SDHB low expressed group compared to SDHB high expressed tumors. Our data suggest that the SDH-associated malignant potential of Pheo/PGL is strongly dependent on GLS-1 expression and glutaminases may be novel targets for therapy.

Abstract P3-02-05: Targeting glutamine metabolism inhibits GD2+ breast cancer stem cell function in triple negative breast cancer

Abstract Breast cancer stem cells (BSCs) constitute a fraction of primary tumor cells that exhibit drug resistance and have metastatic potential. Ganglioside GD2 has been shown by us and others as a marker for BCSCs. Nutrient deprivation-associated metabolic stress observed during tumor progression is reportedly associated with the cancer stem cell phenotype, and we have shown that oxidative stress caused by serum deprivation induces GD2 expression in vitro and in vivo. To identify metabolic signatures associated with GD2+ cells and their potential adaptive mechanisms to oxidative stress, global metabolic profiling was performed using a mass spectroscopy-based approach. We found that metabolites associated with amino acid metabolism, particularly glutathione metabolism, to be most highly upregulated in GD2+ compared to GD2− cells. In addition, glutathione biosynthesis and oxidation to glutathione disulfide was also increased in cells undergoing oxidative stress. These data suggest that the glutathione-mediated detoxification pathways play a key role in GD2+ or basal type breast cancer cells. As glutamine is a major precursor molecule for glutathione biosynthesis, we hypothesized that glutamine, at least in part, regulates cellular redox tone and the GD2+ phenotype in breast cancer cells. To test our hypothesis, we cultured breast cancer cell lines MDA-MB-231 and SUM159 in media containing 0%, 0.5%, 1%, 2%, or 4% of L-glutamine for 3 days. Flow cytometry analysis revealed that the percentage of GD2+ increased from 6% to 12% and from 12% to 22% in MDA-MB-231 and SUM159 cells, respectively when the cells were cultured at 0 or 4% glutamine. Glutaminase, an enzyme that converts glutamine to glutamate, which further leads to glutathione biosynthesis, has been shown to be overexpressed in basal-type breast cancer cells. To investigate the role of glutaminase on stem cell redox homeostasis and phenotype, we treated TNBC cell lines MDA-MB-231 and SUM159 cells with glutaminase inhibitors CB-839 or 968 compound for 3 days. Flow analysis revealed 70-80% reduction in GD2 expression after treatment in a dose dependent manner. In addition, treatment with CB-839 inhibited cell proliferation by over 80% in a dose-dependent manner in MDA-MB-231 cells and SUM159 cells (p<0.001). Next, to determine the effect of glutaminase inhibition on BCSC function, we cultured MDA-MB-231 and SUM-159 cells in low adherent cell culture dishes with or without glutaminase inhibitors 968 or CB-839 for 7 days. Interestingly, we observed a 40-50% reduction in mammosphere formation in both treated cell lines compared to the untreated controls, suggesting that inhibition of glutaminase is critical for BCSC function. In conclusion, the anti-oxidant glutathione is highly upregulated in GD2+ BCSCs, and this appears to be associated with the concentration of glutamine, and appears to regulate, perhaps via the regulation of redox homeostaisis, GD2 expression in BCSCs. Targeting glutaminase and redox homeostasis using CB-839 or CB968 inhibits BCSC as measured by GD2 positivity and mammosphere formation. Glutaminase inhibition in combination with chemotherapy could be a valuable future therapeutic strategy for targeting BCSCs in TNBC. Citation Format: Stanley Ly, Khoa Nguyen, Michael Andreeff, Venkata Lokesh Battula. Targeting glutamine metabolism inhibits GD2+ breast cancer stem cell function in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-02-05.

Intratumoral heterogeneity of glutaminase and lactate dehydrogenase A protein expression in colorectal cancer.

The high expression of metabolic enzymes, including glutaminase (GA) and lactate dehydrogenase A (LDHA), which contribute to bioenergetics and biosynthesis of mammalian cells, has been identified in a variety of cancer types. The current study indicated intratumoral heterogeneity with respect to protein expression of the metabolic enzymes in colorectal cancer (CRC). GA protein expression was determined using immunohistochemistry in 98 cases of surgically resected T3 CRC. A total of 75 cases (74%) exhibited moderate to strong immunopositivity of GA based on whole-section examination. A significant correlation was demonstrated between GA expression and clinicopathological features, including histological type and tumor budding in a patient population. Detailed histological analysis revealed the upregulation of GA protein expression at the invasive margin, including tumor budding of CRC tissues. Semi-quantitative examination revealed a significant difference in immunoexpression level of GA between the invasive margin and central CRC. However, LDHA expression exhibited an opposite pattern, with expression elevated at the center and significantly decreased at the tumors invasive margin. Immunohistochemical expression of another glycolytic enzyme hexokinase II was equivalent in both regions. Furthermore, gene silencing of GLS1, which encodes GA protein, and GA inhibitor treatment significantly inhibited cell growth of CRC cell lines. Therefore, the results of the present study demonstrated that the alteration in GA and LDHA expression is more prominent at the invasive margin, which involves tumor budding in CRC.

