RESUMEN
Cancer cells reprogram their metabolism to promote growth and proliferation. The genetic evidence pointing to the importance of the amino acid serine in tumorigenesis is striking. The gene encoding the enzyme 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the first committed step of serine biosynthesis, is overexpressed in tumors and cancer cell lines via focal amplification and nuclear factor erythroid-2-related factor 2 (NRF2)-mediated up-regulation. PHGDH-overexpressing cells are exquisitely sensitive to genetic ablation of the pathway. Here, we report the discovery of a selective small molecule inhibitor of PHGDH, CBR-5884, identified by screening a library of 800,000 drug-like compounds. CBR-5884 inhibited de novo serine synthesis in cancer cells and was selectively toxic to cancer cell lines with high serine biosynthetic activity. Biochemical characterization of the inhibitor revealed that it was a noncompetitive inhibitor that showed a time-dependent onset of inhibition and disrupted the oligomerization state of PHGDH. The identification of a small molecule inhibitor of PHGDH not only enables thorough preclinical evaluation of PHGDH as a target in cancers, but also provides a tool with which to study serine metabolism.
Asunto(s)
Neoplasias/metabolismo , Fosfoglicerato-Deshidrogenasa/antagonistas & inhibidores , Serina/biosíntesis , Línea Celular Tumoral , Proliferación Celular , Humanos , Neoplasias/patologíaRESUMEN
Pancreatic cancer cells are characterized by deregulated metabolic programs that facilitate growth and resistance to oxidative stress. Among these programs, pancreatic cancers preferentially utilize a metabolic pathway through the enzyme aspartate aminotransferase 1 [also known as glutamate oxaloacetate transaminase 1 (GOT1)] to support cellular redox homeostasis. As such, small molecule inhibitors that target GOT1 could serve as starting points for the development of new therapies for pancreatic cancer. We ran a high-throughput screen for inhibitors of GOT1 and identified a small molecule, iGOT1-01, with in vitro GOT1 inhibitor activity. Application in pancreatic cancer cells revealed metabolic and growth inhibitory activity reflecting a promiscuous inhibitory profile. We then performed an in silico docking analysis to study inhibitor-GOT1 interactions with iGOT1-01 analogues that possess improved solubility and potency properties. These results suggested that the GOT1 inhibitor competed for binding to the pyridoxal 5-phosphate (PLP) cofactor site of GOT1. To analyze how the GOT1 inhibitor bound to GOT1, a series of GOT1 mutant enzymes that abolished PLP binding were generated. Application of the mutants in X-ray crystallography and thermal shift assays again suggested but were unable to formally conclude that the GOT1 inhibitor bound to the PLP site. Mutational studies revealed the relationship between PLP binding and the thermal stability of GOT1 while highlighting the essential nature of several residues for GOT1 catalytic activity. Insight into the mode of action of GOT1 inhibitors may provide leads to the development of drugs that target redox balance in pancreatic cancer.
Asunto(s)
Aspartato Aminotransferasa Citoplasmática/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/patología , Inhibidores Enzimáticos/farmacología , Mutación , Neoplasias Pancreáticas/patología , Aspartato Aminotransferasa Citoplasmática/genética , Aspartato Aminotransferasa Citoplasmática/metabolismo , Sitios de Unión , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Metabolómica , Modelos Moleculares , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Conformación Proteica , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
On-patent and off-patent drugs with previously unrecognized anticancer activity could be rapidly repurposed for this new indication given their prior toxicity testing. To identify such compounds, we conducted chemical screens and identified the antihelmintic flubendazole. Flubendazole induced cell death in leukemia and myeloma cell lines and primary patient samples at nanomolar concentrations. Moreover, it delayed tumor growth in leukemia and myeloma xenografts without evidence of toxicity. Mechanistically, flubendazole inhibited tubulin polymerization by binding tubulin at a site distinct from vinblastine. In addition, cells resistant to vinblastine because of overexpression of P-glycoprotein remained fully sensitive to flubendazole, indicating that flubendazole can overcome some forms of vinblastine resistance. Given the different mechanisms of action, we evaluated the combination of flubendazole and vinblastine in vitro and in vivo. Flubendazole synergized with vinblastine to reduce the viability of OCI-AML2 cells. In addition, combinations of flubendazole with vinblastine or vincristine in a leukemia xenograft model delayed tumor growth more than either drug alone. Therefore, flubendazole is a novel microtubule inhibitor that displays preclinical activity in leukemia and myeloma.
