RESUMO
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.
Assuntos
Anilidas/administração & dosagem , Benzenoacetamidas/administração & dosagem , Carcinoma de Células Renais/tratamento farmacológico , Everolimo/administração & dosagem , Neoplasias Renais/tratamento farmacológico , Piridinas/administração & dosagem , Tiadiazóis/administração & dosagem , Anilidas/farmacologia , Animais , Benzenoacetamidas/farmacologia , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Everolimo/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glutaminase/antagonistas & inibidores , Glutamina/metabolismo , Humanos , Neoplasias Renais/metabolismo , Camundongos , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tiadiazóis/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Elevation of L-2-hydroxylgutarate (L-2-HG) in renal cell carcinoma (RCC) is due in part to reduced expression of L-2-HG dehydrogenase (L2HGDH). However, the contribution of L-2-HG to renal carcinogenesis and insight into the biochemistry and targets of this small molecule remains to be elucidated. EXPERIMENTAL DESIGN: Genetic and pharmacologic approaches to modulate L-2-HG levels were assessed for effects on in vitro and in vivo phenotypes. Metabolomics was used to dissect the biochemical mechanisms that promote L-2-HG accumulation in RCC cells. Transcriptomic analysis was utilized to identify relevant targets of L-2-HG. Finally, bioinformatic and metabolomic analyses were used to assess the L-2-HG/L2HGDH axis as a function of patient outcome and cancer progression. RESULTS: L2HGDH suppresses both in vitro cell migration and in vivo tumor growth and these effects are mediated by L2HGDH's catalytic activity. Biochemical studies indicate that glutamine is the predominant carbon source for L-2-HG via the activity of malate dehydrogenase 2 (MDH2). Inhibition of the glutamine-MDH2 axis suppresses in vitro phenotypes in an L-2-HG-dependent manner. Moreover, in vivo growth of RCC cells with basal elevation of L-2-HG is suppressed by glutaminase inhibition. Transcriptomic and functional analyses demonstrate that the histone demethylase KDM6A is a target of L-2-HG in RCC. Finally, increased L-2-HG levels, L2HGDH copy loss, and lower L2HGDH expression are associated with tumor progression and/or worsened prognosis in patients with RCC. CONCLUSIONS: Collectively, our studies provide biochemical and mechanistic insight into the biology of this small molecule and provide new opportunities for treating L-2-HG-driven kidney cancers.
Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Epigênese Genética , Glutaratos/metabolismo , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Oxirredutases do Álcool/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular/genética , Modelos Animais de Doenças , Expressão Gênica , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/patologia , Metilação , Terapia de Alvo Molecular , Fenótipo , RNA Interferente Pequeno/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Myeloid cells are an abundant leukocyte in many types of tumors and contribute to immune evasion. Expression of the enzyme arginase 1 (Arg1) is a defining feature of immunosuppressive myeloid cells and leads to depletion of L-arginine, a nutrient required for T cell and natural killer (NK) cell proliferation. Here we use CB-1158, a potent and orally-bioavailable small-molecule inhibitor of arginase, to investigate the role of Arg1 in regulating anti-tumor immunity. METHODS: CB-1158 was tested for the ability to block myeloid cell-mediated inhibition of T cell proliferation in vitro, and for tumor growth inhibition in syngeneic mouse models of cancer as a single agent and in combination with other therapies. Tumors from animals treated with CB-1158 were profiled for changes in immune cell subsets, expression of immune-related genes, and cytokines. Human tumor tissue microarrays were probed for Arg1 expression by immunohistochemistry and immunofluorescence. Cancer patient plasma samples were assessed for Arg1 protein and L-arginine by ELISA and mass spectrometry, respectively. RESULTS: CB-1158 blocked myeloid cell-mediated suppression of T cell proliferation in vitro and reduced tumor growth in multiple mouse models of cancer, as a single agent and in combination with checkpoint blockade, adoptive T cell therapy, adoptive NK cell therapy, and the chemotherapy agent gemcitabine. Profiling of the tumor microenvironment revealed that CB-1158 increased tumor-infiltrating CD8+ T cells and NK cells, inflammatory cytokines, and expression of interferon-inducible genes. Patient tumor samples from multiple histologies expressed an abundance of tumor-infiltrating Arg1+ myeloid cells. Plasma samples from cancer patients exhibited elevated Arg1 and reduced L-arginine compared to healthy volunteers. CONCLUSIONS: These results demonstrate that Arg1 is a key mediator of immune suppression and that inhibiting Arg1 with CB-1158 shifts the immune landscape toward a pro-inflammatory environment, blunting myeloid cell-mediated immune evasion and reducing tumor growth. Furthermore, our results suggest that arginase blockade by CB-1158 may be an effective therapy in multiple types of cancer and combining CB-1158 with standard-of-care chemotherapy or other immunotherapies may yield improved clinical responses.
