Glutaminase Gene Research Articles

Inhibition of PIM Kinases Targets Synthetic Vulnerabilities and Enhances Antigen Presentation in B-Cell Lymphoma

The PIM kinases are highly expressed in activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). Oncogenic cooperation between PIMs and MYC has been demonstrated. Transgenic mice co-expressing Em-PIM and Em-MYC showed accelerated lymphomagenesis. Conversely, knockdown of PIMs dramatically decreased cMYC levels and lowered tumor incidence. Based on these preclinical data, a treatment strategy aiming at disrupting the oncogenic cooperation between PIMs and MYC may improve the outcome of DLBCL. Therefore, we treated a panel of DLBCL cell lines with increasing dose of the clinically relevant pan-PIM inhibitor (PIMi) AZD1208 (from 0.1 to 10μM) for 48 hours (Hrs), which resulted in a dose-dependent growth inhibition with a stronger efficacy in ABC DLBCL cell lines. (Figure 1A)The analysis of a CRISPR loss-of-function screening in three ABC (LY3, TMD8, HBL1) and three GCB (SUDHL-4, Pfeiffer, BJAB) DLBCL cell lines (Reddy et al, 2017) showed that PIM2 silencing led to significantly decreased viability irrespective of cell-of-origin (Figure 1B), suggesting that this oncogene is essential for cell proliferation in DLBCLs. To identify the genes through which PIMs drive the lymphoma phenotype we performed gene expression profiling using 4 ABC DLBCL cell lines (RIVA, TMD8, SUDHL-2, U2932) treated with either DMSO or AZD1208 at 1μM for 4, 8 and 12 Hrs. We observed induction of 3,439 genes whereas 2,473 genes were downregulated. (Figure 1C) Gene pathway analysis showed that AZD1208 led to downregulation of genes regulated by MYC, including its known downstream p53 and NFKB target genes. On the other hand, AZD1208 treatment broadly induced MHC class II and antigen presentation genes as well as PI3K/AKT, cell cycle and glutaminase genes. (Figure 1D) Using a high-throughput screening approach, we found that the inhibitors of cell cycle (such as the BCL2 inhibitor venetoclax/ABT199 and the PLK4 inhibitor CFI-400945) and of glutaminase (CB839) enhanced the antiproliferative effect of AZD1208, whereas combinations with the PI3K/AKT/mTOR inhibitors had negligible synergistic effect. (Figure 1E) In conclusion, our study revealed previously unknown mechanisms of action of PIM inhibitors and provides a framework for future combination strategies. Disclosures Younes: Xynomics: Consultancy; Biopath: Consultancy; Genentech: Research Funding; AstraZeneca: Research Funding; Syndax: Research Funding; BMS: Research Funding; HCM: Consultancy; Celgene: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Takeda: Honoraria; Roche: Consultancy, Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Curis: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Abbvie: Honoraria; Pharmacyclics: Research Funding. Bertoni:Nordic Nanovector ASA: Research Funding; Acerta: Research Funding; ADC Therapeutics: Research Funding; Bayer AG: Research Funding; Cellestia: Research Funding; CTI Life Sciences: Research Funding; EMD Serono: Research Funding; Helsinn: Consultancy, Research Funding; ImmunoGen: Research Funding; Menarini Ricerche: Consultancy, Research Funding; NEOMED Therapeutics 1: Research Funding; Oncology Therapeutic Development: Research Funding; PIQUR Therapeutics AG: Other: travel grant, Research Funding; HTG: Other: Expert Statements ; Amgen: Other: travel grants; Astra Zeneca: Other: travel grants; Jazz Pharmaceuticals: Other: travel grants.

Dual inhibition of glutaminase and carnitine palmitoyltransferase decreases growth and migration of glutaminase inhibition–resistant triple-negative breast cancer cells

