glutaminase and Kidney-Neoplasms

Glutaminase is a key enzyme, which supports elevated dependency of tumors on glutamine-dependent biosynthesis of metabolic intermediates. Dual targeting of glucose and glutamine metabolism by the mTOR inhibitor everolimus plus the oral glutaminase inhibitor telaglenastat showed preclinical synergistic anticancer effects, which translated to encouraging safety and efficacy findings in a phase I trial of 2L+ renal cell carcinoma (RCC). This study evaluated telaglenastat plus everolimus (TelaE) versus placebo plus everolimus (PboE) in patients with advanced/metastatic RCC (mRCC) in the 3L+ setting (NCT03163667).. Eligible patients with mRCC, previously treated with at least two prior lines of therapy [including ≥1 VEGFR-targeted tyrosine kinase inhibitor (TKI)] were randomized 2:1 to receive E, plus Tela or Pbo, until disease progression or unacceptable toxicity. Primary endpoint was investigator-assessed progression-free survival (PFS; one-sided α <0.2).. Sixty-nine patients were randomized (46 TelaE, 23 PboE). Patients had a median three prior lines of therapy, including TKIs (100%) and checkpoint inhibitors (88%). At median follow-up of 7.5 months, median PFS was 3.8 months for TelaE versus 1.9 months for PboE [HR, 0.64; 95% confidence interval (CI), 0.34-1.20; one-sided P = 0.079]. One TelaE patient had a partial response and 26 had stable disease (SD). Eleven patients on PboE had SD. Treatment-emergent adverse events included fatigue, anemia, cough, dyspnea, elevated serum creatinine, and diarrhea; grade 3 to 4 events occurred in 74% TelaE patients versus 61% PboE.. TelaE was well tolerated and improved PFS versus PboE in patients with mRCC previously treated with TKIs and checkpoint inhibitors.

Topics: Angiogenesis Inhibitors; Carcinoma, Renal Cell; Everolimus; Glutaminase; Glutamine; Humans; Kidney Neoplasms; Protein Kinase Inhibitors; Sirolimus

Topics: Biomarkers, Tumor; Carcinoma, Renal Cell; Glutaminase; Humans; Kidney Neoplasms

Targeting metabolic vulnerabilities has been proposed as a therapeutic strategy in renal cell carcinoma (RCC). Here, we analyzed the metabolism of patient-derived xenografts (tumorgrafts) from diverse subtypes of RCC. Tumorgrafts from

Topics: Animals; Carcinoma, Renal Cell; Glutaminase; Glutamine; Humans; Isocitrate Dehydrogenase; Kidney Neoplasms; Mice

Dysregulated metabolism is a hallmark of cancer that manifests through alterations in bioenergetic and biosynthetic pathways to enable tumor cell proliferation and survival. Tumor cells exhibit high rates of glycolysis, a phenomenon known as the Warburg effect, and an increase in glutamine consumption to support the tricarboxylic acid (TCA) cycle. Renal cell carcinoma (RCC) tumors express high levels of glutaminase (GLS), the enzyme required for the first step in metabolic conversion of glutamine to glutamate and the entry of glutamine into the TCA cycle. We found that RCC cells are highly dependent on glutamine for proliferation, and this dependence strongly correlated with sensitivity to telaglenstat (CB-839), an investigational, first-in-class, selective, orally bioavailable GLS inhibitor. Metabolic profiling of RCC cell lines treated with telaglenastat revealed a decrease in glutamine consumption, which was concomitant with a decrease in the production of glutamate and other glutamine-derived metabolites, consistent with GLS inhibition. Treatment of RCC cells with signal transduction inhibitors everolimus (mTOR inhibitor) or cabozantinib (VEGFR/MET/AXL inhibitor) in combination with telaglenastat resulted in decreased consumption of both glucose and glutamine and synergistic anti-proliferative effects. Treatment of mice bearing Caki-1 RCC xenograft tumors with cabozantinib plus telaglenastat resulted in reduced tumor growth compared to either agent alone. Enhanced anti-tumor activity was also observed with the combination of everolimus plus telaglenastat. Collectively, our results demonstrate potent, synergistic, anti-tumor activity of telaglenastat plus signal transduction inhibitors cabozantinib or everolimus via a mechanism involving dual inhibition of glucose and glutamine consumption.