Asunto(s)
Antinematodos/farmacología , Leucemia/tratamiento farmacológico , Mebendazol/análogos & derivados , Microtúbulos/metabolismo , Mieloma Múltiple/tratamiento farmacológico , Alcaloides de la Vinca/farmacología , Animales , Antinematodos/agonistas , Antinematodos/uso terapéutico , Antineoplásicos Fitogénicos/agonistas , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Muerte Celular , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Células HeLa , Humanos , Leucemia/metabolismo , Masculino , Mebendazol/agonistas , Mebendazol/farmacología , Mebendazol/uso terapéutico , Ratones , Mieloma Múltiple/metabolismo , Células U937 , Vinblastina/agonistas , Vinblastina/farmacología , Vinblastina/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
The proteasomal pathway of protein degradation involves 2 discrete steps: ubiquitination and degradation. Here, we evaluated the effects of inhibiting the ubiquitination pathway at the level of the ubiquitin-activating enzyme UBA1 (E1). By immunoblotting, leukemia cell lines and primary patient samples had increased protein ubiquitination. Therefore, we examined the effects of genetic and chemical inhibition of the E1 enzyme. Knockdown of E1 decreased the abundance of ubiquitinated proteins in leukemia and myeloma cells and induced cell death. To further investigate effects of E1 inhibition in malignancy, we discovered a novel small molecule inhibitor, 3,5-dioxopyrazolidine compound, 1-(3-chloro-4-fluorophenyl)-4-[(5-nitro-2-furyl)methylene]-3,5-pyrazolidinedione (PYZD-4409). PYZD-4409 induced cell death in malignant cells and preferentially inhibited the clonogenic growth of primary acute myeloid leukemia cells compared with normal hematopoietic cells. Mechanistically, genetic or chemical inhibition of E1 increased expression of E1 stress markers. Moreover, BI-1 overexpression blocked cell death after E1 inhibition, suggesting ER stress is functionally important for cell death after E1 inhibition. Finally, in a mouse model of leukemia, intraperitoneal administration of PYZD-4409 decreased tumor weight and volume compared with control without untoward toxicity. Thus, our work highlights the E1 enzyme as a novel target for the treatment of hematologic malignancies.
Asunto(s)
Leucemia/enzimología , Leucemia/terapia , Mieloma Múltiple/enzimología , Mieloma Múltiple/terapia , Enzimas Activadoras de Ubiquitina/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ciclina D3/metabolismo , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/patología , Inhibidores Enzimáticos/farmacología , Técnicas de Silenciamiento del Gen , Sistema Hematopoyético/citología , Sistema Hematopoyético/efectos de los fármacos , Humanos , Ratones , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Estrés Fisiológico/efectos de los fármacos , Factores de Tiempo , Proteína p53 Supresora de Tumor/metabolismo , Enzimas Activadoras de Ubiquitina/antagonistas & inhibidores , Ubiquitinación/efectos de los fármacosRESUMEN
D-cyclins are regulators of cell division that act in a complex with cyclin-dependent kinases to commit cells to a program of DNA replication. D-cyclins are overexpressed in many tumors, including multiple myeloma and leukemia, and contribute to disease progression and chemoresistance. To better understand the role and impact of D-cyclins in hematologic malignancies, we conducted a high throughput screen for inhibitors of the cyclin D2 promoter and identified the drug cyproheptadine. In myeloma and leukemia cells, cyproheptadine decreased expression of cyclins D1, D2, and D3 and arrested these cells in the G(0)/G(1) phase. After D-cyclin suppression, cyproheptadine induced apoptosis in myeloma and leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. In mouse models of myeloma and leukemia, cyproheptadine inhibited tumor growth without significant toxicity. Cyproheptadine-induced apoptosis was preceded by activation of the mitochondrial pathway of caspase activation and was independent of the drug's known activity as an H1 histamine and serotonin receptor antagonist. Thus, cyproheptadine represents a lead for a novel therapeutic agent for the treatment of malignancy. Because the drug is well tolerated and already approved in multiple countries for clinical use as an antihistamine and appetite stimulant, it could be moved directly into clinical trials for cancer.