Triple-negative breast cancers (TNBCs) lack progesterone and estrogen receptors and do not have amplified human epidermal growth factor receptor 2, the main therapeutic targets for managing breast cancer. TNBCs have an altered metabolism, including an increased Warburg effect and glutamine dependence, making the glutaminase inhibitor CB-839 therapeutically promising for this tumor type. Accordingly, CB-839 is currently in phase I/II clinical trials. However, not all TNBCs respond to CB-839 treatment, and the tumor resistance mechanism is not yet fully understood. Here we classified cell lines as CB-839-sensitive or -resistant according to their growth responses to CB-839. Compared with sensitive cells, resistant cells were less glutaminolytic and, upon CB-839 treatment, exhibited a smaller decrease in ATP content and less mitochondrial fragmentation, an indicator of poor mitochondrial health. Transcriptional analyses revealed that the expression levels of genes linked to lipid metabolism were altered between sensitive and resistant cells and between breast cancer tissues (available from The Cancer Genome Atlas project) with low versus high glutaminase (GLS) gene expression. Of note, CB-839-resistant TNBC cells had increased carnitine palmitoyltransferase 2 (CPT2) protein and CPT1 activity levels. In agreement, CB-839-resistant TNBC cells mobilized more fatty acids into mitochondria for oxidation, which responded to AMP-activated protein kinase and acetyl-CoA carboxylase signaling. Moreover, chemical inhibition of both glutaminase and CPT1 decreased cell proliferation and migration of CB-839-resistant cells compared with single inhibition of each enzyme. We propose that dual targeting of glutaminase and CPT1 activities may have therapeutic relevance for managing CB-839-resistant tumors.

Improving the Production of Salt-Tolerant Glutaminase by Integrating Multiple Copies of Mglu into the Protease and 16S rDNA Genes of Bacillus subtilis 168.

In this study, the Micrococcus luteus K-3 glutaminase was successfully over-expressed in the GRAS (Generally Recognized as Safe) Bacillus subtilis strain 168 by integration of the Mglu gene in the 16S rDNA locus. This was done in order to screen a strain producing high levels of recombinant glutaminase from the selected candidates. The transcription of the glutaminase genes in the B. subtilis 168 chromosome and the expression of glutaminase protein was further assessed by qPCR, SDS-PAGE analysis and an enzyme activity assay. To further increase the production of glutaminase, the nprB and nprE genes, which encode specific proteases, were disrupted by integration of the Mglu gene. After continuous cell culturing without the addition of antibiotics, the integrated recombinant strains showed excellent genetic stability, demonstrating favorable industrialization potential. After the fermentation temperature was optimized, a 5-L bioreactor was used for fed-batch fermentation of the recombinant glutaminase producing strain at 24 °C, and the highest enzyme activity achieved was approximately 357.6 U/mL.

Enhanced thermostability of halo-tolerant glutaminase from Bacillus licheniformis ATCC 14580 by immobilization onto nano magnetic cellulose sheet and its application in production of glutamic acid

A halo-tolerant glutaminase gene (BlglsA) was isolated from Bacillus licheniformis. Heterologous expression of BlglsA revealed that it encodes for a 36 kDa protein containing 327 amino acid residues. The purified enzyme showed optimal activity at a pH of 9.5 while 35 °C was found to be the optimum temperature. The enzyme retained about 92 and 97% stability at pH 12 and temperature (40 °C) respectively. Subsequent immobilization of BlglsA on nano magnetic cellulose sheet (NMCS) led to an enhanced tolerance to higher temperature. NMCS-BlglsA showed optimum activity at 45 °C, although it was stable even at 60 °C. NaCl tolerance (≥90% in 0.3 M) was almost similar to BlglsA and NMCS-BlglsA. The metal ions Fe2+ (5 mM) and Mn2+ (2.5 mM) improved the BlglsA relative activity by 61 and 48%, respectively. In contrast, 5 mM Mn2+ was found suitable to enhance the activity of NMCS-BlglsA up to 72%. The production of glutamic acid by NMCS-BlglsA was 1.61 g/l in 48 h. Reusability test of NMCS-BlglsA showed 76 and 35% retention of the actual activity after 4th and 7th cycle, respectively. Such remarkable biochemical properties of NMCS-BlglsA make it an attractive enzyme for food industries.

In Silico Preliminary Association of Ammonia Metabolism Genes GLS, CPS1, and GLUL with Risk of Alzheimer's Disease, Major Depressive Disorder, and Type 2 Diabetes.