Topics: Anilides; Animals; Benzeneacetamides; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Synergism; Everolimus; Female; Gene Expression Regulation, Neoplastic; Glucose; Glutaminase; Glutamine; Humans; Kidney Neoplasms; Mice; Pyridines; Signal Transduction; Thiadiazoles; Xenograft Model Antitumor Assays

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is characterized by germline mutations of the FH gene that encodes for the TCA cycle enzyme, fumarate hydratase. HLRCC patients are at risk for the development of an aggressive form of type 2 papillary renal cell carcinoma. By studying the mechanism of action of marizomib, a proteasome inhibitor able to cross the blood-brain barrier, we found that it modulates the metabolism of HLRCC cells. Marizomib decreased glycolysis in vitro and in vivo by downregulating p62 and c-Myc. C-Myc downregulation decreased the expression of lactate dehydrogenase A, the enzyme catalyzing the conversion of pyruvate to lactate. In addition, proteasomal inhibition lowered the expression of the glutaminases GLS and GLS2, which support glutamine metabolism and the maintenance of the redox balance. Thus, in HLRCC cells, proteasome inhibition disrupts glucose and glutamine metabolism, restricting nutrients and lowering the cells' anti-oxidant response capacity. Although the cytotoxicity induced by proteasome inhibitors is complex, the understanding of their metabolic effects in HLRCC may lead to the development of effective therapeutic strategies or to the development of markers of efficacy.

Topics: Animals; Carcinoma, Renal Cell; Cell Line, Tumor; Female; Fumarate Hydratase; Gene Expression Regulation, Neoplastic; Germ-Line Mutation; Glutaminase; Glycolysis; Humans; Kidney Neoplasms; Lactate Dehydrogenase 5; Lactones; Leiomyomatosis; Mice; Mice, Nude; Neoplastic Syndromes, Hereditary; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-myc; Pyrroles; Sequestosome-1 Protein; Signal Transduction; Skin Neoplasms; Uterine Neoplasms; Xenograft Model Antitumor Assays

Many cancer-associated mutations that deregulate cellular metabolic responses to hypoxia also reprogram carbon metabolism to promote utilization of glutamine. In renal cell carcinoma (RCC), cells deficient in the von Hippel-Lindau (VHL) tumor suppressor gene use glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate (αKG). Glutamine can also generate aspartate, the carbon source for pyrimidine biosynthesis, and glutathione for redox balance. Here we have shown that VHL-/- RCC cells rely on RC-derived aspartate to maintain de novo pyrimidine biosynthesis. Glutaminase 1 (GLS1) inhibitors depleted pyrimidines and increased ROS in VHL-/- cells but not in VHL+/+ cells, which utilized glucose oxidation for glutamate and aspartate production. GLS1 inhibitor-induced nucleoside depletion and ROS enhancement led to DNA replication stress and activation of an intra-S phase checkpoint, and suppressed the growth of VHL-/- RCC cells. These effects were rescued by administration of glutamate, αKG, or nucleobases with N-acetylcysteine. Further, we observed that the poly(ADP-ribose) polymerase (PARP) inhibitor olaparib synergizes with GLS1 inhibitors to suppress the growth of VHL-/- cells in vitro and in vivo. This work describes a mechanism that explains the sensitivity of RCC tumor growth to GLS1 inhibitors and supports the development of therapeutic strategies for targeting VHL-deficient RCC.

Topics: Animals; Carcinoma, Renal Cell; Glutamates; Glutaminase; Glutamine; Humans; Kidney Neoplasms; Mice; Mice, Nude; Phthalazines; Piperazines; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; S Phase Cell Cycle Checkpoints; Von Hippel-Lindau Tumor Suppressor Protein; Xenograft Model Antitumor Assays

Many cancers appear to activate intrinsic antioxidant systems as a means to counteract oxidative stress. Some cancers, such as clear cell renal cell carcinoma (ccRCC), require exogenous glutamine for growth and exhibit reprogrammed glutamine metabolism, at least in part due to the glutathione pathway, an efficient cellular buffering system that counteracts reactive oxygen species and other oxidants. We show here that ccRCC xenograft tumors under the renal capsule exhibit enhanced oxidative stress compared with adjacent normal tissue and the contralateral kidney. Upon glutaminase inhibition with CB-839 or BPTES, the RCC cell lines SN12PM-6-1 (SN12) and 786-O exhibited decreased survival and pronounced apoptosis associated with a decreased GSH/GSSG ratio, augmented nuclear factor erythroid-related factor 2, and increased 8-oxo-7,8-dihydro-2'-deoxyguanosine, a marker of DNA damage. SN12 tumor xenografts showed decreased growth when treated with CB-839. Furthermore, PET imaging confirmed that ccRCC tumors exhibited increased tumoral uptake of