Asunto(s)
Ciclinas/genética , Ciproheptadina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Mieloma Múltiple/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Ciclina D1/genética , Ciclina D2 , Ciclina D3 , Ciproheptadina/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Leucemia Mieloide Aguda/patología , Ratones , Mieloma Múltiple/patologíaRESUMEN
Quinolines are a class of chemical compounds with emerging anti-cancer properties. Here, we tested the activity of series of quinolines and quinoline-like molecules for anti-cancer activity and identified a novel diquinoline, 1-methyl-2-[3-(1-methyl-1,2-dihydroquinolin-2-yliden)prop-1-enyl]quinolinium iodide (Q(2)). Q(2 )induced cell death in leukemia, myeloma, and solid tumor cell lines with LD50s in the low to submicromolar range. Moreover, Q(2) induced cell death in primary acute myeloid leukemia (AML) cells preferentially over normal hematopoietic cells. In a mouse model of leukemia, Q(2) delayed tumor growth. Mechanistically, Q(2) induced cell death through caspase independent mechanisms. By electron microscopy, Q(2) increased cytoplasmic vacuolization and mitochondrial swelling. Potentially consistent with the induction of autophagic cell death, Q(2) treatment led to a punctate distribution of LC3 and increased MDC staining. Thus, Q(2) is a novel quinolinium with preclinical activity in malignancies such as leukemia and myeloma and warrants further investigation.
Asunto(s)
Muerte Celular/efectos de los fármacos , Compuestos de Quinolinio/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Autofagia/efectos de los fármacos , Caspasas/fisiología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células HeLa , Humanos , Células Jurkat , Dosificación Letal Mediana , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Mieloide Aguda/tratamiento farmacológico , Ratones , Ratones Endogámicos DBA , Compuestos de Quinolinio/uso terapéutico , Células Tumorales Cultivadas , Células U937RESUMEN
Dysregulation of iron homeostasis is implicated in Alzheimer's disease (AD). In this pilot study, common variants of the apolipoprotein E (APOE) and HFE genes resulting in the iron overload disorder of hereditary hemochromatosis (C282Y, H63D and S65C) were evaluated as factors in sporadic AD in an Ontario sample in which folic acid fortification has been mandatory since 1998. Laboratory studies also were done to search for genetic effects on blood markers of iron status, red cell folates and serum B12. Participants included 58 healthy volunteers (25 males, 33 females) and 54 patients with probable AD (20 males, 34 females). Statistical analyses were interpreted at the 95% confidence level. Contingency table and odds ratio analyses supported the hypothesis that in females of the given age range, E4 significantly predisposed to AD in the presence but not absence of H63D. In males, E4 significantly predisposed to AD in the absence of H63D, and H63D in the absence of E4 appeared protective against AD. Among E4+ AD patients, H63D was associated with significant lowering of red cell folate concentration, possibly as the result of excessive oxidative stress. However, folate levels in the lowest population quartile did not affect the risk of AD. A model is presented to explain the experimental findings.
Asunto(s)
Alelos , Enfermedad de Alzheimer/genética , Apolipoproteína E4/genética , Análisis Mutacional de ADN , Ácido Fólico/administración & dosificación , Variación Genética/genética , Hemocromatosis/genética , Antígenos de Histocompatibilidad Clase I/genética , Sobrecarga de Hierro/genética , Proteínas de la Membrana/genética , Anciano de 80 o más Años , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/tratamiento farmacológico , Biomarcadores/sangre , Eritrocitos/metabolismo , Femenino , Ácido Fólico/sangre , Predisposición Genética a la Enfermedad/genética , Genotipo , Hemocromatosis/sangre , Proteína de la Hemocromatosis , Humanos , Sobrecarga de Hierro/sangre , Masculino , Escala del Estado Mental , Persona de Mediana Edad , Ontario , Factores Sexuales , Vitamina B 12/sangreRESUMEN
Oncogenic signaling promotes tumor invasion and metastasis, in part, by increasing the expression of tri- and tetra- branched N-glycans. The branched N-glycans bind to galectins forming a multivalent lattice that enhances cell surface residency of growth factor receptors, and focal adhesion turnover. N-acetylglucosaminyltransferase I (MGAT1), the first branching enzyme in the pathway, is required for the addition of all subsequent branches. Here we have introduced MGAT1 shRNA into human HeLa cervical and PC-3-Yellow prostate tumor cells lines, generating cell lines with reduced transcript, enzyme activity and branched N-glycans at the cell surface. MGAT1 knockdown inhibited HeLa cell migration and invasion, but did not alter cell proliferation rates. Swainsonine, an inhibitor of α-mannosidase II immediately downstream of MGAT1, also inhibited cell invasion and was not additive with MGAT1 shRNA, consistent with a common mechanism of action. Focal adhesion and microfilament organization in MGAT1 knockdown cells also indicate a less motile phenotype. In vivo, MGAT1 knockdown in the PC-3-Yellow orthotopic prostate cancer xenograft model significantly decreased primary tumor growth and the incidence of lung metastases. Our results demonstrate that blocking MGAT1 is a potential target for anti-cancer therapy.