Ammonia is a toxic by-product of protein catabolism and is involved in changes in glutamate metabolism. Therefore, ammonia metabolism genes may link a range of diseases involving glutamate signaling such as Alzheimer's disease (AD), major depressive disorder (MDD), and type 2 diabetes (T2D). We analyzed data from a National Institute on Aging study with a family-based design to determine if 45 single nucleotide polymorphisms (SNPs) in glutaminase (GLS), carbamoyl phosphate synthetase 1 (CPS1), or glutamate-ammonia ligase (GLUL) genes were associated with AD, MDD, or T2D using PLINK software. HAPLOVIEW software was used to calculate linkage disequilibrium measures for the SNPs. Next, we analyzed the associated variations for potential effects on transcriptional control sites to identify possible functional effects of the SNPs. Of the SNPs that passed the quality control tests, four SNPs in the GLS gene were significantly associated with AD, two SNPs in the GLS gene were associated with T2D, and one SNP in the GLUL gene and three SNPs in the CPS1 gene were associated with MDD before Bonferroni correction. The in silico bioinformatic analysis suggested probable functional roles for six associated SNPs. Glutamate signaling pathways have been implicated in all these diseases, and other studies have detected similar brain pathologies such as cortical thinning in AD, MDD, and T2D. Taken together, these data potentially link GLS with AD, GLS with T2D, and CPS1 and GLUL with MDD and stimulate the generation of testable hypotheses that may help explain the molecular basis of pathologies shared by these disorders.

Glutaminase and MMP-9 Downregulation in Cortex and Hippocampus of LPA1 Receptor Null Mice Correlate with Altered Dendritic Spine Plasticity.

Lysophosphatidic acid (LPA) is an extracellular lipid mediator that regulates nervous system development and functions acting through G protein-coupled receptors (GPCRs). Here we explore the crosstalk between LPA1 receptor and glutamatergic transmission by examining expression of glutaminase (GA) isoforms in different brain areas isolated from wild-type (WT) and KOLPA1 mice. Silencing of LPA1 receptor induced a severe down-regulation of Gls-encoded long glutaminase protein variant (KGA) (glutaminase gene encoding the kidney-type isoforms, GLS) protein expression in several brain regions, particularly in brain cortex and hippocampus. Immunohistochemical assessment of protein levels for the second type of glutaminase (GA) isoform, glutaminase gene encoding the liver-type isoforms (GLS2), did not detect substantial differences with regard to WT animals. The regional mRNA levels of GLS were determined by real time RT-PCR and did not show significant variations, except for prefrontal and motor cortex values which clearly diminished in KO mice. Total GA activity was also significantly reduced in prefrontal and motor cortex, but remained essentially unchanged in the hippocampus and rest of brain regions examined, suggesting activation of genetic compensatory mechanisms and/or post-translational modifications to compensate for KGA protein deficit. Remarkably, Golgi staining of hippocampal regions showed an altered morphology of glutamatergic pyramidal cells dendritic spines towards a less mature filopodia-like phenotype, as compared with WT littermates. This structural change correlated with a strong decrease of active matrix-metalloproteinase (MMP) 9 in cerebral cortex and hippocampus of KOLPA1 mice. Taken together, these results demonstrate that LPA signaling through LPA1 influence expression of the main isoenzyme of glutamate biosynthesis with strong repercussions on dendritic spines maturation, which may partially explain the cognitive and learning defects previously reported for this colony of KOLPA1 mice.

A conserved neuronal DAF-16/FoxO plays an important role in conveying pheromone signals to elicit repulsion behavior in Caenorhabditis elegans

Animals use pheromones as a conspecific chemical language to respond appropriately to environmental changes. The soil nematode Caenorhabditis elegans secretes ascaroside pheromones throughout the lifecycle, which influences entry into dauer phase in early larvae, in addition to sexual attraction and aggregation. In adult hermaphrodites, pheromone sensory signals perceived by worms usually elicit repulsion as an initial behavioral signature. However, the molecular mechanisms underlying neuronal pheromone sensory process from perception to repulsion in adult hermaphrodites remain poorly understood. Here, we show that pheromone signals perceived by GPA-3 is conveyed through glutamatergic neurotransmission in which neuronal DAF-16/FoxO plays an important modulatory role by controlling glutaminase gene expression. We further provide evidence that this modulatory role for DAF-16/FoxO seems to be conserved evolutionarily by electro-physiological study in mouse primary hippocampal neurons that are responsible for glutamatergic neurotransmission. These findings provide the basis for understanding the nematode pheromone signaling, which seems crucial for adaptation of adult hermaphrodites to changes in environmental condition for survival.