Glutaminase 1 Regulates Neuroinflammation After Cerebral Ischemia Through Enhancing Microglial Activation and Pro-Inflammatory Exosome Release.

Cerebral ischemia induces a robust neuroinflammatory response that is largely mediated by the activation of CNS resident microglia. Activated microglia produce pro-inflammatory molecules to cause neuronal damage. Identifying regulators of microglial activation bears great potential in discovering promising candidates for neuroprotection post cerebral ischemia. Previous studies demonstrate abnormal elevation of glutaminase 1 (GLS1) in microglia in chronic CNS disorders including Alzheimer's disease and HIV-associated neurocognitive disorders. Ectopic expression of GLS1 induced microglia polarization into pro-inflammatory phenotype and exosome release in vitro. However, whether GLS1 is involved in neuroinflammation in acute brain injury remains unknown. Here, we observed activation of microglia, elevation of GLS1 expression, and accumulation of pro-inflammatory exosomes in rat brains 72 h post focal cerebral ischemia. Treatment with CB839, a glutaminase inhibitor, reversed ischemia-induced microglial activation, inflammatory response, and exosome release. Furthermore, we found that the application of exosome secretion inhibitor, GW4869, displayed similar anti-inflammatory effects to that of CB839, suggesting GLS1-mediated exosome release may play an important role in the formation of neuroinflammatory microenvironment. Therefore, GLS1 may serve as a key mediator and promising target of neuroinflammatory response in cerebral ischemia.

Targeting glutamine metabolism slows soft tissue sarcoma growth

Tumour cells frequently utilize glutamine to meet bioenergetic and biosynthetic demands of rapid cell growth. However, glutamine dependence can be highly variable between in vitro and in vivo settings, based on surrounding microenvironments and complex adaptive responses to glutamine deprivation. Soft tissue sarcomas (STSs) are mesenchymal tumours where cytotoxic chemotherapy remains the primary approach for metastatic or unresectable disease. Therefore, it is critical to identify alternate therapies to improve patient outcomes. Using autochthonous STS murine models and unbiased metabolomics, we demonstrate that glutamine metabolism supports sarcomagenesis. STS subtypes expressing elevated glutaminase (GLS) levels are highly sensitive to glutamine starvation. In contrast to previous studies, treatment of autochthonous tumour-bearing animals with Telaglenastat (CB-839), an orally bioavailable GLS inhibitor, successfully inhibits undifferentiated pleomorphic sarcoma (UPS) tumour growth. We reveal glutamine metabolism as critical for sarcomagenesis, with CB-839 exhibiting potent therapeutic potential.

Inhibition of glutaminase to reverse fibrosis in iatrogenic laryngotracheal stenosis.

Glutamine metabolism is a critical energy source for iatrogenic laryngotracheal stenosis (iLTS) scar fibroblasts, and glutaminase (GLS) is an essential enzyme converting glutamine to glutamate. We hypothesize that the GLS-specific inhibitor BPTES will block glutaminolysis and reduce iLTS scar fibroblast proliferation, collagen deposition, and fibroblast metabolism in vitro. Test-tube Lab Research. Immunohistochemistry of a cricotracheal resection (n = 1) and a normal airway specimen (n = 1) were assessed for GLS expression. GLS expression was assessed in brush biopsies of subglottic/tracheal fibrosis and normal airway from patients with iLTS (n = 6). Fibroblasts were isolated and cultured from biopsies of subglottic/tracheal fibrosis (n = 6). Fibroblast were treated with BPTES and BPTES + dimethyl α-ketoglutarate (DMK), an analogue of the downstream product of GLS. Fibroblast proliferation, gene expression, protein production, and metabolism were assessed in all treatment conditions and compared to control. GLS was overexpressed in brush biopsies of iLTS scar specimens (P = .029) compared to normal controls. In vitro, BPTES inhibited iLTS scar fibroblast proliferation (P = .007), collagen I (Col I) (P < .0001), collagen III (P = .004), and α-smooth muscle actin (P = .0025) gene expression and protein production (P = .031). Metabolic analysis demonstrated that BPTES reduced glycolytic reserve (P = .007) but had no effects on mitochondrial oxidative phosphorylation. DMK rescued BPTES inhibition of Col I gene expression (P = .0018) and protein production (P = .021). GLS is overexpressed in iLTS scar. Blockage of GLS with BPTES significantly inhibits iLTS scar fibroblasts proliferation and function, demonstrating a critical role for GLS in iLTS. Targeting GLS to inhibit glutaminolysis may be a successful strategy to reverse scar formation in the airway. NA Laryngoscope, 2020.