Assuntos
Arginase/metabolismo , Células Mieloides/citologia , Neoplasias/tratamento farmacológico , Pirrolidinas/administração & dosagem , Bibliotecas de Moléculas Pequenas/administração & dosagem , Microambiente Tumoral/efeitos dos fármacos , Animais , Arginase/antagonistas & inibidores , Arginina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Células Hep G2 , Humanos , Células K562 , Masculino , Camundongos , Células Mieloides/efeitos dos fármacos , Células Mieloides/enzimologia , Neoplasias/imunologia , Neoplasias/metabolismo , Pirrolidinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer cells exhibit increased use of nutrients, including glucose and glutamine, to support the bioenergetic and biosynthetic demands of proliferation. We tested the small-molecule inhibitor of glutaminase CB-839 in combination with erlotinib on epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) as a therapeutic strategy to simultaneously impair cancer glucose and glutamine utilization and thereby suppress tumor growth. Here, we show that CB-839 cooperates with erlotinib to drive energetic stress and activate the AMP-activated protein kinase (AMPK) pathway in EGFR (del19) lung tumors. Tumor cells undergo metabolic crisis and cell death, resulting in rapid tumor regression in vivo in mouse NSCLC xenografts. Consistently, positron emission tomography (PET) imaging with 18F-fluoro-2-deoxyglucose (18F-FDG) and 11C-glutamine (11C-Gln) of xenografts indicated reduced glucose and glutamine uptake in tumors following treatment with CB-839 + erlotinib. Therefore, PET imaging with 18F-FDG and 11C-Gln tracers can be used to non-invasively measure metabolic response to CB-839 and erlotinib combination therapy.
Assuntos
Apoptose/efeitos dos fármacos , Benzenoacetamidas/toxicidade , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib/toxicidade , Glutaminase/antagonistas & inibidores , Tiadiazóis/toxicidade , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Autofagia/efeitos dos fármacos , Benzenoacetamidas/uso terapêutico , Radioisótopos de Carbono/química , Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/uso terapêutico , Fluordesoxiglucose F18/química , Glutaminase/metabolismo , Glutamina/química , Glutamina/metabolismo , Humanos , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Camundongos SCID , Mutação , Interferência de RNA , Compostos Radiofarmacêuticos/química , Tiadiazóis/uso terapêutico , Transplante HeterólogoRESUMO
Intermittent claudication is a form of exercise intolerance characterized by muscle pain during walking in patients with peripheral artery disease (PAD). Endothelial cell and muscle dysfunction are thought to be important contributors to the etiology of this disease, but a lack of preclinical models that incorporate these elements and measure exercise performance as a primary end point has slowed progress in finding new treatment options for these patients. We sought to develop an animal model of peripheral vascular insufficiency in which microvascular dysfunction and exercise intolerance were defining features. We further set out to determine if pharmacological activation of 5'-AMP-activated protein kinase (AMPK) might counteract any of these functional deficits. Mice aged on a high-fat diet demonstrate many functional and molecular characteristics of PAD, including the sequential development of peripheral vascular insufficiency, increased muscle fatigability, and progressive exercise intolerance. These changes occur gradually and are associated with alterations in nitric oxide bioavailability. Treatment of animals with an AMPK activator, R118, increased voluntary wheel running activity, decreased muscle fatigability, and prevented the progressive decrease in treadmill exercise capacity. These functional performance benefits were accompanied by improved mitochondrial function, the normalization of perfusion in exercising muscle, increased nitric oxide bioavailability, and decreased circulating levels of the endogenous endothelial nitric oxide synthase inhibitor asymmetric dimethylarginine. These data suggest that aged, obese mice represent a novel model for studying exercise intolerance associated with peripheral vascular insufficiency, and pharmacological activation of AMPK may be a suitable treatment for intermittent claudication associated with PAD.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Dieta Hiperlipídica , Ativadores de Enzimas/administração & dosagem , Obesidade/complicações , Doenças Vasculares Periféricas/fisiopatologia , Esforço Físico/fisiologia , Envelhecimento , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Apolipoproteínas E/fisiologia , Arginina/análogos & derivados , Arginina/sangue , Cilostazol , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Humanos , Claudicação Intermitente/complicações , Claudicação Intermitente/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fadiga Muscular/efeitos dos fármacos , Músculo Esquelético/irrigação sanguínea , Óxido Nítrico Sintase Tipo III/metabolismo , Doenças Vasculares Periféricas/etiologia , Inibidores da Fosfodiesterase 3/administração & dosagem , Tetrazóis/administração & dosagem , VasodilatadoresRESUMO