Dopamine neuron dependent behaviors mediated by glutamate cotransmission

Dopamine neurons in the ventral tegmental area use glutamate as a cotransmitter. To elucidate the behavioral role of the cotransmission, we targeted the glutamate-recycling enzyme glutaminase (gene Gls1). In mice with a dopamine transporter (Slc6a3)-driven conditional heterozygous (cHET) reduction of Gls1 in their dopamine neurons, dopamine neuron survival and transmission were unaffected, while glutamate cotransmission at phasic firing frequencies was reduced, enabling a selective focus on the cotransmission. The mice showed normal emotional and motor behaviors, and an unaffected response to acute amphetamine. Strikingly, amphetamine sensitization was reduced and latent inhibition potentiated. These behavioral effects, also seen in global GLS1 HETs with a schizophrenia resilience phenotype, were not seen in mice with an Emx1-driven forebrain reduction affecting most brain glutamatergic neurons. Thus, a reduction in dopamine neuron glutamate cotransmission appears to mediate significant components of the GLS1 HET schizophrenia resilience phenotype, and glutamate cotransmission appears to be important in attribution of motivational salience.

Design, Synthesis, and Evaluation of Thiazolidine-2,4-dione Derivatives as a Novel Class of Glutaminase Inhibitors.

Humans have two glutaminase genes, GLS (GLS1) and GLS2, each of which has two alternative transcripts: the kidney isoform (KGA) and glutaminase C (GAC) for GLS, and the liver isoform (LGA) and glutaminase B (GAB) for GLS2. Initial hit compound (Z)-5-((1-(4-bromophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methylene)thiazolidine-2,4-dione (2), a thiazolidine-2,4-dione, was obtained from a high throughput screening of 40 000 compounds against KGA. Subsequently, a series of thiazolidine-2,4-dione derivatives was synthesized. Most of these were found to inhibit KGA and GAC with comparable activities, were less potent inhibitors of GAB, and were moderately selective for GLS1 over GLS2. The relationships between chemical structure, activity, and selectivity were investigated. The lead compounds obtained were found to (1) offer in vitro cellular activities for inhibiting cell growth, clonogenicity, and cellular glutamate production, (2) exhibit high concentrations of exposure in plasma by a pharmacokinetic study, and (3) reduce the tumor size of xenografted human pancreatic AsPC-1 carcinoma cells in mice.

Validation study associating glutaminase promoter variations with hepatic encephalopathy in East Asian populations.

In a recent study, microsatellite variations (GCA tandem repeats) in the promoter region of the (kidney-type) glutaminase gene were associated with the development of hepatic encephalopathy (HE) in Spanish patients with cirrhosis. The objective of this study was to validate the relation between microsatellite variations in the glutaminase promoter region and the development of overt HE in Korean patients with liver cirrhosis. We performed a prospective cohort study of 154 cirrhotic patients who underwent a glutaminase microsatellite study without previous overt HE history at baseline. The primary end point was the first episode of overt HE. The microsatellite length was categorized into three groups based on its repeated number, with a cutoff value of 14; 65 (42.2%), 70 (45.5%), and 19 (12.3%) patients had the short-short, short-long, and long-long alleles, respectively. Over a median 3.5 years of follow-up (range = 0.1-4.4), overt HE developed in 28 patients (18.2%). The 3-year cumulative incidence of overt HE was 18.4%. Multivariate Cox model indicated that past hepatocellular carcinoma history, alcoholic etiology for cirrhosis, higher Model for End-Stage Liver Disease scores and their deterioration, and serum ammonium levels were independently associated with HE development. However, microsatellite length was not associated with the development of overt HE. In Korean patients with cirrhosis, microsatellite variations in the glutaminase promoter region were not associated with development of overt HE. Thus, additional studies are needed to identify other genetic factors related to glutaminase activity in Asians with overt HE.

New technologies - new insights into the pathogenesis of hepatic encephalopathy.