β1-Integrin- and KV1.3 channel-dependent signaling stimulates glutamate release from Th17 cells.

Although the impact of Th17 cells on autoimmunity is undisputable, their pathogenic effector mechanism is still enigmatic. We discovered soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex proteins in Th17 cells that enable a vesicular glutamate release pathway that induces local intracytoplasmic calcium release and subsequent damage in neurons. This pathway is glutamine dependent and triggered by binding of β1-integrin to vascular cell adhesion molecule 1 (VCAM-1) on neurons in the inflammatory context. Glutamate secretion could be blocked by inhibiting either glutaminase or KV1.3 channels, which are known to be linked to integrin expression and highly expressed on stimulated T cells. Although KV1.3 is not expressed in CNS tissue, intrathecal administration of a KV1.3 channel blocker or a glutaminase inhibitor ameliorated disability in experimental neuroinflammation. In humans, T cells from patients with multiple sclerosis secreted higher levels of glutamate, and cerebrospinal fluid glutamine levels were increased. Altogether, our findings demonstrate that β1-integrin- and KV1.3 channel-dependent signaling stimulates glutamate release from Th17 cells upon direct cell-cell contact between Th17 cells and neurons.

Dendrimer-conjugated glutaminase inhibitor selectively targets microglial glutaminase in a mouse model of Rett syndrome.

Background: Elevated glutamate production and release from glial cells is a common feature of many CNS disorders. Inhibitors of glutaminase (GLS), the enzyme responsible for converting glutamine to glutamate have been developed to target glutamate overproduction. However, many GLS inhibitors have poor aqueous solubility, are unable to cross the blood brain barrier, or demonstrate significant toxicity when given systemically, precluding translation. Enhanced aqueous solubility and systemic therapy targeted to activated glia may address this challenge. Here we examine the impact of microglial-targeted GLS inhibition in a mouse model of Rett syndrome (RTT), a developmental disorder with no viable therapies, manifesting profound central nervous system effects, in which elevated glutamatergic tone, upregulation of microglial GLS, oxidative stress and neuroimmune dysregulation are key features.Methods: To enable this, we conjugated a potent glutaminase inhibitor, N-(5-{2-[2-(5-amino-[1,3,4]thiadiazol-2-yl)-ethylsulfanyl]-ethyl}-[1,3,4]thiadiazol-2-yl)-2-phenyl-acetamide (JHU29) to a generation 4 hydroxyl PAMAM dendrimer (D-JHU29). We then examined the effect of D-JHU29 in organotypic slice culture on glutamate release. We also examined GLS activity in microglial and non-microglial cells, and neurobehavioral phenotype after systemic administration of D-JHU29 in a mouse model of RTT.Results: We report successful conjugation of JHU29 to dendrimer resulting in enhanced water solubility compared to free JHU29. D-JHU29 reduced the excessive glutamate release observed in tissue culture slices in a clinically relevant Mecp2-knockout (KO) RTT mouse. Microglia isolated from Mecp2-KO mice demonstrated upregulation of GLS activity that normalized to wild-type levels following systemic treatment with D-JHU29. Neurobehavioral assessments in D-JHU29 treated Mecp2-KO mice revealed selective improvements in mobility.Conclusion: These findings demonstrate that glutaminase inhibitors conjugated to dendrimers are a viable mechanism to selectively inhibit microglial GLS to reduce glutamate production and improve mobility in a mouse model of RTT, with broader implications for selectively targeting this pathway in other neurodegenerative disorders.

Patents targeting the Warburg effect for cancer therapy: an interview with William P Katt.

Dr William Katt is a multidisciplinary scientist with particular focus in computational, synthetic and biological chemistry. He obtained his undergraduate degree at Rensselaer Polytechnic Institute and performed his graduate studies at Yale University, where he focused on designing small-molecule inhibitors of the Rho/Rho GEF interaction. Following those studies, Dr Katt accepted a fellowship from the American Cancer Society which funded his work at Cornell University, where he investigated small-molecule inhibitors of the enzyme glutaminase, a key player in cancer metabolism. Today, Dr Katt is a research associate at Cornell and maintains a number of collaborations with researchers across the nation examining glutaminase, cancer stem cells, nano-therapeutics and more, with the goal of developing therapeutic approaches that will eventually help patients in the clinic.