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antineoplastic Agents; Antioxidants; Apoptosis; Benzeneacetamides; Carcinoma, Renal Cell; Deoxyguanosine; Glutaminase; Glutamine; Humans; Kidney Neoplasms; Mice; NF-E2 Transcription Factor; Oxidative Stress; Reactive Oxygen Species; Thiadiazoles; Xenograft Model Antitumor Assays

Cancer cells employ glutaminolysis to provide a source of intermediates for their upregulated biosynthetic needs. Glutaminase, which catalyzes the conversion of glutamine to glutamate, is gaining increasing attention as a potential drug target. Small-molecule inhibitors such as BPTES and CB-839, which target the allosteric site of glutaminase with high specificity, demonstrate immense promise as anti-tumor drugs. Here, we report the study of a new BPTES analog, N,N'-(5,5'-(trans-cyclohexane-1,3-diyl)bis(1,3,4-tiadiazole-5,2-diyl))bis(2-phenylacetamide) (trans-CBTBP), and compared its inhibitory effect against that of CB-839 and BPTES. We show that CB-839 has a 30- and 50-fold lower IC50 than trans-CBTBP and BPTES, respectively. To explore the structural basis for the differences in their inhibitory efficacy, we solved the complex structures of cKGA with 1S, 3S-CBTBP and CB-839. We found that CB-839 produces a greater degree of interaction with cKGA than 1S, 3S-CBTBP or BPTES. The results of this study will facilitate the rational design of new KGA inhibitors to better treat glutamine-addicted cancers.

Topics: Allosteric Site; Antineoplastic Agents; Cell Proliferation; Glutaminase; HEK293 Cells; Humans; Inhibitory Concentration 50; Kidney; Kidney Neoplasms; Molecular Conformation; Protein Binding; Protein Conformation; Sulfides; Thiadiazoles

The MYC oncogene is frequently mutated and overexpressed in human renal cell carcinoma (RCC). However, there have been no studies on the causative role of MYC or any other oncogene in the initiation or maintenance of kidney tumorigenesis. Here, we show through a conditional transgenic mouse model that the MYC oncogene, but not the RAS oncogene, initiates and maintains RCC. Desorption electrospray ionization-mass-spectrometric imaging was used to obtain chemical maps of metabolites and lipids in the mouse RCC samples. Gene expression analysis revealed that the mouse tumors mimicked human RCC. The data suggested that MYC-induced RCC up-regulated the glutaminolytic pathway instead of the glycolytic pathway. The pharmacologic inhibition of glutamine metabolism with bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide impeded MYC-mediated RCC tumor progression. Our studies demonstrate that MYC overexpression causes RCC and points to the inhibition of glutamine metabolism as a potential therapeutic approach for the treatment of this disease.

Topics: Animals; Carcinoma, Renal Cell; Cell Line, Tumor; Disease Models, Animal; Disease Progression; Enzyme Inhibitors; Genes, myc; Genes, ras; Glutaminase; Glutamine; Humans; Kidney Neoplasms; Lipid Metabolism; Mice; Mice, SCID; Mice, Transgenic; RNA, Messenger; RNA, Neoplasm; Spectrometry, Mass, Electrospray Ionization; Sulfides; Thiadiazoles; Up-Regulation

Since failure of differentiation has been suggested to be involved in the neoplastic process and progression of tumors, we evaluated whether the transcript levels of differentiation markers of proximal renal tubular cells, from which renal cell carcinoma (RCC) arises, could be used as prognostic markers. We used Northern blot analysis to study the expression of aquaporin 1 (aqp1) and carbonic anhydrase IV (ca4) genes in 66 paired samples of primary RCC and non-tumorous kidney tissues. Poor differentiation of tumor cells and non-clear cell-subtype RCC were significantly associated with low levels of aqp1 transcripts. When patients were divided into 2 groups according to level of aqpI transcript in RCC, a low level of aqp1 was significantly associated with unfavorable outcome. Among 18 patients with metastatic RCC and 40 patients with moderately differentiated RCC, those with RCC expressing low levels of aqpl mRNA demonstrated poorer survival than those with RCC expressing relatively high levels of aqp1. Similarly, decreased expression of ca4 mRNA in RCC was associated with poor survival. On multivariate analysis, transcript levels of aqpI and stage of the tumor were the independent factors predicting disease-specific survival. Transcript levels of aqp1 may serve as a new molecular prognostic marker in patients with RCC following nephrectomy.