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome which frequently accompanies acute or chronic liver disease. It is characterized by a variety of symptoms of different severity such as cognitive deficits and impaired motor functions. Currently, HE is seen as a consequence of a low grade cerebral oedema associated with the formation of cerebral oxidative stress and deranged cerebral oscillatory networks. However, the pathogenesis of HE is still incompletely understood as liver dysfunction triggers exceptionally complex metabolic derangements in the body which need to be investigated by appropriate technologies. This review summarizes technological approaches presented at the ISHEN conference 2014 in London which may help to gain new insights into the pathogenesis of HE. Dynamic in vivo 13C nuclear magnetic resonance spectroscopy was performed to analyse effects of chronic liver failure in rats on brain energy metabolism. By using a genomics approach, microRNA expression changes were identified in plasma of animals with acute liver failure which may be involved in interorgan interactions and which may serve as organ-specific biomarkers for tissue damage during acute liver failure. Genomics were also applied to analyse glutaminase gene polymorphisms in patients with liver cirrhosis indicating that haplotype-dependent glutaminase activity is an important pathogenic factor in HE. Metabonomics represents a promising approach to better understand HE, by capturing the systems level metabolic changes associated with disease in individuals, and enabling monitoring of metabolic phenotypes in real time, over a time course and in response to treatment, to better inform clinical decision making. Targeted fluxomics allow the determination of metabolic reaction rates thereby discriminating metabolite level changes in HE in terms of production, consumption and clearance.

PWE-033 Promoter Region Variations in The Glutaminase Gene as A Risk Factor for The Development Hepatic Encephalopathy in Patients with Cirrhosis: Abstract PWE-033 Table 1

Introduction Hepatic encephalopathy (HE) is the commonest complication of cirrhosis but its development is not invariable; thus approximately 20% of individuals remain free of neuropsychiatric complications and patients with minimal HE do not invariably develop overt HE. It is possible that genetic factors, most likely resulting in deregulation of ammonia metabolism, might determine an individual’s susceptibility to develop this complication. In 2010, Romero-Gomez and colleagues, 1 identified a functional microsatellite consisting of GCA repeats near the promoter region of the glutaminase gene, GLS1, and proposed that patients with cirrhosis who were homozygous for the long microsatellite allele were more likely to develop overt HE. The aim of this study was to further investigate the association between GLS1 promoter microsatellite variants and HE. Methods The study population comprised 110 patients with cirrhosis, of British/Irish ancestry, in whom neuropsychiatic status had remained stable during monitoring for a minimum of 5 years, except in those treated for overt HE. Patients were classified using clinical, neurophysiological and neuropsychometric variables as: unimpaired (51; 46%) or as having minimal (24; 22%) or overt (35; 32%) HE. The control population comprised 325 ancestrally-matched healthy controls. DNA was genotyped using gel electrophoresis and verified by Sanger sequencing. Alleles were stratified as long (≥14 GCA repeats) or short ( Results There were no significant differences in allele frequencies or genotype distributions between cases and controls. The proportion of long-short heterozygotes was significantly higher in the unimpaired patients (62.7%) than in those with either minimal HE (37.5%) or overt HE (37.1%) (p = 0.029; Table). Conclusion There was no evidence that the long-long genotype is a risk factor for HE in the present study. However, there was evidence of possible heterozygote advantage resulting in significant protection against the development of this syndrome. More studies are needed to confirm or refute these findings. Reference 1 Romero-Gomez M, et al . Variations in the promoter region of the glutaminase gene and the development of hepatic encephalopathy in patients with cirrhosis: a cohort study. Ann Intern Med 2010; 153 :281–8. Disclosure of Interest None Declared