Modulation of mitochondrial function through inhibiting respiratory complex I activates a key sensor of cellular energy status, the 5'-AMP-activated protein kinase (AMPK). Activation of AMPK results in the mobilization of nutrient uptake and catabolism for mitochondrial ATP generation to restore energy homeostasis. How these nutrient pathways are affected in the presence of a potent modulator of mitochondrial function and the role of AMPK activation in these effects remain unclear. We have identified a molecule, named R419, that activates AMPK in vitro via complex I inhibition at much lower concentrations than metformin (IC50 100 nM vs 27 mM, respectively). R419 potently increased myocyte glucose uptake that was dependent on AMPK activation, while its ability to suppress hepatic glucose production in vitro was not. In addition, R419 treatment of mouse primary hepatocytes increased fatty acid oxidation and inhibited lipogenesis in an AMPK-dependent fashion. We have performed an extensive metabolic characterization of its effects in the db/db mouse diabetes model. In vivo metabolite profiling of R419-treated db/db mice showed a clear upregulation of fatty acid oxidation and catabolism of branched chain amino acids. Additionally, analyses performed using both (13)C-palmitate and (13)C-glucose tracers revealed that R419 induces complete oxidation of both glucose and palmitate to CO2 in skeletal muscle, liver, and adipose tissue, confirming that the compound increases mitochondrial function in vivo. Taken together, our results show that R419 is a potent inhibitor of complex I and modulates mitochondrial function in vitro and in diabetic animals in vivo. R419 may serve as a valuable molecular tool for investigating the impact of modulating mitochondrial function on nutrient metabolism in multiple tissues and on glucose and lipid homeostasis in diabetic animal models.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Diabetes Mellitus Experimental/metabolismo , Mitocôndrias Hepáticas/metabolismo , Células Musculares/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Diabetes Mellitus Experimental/patologia , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos/metabolismo , Glucose/metabolismo , Células Hep G2 , Humanos , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Camundongos , Mitocôndrias Hepáticas/patologia , Células Musculares/patologia , Oxirredução/efeitos dos fármacos , Palmitatos/farmacologia , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Accumulating evidence suggests important roles for the receptor tyrosine kinase Axl in cancer progression, invasion, metastasis, drug resistance, and patient mortality, highlighting Axl as an attractive target for therapeutic development. We have generated and characterized a potent and selective small-molecule inhibitor, R428, that blocks the catalytic and procancerous activities of Axl. R428 inhibits Axl with low nanomolar activity and blocked Axl-dependent events, including Akt phosphorylation, breast cancer cell invasion, and proinflammatory cytokine production. Pharmacologic investigations revealed favorable exposure after oral administration such that R428-treated tumors displayed a dose-dependent reduction in expression of the cytokine granulocyte macrophage colony-stimulating factor and the epithelial-mesenchymal transition transcriptional regulator Snail. In support of an earlier study, R428 inhibited angiogenesis in corneal micropocket and tumor models. R428 administration reduced metastatic burden and extended survival in MDA-MB-231 intracardiac and 4T1 orthotopic (median survival, >80 days compared with 52 days; P < 0.05) mouse models of breast cancer metastasis. Additionally, R428 synergized with cisplatin to enhance suppression of liver micrometastasis. Our results show that Axl signaling regulates breast cancer metastasis at multiple levels in tumor cells and tumor stromal cells and that selective Axl blockade confers therapeutic value in prolonging survival of animals bearing metastatic tumors.
Assuntos
Benzocicloeptenos/farmacologia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Carcinoma/mortalidade , Carcinoma/patologia , Proteínas Oncogênicas/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Triazóis/farmacologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzocicloeptenos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Carcinoma/tratamento farmacológico , Feminino , Células HeLa , Humanos , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas , Análise de Sobrevida , Triazóis/uso terapêutico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Receptor Tirosina Quinase AxlRESUMO
Survival factors play critical roles in regulating cell growth in normal and cancer cells. We designed a genetic screen to identify survival factors which protect tumor cells from apoptosis. A retroviral expression library of random cDNA fragments was constructed from cancer cells and used to transduce the colon carcinoma cell line HCT116. Recipient cells were functionally selected for induction of caspase 3-mediated apoptosis. Analyses of over 10,000 putative genetic suppression elements (GSEs) sequences revealed cognate gene candidates that are implicated in apoptosis. We further analysed 26 genes encoding cell surface and secreted proteins that can potentially serve as targets for therapeutic antibodies. Tetracycline-inducible GSEs from several gene candidates induced apoptosis in stable HCT 116 cell lines. Similar phenotypes were caused by RNAi derived from the same genes. Our data suggest requirement for the cell surface targets IGF2R, L1CAM and SLC31A1 in tumor cell growth in vitro, and suggests that IGF2R is required for xenograft tumor growth in a mouse model.