Asunto(s)
N-Acetilglucosaminiltransferasas/genética , Neoplasias/genética , Neoplasias/terapia , ARN Interferente Pequeño/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Progresión de la Enfermedad , Inhibidores Enzimáticos/farmacología , Femenino , Células HeLa , Humanos , Masculino , Ratones , N-Acetilglucosaminiltransferasas/metabolismo , Metástasis de la Neoplasia , Trasplante de Neoplasias , Fenotipo , Polisacáridos/química , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/terapia , Transducción de Señal , Swainsonina/farmacologíaRESUMEN
Normal epithelial cells undergo apoptosis upon detachment from the extracellular matrix, a process termed "anoikis." However, malignant epithelial cells with metastatic potential resist anoikis and can survive in an anchorage-independent fashion. Molecules that sensitize resistant cells to anoikis will be useful chemical probes to understand this pathway. To identify novel anoikis sensitizers in anoikis-resistant PPC-1 prostate adenocarcinoma cells, a library of 2,000 off-patent drugs and natural products was screened for their ability to preferentially induce cell death in suspension over adherent culture conditions. This screen identified five members of the family of cardiac glycosides as anoikis sensitizers, including ouabain, peruvoside, digoxin, digitoxin, and strophanthidin. We conducted further studies with ouabain to discern the mechanism of cardiac glycoside-induced anoikis sensitization. Ouabain initiated anoikis through the mitochondrial pathway of caspase activation. In addition, ouabain sensitized cells to anoikis by inhibiting its known target, the Na(+)/K(+) ATPase pump, and inducing hypoosmotic stress. Resistance to anoikis permits cancer cells to survive in the circulation and facilitates their metastasis to distant organs, so we tested the effects of Na(+)/K(+) ATPase inhibition on distant tumor formation in mouse models. In these mouse models, ouabain inhibited tumor metastases but did not alter the growth of subcutaneous tumors. Thus, we have identified a novel mechanism to sensitize resistant cells to anoikis and decrease tumor metastasis. These results suggest a potential mechanism for the observed clinical reduction in metastasis and relapse in breast cancer patients who have undergone treatments with cardiac glycosides.
Asunto(s)
Anoicis/efectos de los fármacos , Glicósidos Cardíacos/farmacología , Inhibidores Enzimáticos/farmacología , Neoplasias/tratamiento farmacológico , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Animales , Anoicis/fisiología , Inhibidores de Caspasas , Caspasas/metabolismo , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Activación Enzimática/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Metástasis de la Neoplasia , Neoplasias/enzimología , Neoplasias/patología , Presión Osmótica/efectos de los fármacos , Ouabaína/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Aberrant N-linked glycans promote the malignant potential of cells by enhancing the epithelial-to-mesenchymal transition and the invasive phenotype. To identify small molecule inhibitors of N-glycan biosynthesis, we developed a chemical screen based on the ability of the tetravalent plant lectin L-phytohemagglutinin (L-PHA) to bind and crosslink surface glycoproteins with beta1,6GlcNAc-branched complex type N-glycans and thereby induce agglutination and cell death. In this screen, Jurkat cells were treated with a library of off-patent chemicals (n = 1,280) to identify molecules that blocked L-PHA-induced death. The most potent hit from this screen was the cardiac glycoside (CG) dihydroouabain. In secondary assays, a panel of CGs was tested for their effects on L-PHA-induced agglutination and cell death. All of the CGs tested inhibited L-PHA-induced death in Jurkat cells, and the most potent CG tested was digoxin with an EC(50) of 60 +/- 20 nmol/L. Digoxin also increased the fraction of some concanavalin A-binding N-glycans. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, digoxin specifically increased GlcNAc(1)Man(3)GlcNAc(2)Fuc(1) and GlcNAc(2)Man(3)GlcNAc(2)Fuc(1) oligosaccharides demonstrating an impairment of the N-glycan pathway. Consistent with this effect on the N-glycan pathway, digoxin inhibited N-glycosylation-mediated processes of tumor cell migration and invasion. Furthermore, digoxin prevented distant tumor formation in two mouse models of metastatic prostate cancer. Thus, taken together, our high throughput screen identified CGs as modifiers of the N-glycan pathway. These molecules can be used as tools to better understand the role of N-glycans in normal and malignant cells. Moreover, these results may partly explain the anticancer effect of CGs in cardiovascular patients.