Unveiling alterative splice diversity from human oligodendrocyte proteome data

Oligodendrocytes produce and maintain the myelin sheath of axons in the central nervous system. Because misassembled myelin sheaths have been associated with brain disorders such as multiple sclerosis and schizophrenia, recent advances have been made towards the description of the oligodendrocyte proteome. The identification of splice variants represented in the proteome is as important as determining the level of oligodendrocyte-associated proteins. Here, we used an oligodendrocyte proteome dataset deposited in ProteomeXchange to search against a customized protein sequence file containing computationally predicted splice variants. Our approach resulted in the identification of 39 splice variants, including one variant from the GTPase KRAS gene and another from the human glutaminase gene family. We also detected the mRNA expression of five selected splice variants and demonstrated that a fraction of these have their canonical proteins participating in direct protein-protein interactions. In conclusion, we believe our findings contribute to the molecular characterization of oligodendrocytes and may encourage other research groups working with central nervous system disorders to investigate the biological significance of these splice variants. The splice variants identified in this study may encode proteins that could be targeted in novel treatment strategies and diagnostic methods. SignificanceSeveral disorders of the central nervous system (CNS) are associated with misassembled myelin sheaths, which are produced and maintained by oligodendrocytes (OL). Recently, the OL proteome has been explored to identify key proteins and molecular functions associated with CNS disorders. We developed an innovative approach to select, with a higher level of confidence, a relevant list of splice variants from a proteome dataset and detected the mRNA expression of five selected variants: EEF1D, KRAS, MFF, SDR39U1, and SUGT1. We also described splice variants extracted from OL proteome data. Among the splice variants identified, some are from genes previously linked to CNS and related disorders. Our findings may contribute to oligodendrocyte characterization and encourage other research groups to investigate the biological role of splice variants and to improve current treatments and diagnostic methods for CNS disorders.

Cloning and Expression of Glutaminase New Gene in the Sauce Billet Metagenomic

Aim to study the Glutaminase gene structure and function the prokaryotic expression vector pEAZY E1-glsA927 is construnted from the new genes glutaminase named glsA927 which is cloned in the traditional sauce billet microbial metagenomic and connected to pEAZY E1 carrier. After the recombinant plasmid is passed into E. coli BL21 and induced by gene expression and after purified by the Ni column, express product detect the enzyme activity by means of bacteria glutaminase activity quantitative detection kit. Sequence analysis results show that the gene length of glsA927 is 927 bp, which has the highest sequence homology (AF057158.1), 94.82%, reported on GENBANK, with 16 different amino acids. Protein expression analysis results show that when the concentration of IPTG tendency is 0.2 mmol/L, protein expression of induced 4 h has the highest amount. The Ni ion affinity chromatography purification of recombinant glutaminase ihs-glsA927, preliminarily determine glutaminase activity to a maximum of 468.7 U mu/g, SDS-PAG and Western results show that recombinant glutaminase His-glsA927 which is purified by the Ni ion affinity chromatography could be preliminarily determined that the glutaminase activity can reach as much as 468.7 U/&mu g and not easy to be affected by the concentration of NaCl.

The oncogenic transcription factor c-Jun regulates glutaminase expression and sensitizes cells to glutaminase-targeted therapy.

Many transformed cells exhibit altered glucose metabolism and increased utilization of glutamine for anabolic and bioenergetic processes. These metabolic adaptations, which accompany tumorigenesis, are driven by oncogenic signals. Here we report that the transcription factor c-Jun, product of the proto-oncogene JUN, is a key regulator of mitochondrial glutaminase (GLS) levels. Activation of c-Jun downstream of oncogenic Rho GTPase signalling leads to elevated GLS gene expression and glutaminase activity. In human breast cancer cells, GLS protein levels and sensitivity to GLS inhibition correlate strongly with c-Jun levels. We show that c-Jun directly binds to the GLS promoter region, and is sufficient to increase gene expression. Furthermore, ectopic overexpression of c-Jun renders breast cancer cells dependent on GLS activity. These findings reveal a role for c-Jun as a driver of cancer cell metabolic reprogramming, and suggest that cancers overexpressing JUN may be especially sensitive to GLS-targeted therapies.

THU-032 - Variations in the Promoter Region of the Glutaminase Gene and the Development of Hepatic Encephalopathy in Patients with Cirrhosis

THU-032 - Variations in the Promoter Region of the Glutaminase Gene and the Development of Hepatic Encephalopathy in Patients with Cirrhosis

Impaired glutamatergic and GABAergic transmission by amitraz in primary hippocampal cells