Topics: Aquaporin 1; Aquaporins; Blood Group Antigens; Carbonic Anhydrases; Carcinoma, Renal Cell; Female; Glutaminase; Humans; Ion Channels; Kidney Neoplasms; Male; Multivariate Analysis; Nephrectomy; Prognosis; RNA, Messenger; RNA, Neoplasm

Phosphate-dependent glutaminase was purified to homogeneity from isolated mitochondria of Ehrlich ascites-tumour cells. The enzyme had an Mr of 135,000 as judged by chromatography on Sephacryl S-300. SDS/polyacrylamide-gel electrophoresis displayed two protein bands, with Mr values of 64,000 and 56,000. Two major immunoreactive peptides of Mr values of 65,000 and 57,000 were found by immunoblot analysis using anti-(rat kidney glutaminase) antibodies. The concentration-dependences for both glutamine and phosphate were sigmoidal, with S0.5 values of 7.6 mM and 48 mM, and Hill coefficients of 1.5 and 1.6, respectively. The glutaminase pH optimum was 9. The activation energy of the enzymic reaction was 58 kJ/mol. The enzyme showed a high specificity towards glutamine. A possible explanation for the different kinetic behaviour found for purified enzyme and for isolated mitochondria [Kovacević (1974) Cancer Res. 34, 3403-3407] should be that a conformational change occurs when the enzyme is extracted from the mitochondrial inner membrane.

Topics: Animals; Carcinoma, Ehrlich Tumor; Glutaminase; Glutamine; Hydrogen-Ion Concentration; Immunoblotting; Kidney Neoplasms; Kinetics; Mice; Mitochondria; Molecular Weight; Phosphates; Rats

The three kinds of glutamineses in rat kidney and liver were defined in comparative terms by their properties and were separately purified. Substantial purification was obtained by polymerization and depolymerization of the kidney isozyme that is activated by phosphate, These isozymes differ most strikingly in the activators of their reactions:maleate and a high concentration of phosphate, respectively, for the two kidney isozymes, and a low concentration of phosphate for the liver isozyme. The kidney isozyme that is activated by phosphate was also activated by a much lower concentration of ATP and by other complex phosphates. They also differ in physical properties: the maleate-activated isozyme was heat resistant (50 degrees C) and associated with insoluble submitochondrial particles; both phosphate-activated isozymes were heat sensitive and could be solubilized from their respective mitochondria; the phosphate-activated isozyme of kidney polymerized in phosphate-borate solution while that of liver did not. The characteristics of the kidney isozyme that was activated by high phosphate were shared by the glutaminases in adult brain, transplanted tumors, and in fetal liver and kidney. The similarity to the kidney enzyme was confirmed by the use of polymerization in phosphate borate of the isozyme from a mammary carcinoma to effect its purification.

Topics: Age Factors; Animals; Brain; Carcinoma; Enzyme Activation; Female; Glutaminase; Isoenzymes; Kidney; Kidney Neoplasms; Liver; Neoplasms, Experimental; Phosphates; Pregnancy; Rats

Topics: 5-Aminolevulinate Synthetase; Acetamides; Animals; Arginase; Carcinoma, Hepatocellular; DNA Nucleotidyltransferases; Enzyme Induction; Female; Fetus; Glucose; Glutamate-Ammonia Ligase; Glutaminase; Hydrocortisone; Kidney; Kidney Neoplasms; Liver; Liver Neoplasms; Male; Mixed Function Oxygenases; Ornithine; Oxidoreductases; Phosphotransferases; Pyruvate Kinase; Rats; Transaminases; Tryptophan; Tryptophan Oxygenase

Topics: Acidosis; Alkalosis; Ammonia; Animals; Glutaminase; Humans; Hydronephrosis; Kidney; Kidney Failure, Chronic; Kidney Neoplasms; Phosphates; Pyelonephritis; Rabbits; Rats; Tuberculosis, Renal; Ureteral Obstruction