Amitraz is a formamidine pesticide that has been reported to be a neurotoxic compound that induces convulsions among other effects. Excitatory and inhibitory neurotransmission is mediated mainly by glutamate and GABA, respectively, so their alteration could be responsible for induction of seizures. In this regard, amitraz α2 adrenergic agonist action, which has been suggested as likely responsible for this effect, could alter these neurotransmitter systems and lead to seizure induction. Moreover, other amitraz mechanisms such as histamine H1 receptor inhibition could be involved. To confirm if amitraz disrupts glutamatergic/GABAergic transmission by these mechanisms, we evaluated, in primary hippocampal neurons, the effect of amitraz (0.01μM to 100μM) with or without the α2 adrenergic antagonist idazoxan (1μM) and/or the H1 receptor agonist n-methylhistaprodifen (30μM) co-treatment on 4-aminobutyrate aminotransferase, glutamate decarboxylase 65 (GAD 65), succinate-semialdehyde dehydrogenase and glutaminase gene expression and on glutamate and GABA levels after 24h treatment. We observed that amitraz disrupts glutaminase and GAD 65 gene expression, altering glutamatergic and GABAergic transmission. These effects were mediated partially by H1 and α2 receptors suggesting that other mechanisms could be involved. These data could help explain the mechanism by which amitraz induces seizures and provide a therapeutic strategy to protect against this effect in case of poisoning.

A genetic variant in the promoter of phosphate-activated glutaminase is associated with hepatic encephalopathy.

Hepatic encephalopathy (HE) is a serious complication of liver cirrhosis. Recently, a microsatellite in the promoter region of the phosphate-activated glutaminase (GLS) gene was associated with the risk of HE. The aim of the present study was to investigate, using the critical flicker frequency (CFF) test, whether the described GLS variant increases the risk of developing HE in patients with cirrhosis. We recruited 158 patients (66% men; mean age 59years, range 23-86) with liver cirrhosis. Mean model for end-stage liver disease score was 13.8 (range 5-35); 48% of patients presented with Child-Pugh score B or C. The presence and severity of HE were determined by the CFF test, with frequencies ≤39Hz denoting cases. GLS variants were genotyped by sequencing the microsatellite in the promoter region and were classified as short, long or short-long forms (depending on the length of the macrosatellite alleles). In total, 53% of patients had abnormal CFF results (i.e. ≤39Hz; range for entire cohort 26-57). The GLS microsatellite distribution amongst patients was short form (20%), long form (32%) and short-long form (48%) and was consistent with Hardy-Weinberg equilibrium. CFF values differed significantly between groups (P=0.043). Carriers of the GLS long microsatellite had a significantly higher risk of HE according to multivariate analyses (odds ratio3.23, 95%confidence interval1.46-7.13, P=0.004). CFF results were significantly lower amongst carriers of the GLS long microsatellite. Our findings support the role of the GLS long microsatellite in the development of HE; this could be important for identifying susceptible patients and for the prevention of this condition.

Efficacy of Novel Glutaminase Inhibitor CB-839 in Acute Myeloid Leukemia

Inhibition of glutaminase (GLS), the principal enzyme in the glutamine utilization pathway that coverts glutamine (Gln) to glutamate (Glu), is an attractive therapeutic approach in many cancers. Gln plays a unique role in the metabolism of proliferating cancer cells, providing building blocks to sustain cell proliferation and regulating redox homeostasis and signal transduction pathways. Recent findings indicate that leukemic cells depend on Gln as a major carbon source for growth and survival [Willems et al., Blood, 2013]. We previously reported that a subset of acute myeloid leukemia (AML) cell lines are sensitive to Gln deprivation as well as inhibition of glutaminase by the small molecule BPTES [Matre et al. ASH 2013 #606]. Here we report the efficacy of CB-839, a novel, potent, orally bioavailable GLS inhibitor currently under clinical investigation, in the Gln-dependent subset of AML.First, expression of GLS gene splice variants glutaminase C (GAC) and kidney glutaminase (KGA) and of the GLS2 gene was determined through analysis of RNA sequencing data from 173 newly diagnosed AML patients in the TCGA dataset. Of the GLS gene splice variants, the expression levels of GAC were much higher than those of KGA; GLS2 was expressed at low levels. Levels of both GAC and KGA mRNA were significantly higher (two-sample Wilcoxon test) in AML patients with complex cytogenetics and monosomal karyotype (n=31) than in those with diploid AML (n=88, p=0.019 and p=0.01); GAC levels were higher in core-binding factor AML (n=14) than in diploid AML (p=0.018). These findings indicate high expression of the GLSGAC splice variant in specific AML subsets.Analysis of a panel of AML cell lines showed that, in a subset of leukemia cells, CB-839 treatment decreased viable cell number and induced apoptosis. In sensitive cell lines (Molm14, OCI-AML3, MV4;11), CB-839 decreased viable cell number with IC50s between 10nM and 100nM and induced significant apoptosis. HL60, MOLM13, KG1α, and OCI-AML2 cells were less sensitive (IC50 100-1000nM) and responded with minor induction of cell death. CB-839 decreased viability by >40% in blasts from 9 of 20 (45%) primary AML samples.GC- or LC-MS metabolic profiling of OCI-AML3 and THP1 cell lines as well as primary patient samples revealed that GLS inhibition by BPTES or CB-839 was accompanied by concomitant decrease in concentration of downstream GLS metabolites such as glutamate, α-ketoglutarate (a-KG), aspartate, fumarate, and malate. Investigation of the effects of CB-839 on mitochondrial OXPHOS by the Seahorse Bioscience XF96 Analyzer showed that CB-839 exposure for 16 h caused a dose-dependent decrease in maximal respiratory capacity in OCI-AML3 cells, indicating reduced availability of the substrates for OXPHOS. Similar results were obtained upon treatment with BPTES and in AML cells stably transduced with GLS shRNA.Gln, through Glu, is a precursor for cellular α-KG, which can undergo further metabolism through the Krebs cycle or be further metabolized to 2-hydroxyglutarate (2-HG) by mutant isocitrate dehydrogenase (IDH). In THP1 cell lines stably transduced with doxycycline-inducible mutant IDH1-R132H or IDH2-R140Q construct, CB-839 exposure for 4 days reduced intracellular 2-HG oncometabolite levels by >50%. This was associated with induction of differentiation marker CD11b and morphological signs of differentiation in CB-839–treated IDH2-R140Q cells [30%±2% increase in CD11b mean fluorescent intensity (p<0.001)] vs untreated cells; but not in IDH2-WT control cells. Further, IDH2-R140Q THP1 cells were significantly more sensitive to 1mM CB-839 than IDH2-WT cells (61% vs 24% reduction in viable cell numbers). In 4 of 6 IDH1- or IDH2-mutated primary samples, reductions in 2-HG (by 24% [p=0.04]; 31% [p=0.016]; 35% [p=0.033], and 43% [p=0.0056]) were observed upon CB-839 exposure. Preliminary data for an IDH2-R140Q-mutated AML primary sample (n=1) indicate that CB-839 promotes CD11b differentiation in primary AML blasts.In summary, these results indicate that GLS is a relevant therapeutic target in AML, warranting future inclusion of GLS inhibitors in the armamentarium of multi-agent therapeutic approaches. In particular, reduction of production of the oncometabolite 2-HG in conjunction with therapeutic blockade of Gln metabolism may serve as a tailored therapeutic strategy in IDH-mutated AML cells. DisclosuresKonopleva:Calithera Biosciences: Research Funding.

Glutaminase1 heterozygous mice show enhanced trace fear conditioning and Arc/Arg3.1 expression in hippocampus and cingulate cortex

Mice heterozygous for a mutation in the glutaminase (GLS1) gene (GLS1 HZ mice), with reduced glutamate recycling and release, display reduced hippocampal function as well as memory of contextual cues in a delay fear conditioning (FC) paradigm. Here, we asked whether this deficit reflects an inability to process contextual information or a selective alteration in salience attribution. In addition, we asked whether baseline and activity-induced hippocampal activity were diminished in GLS1 HZ mice. For this purpose, we manipulated the relative salience of the conditioned stimulus (CS) and contextual cues in FC tasks, and examined gene expression of the immediate early gene Arc (Arc/Arg3.1) in hippocampus and anterior cingulate cortex (ACC) following trace FC (tFC). The results indicate that GLS1 HZ mice succeed in processing contextual information when the salient CS is absent or less predictive. In addition, in the hippocampus-dependent tFC paradigm GLS1 HZ mice display enhanced CS learning. Furthermore, while baseline arc activation was reduced in GLS1 HZ mice in the hippocampus, in line with previous fMRI findings, it was enhanced in the hippocampus and anterior cingulate cortex following tFC. These findings suggest that GLS1 HZ mice have a pro-cognitive profile in the tFC paradigm, and this phenotype involves activation of both hippocampus and ACC.Taken together with previous work on the GLS1 HZ mouse, this study sheds light on the importance of glutamate transmission to memory processes that require the allocation of attentional resources, and extends our understanding of the underpinnings of attention deficits in SZ.