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1.
Chembiochem ; 22(12): 2107-2110, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33838082

RESUMO

PARP14 is an interferon-stimulated gene that is overexpressed in multiple tumor types, influencing pro-tumor macrophage polarization as well as suppressing the antitumor inflammation response by modulating IFN-γ and IL-4 signaling. PARP14 is a 203 kDa protein that possesses a catalytic domain responsible for the transfer of mono-ADP-ribose to its substrates. PARP14 also contains three macrodomains and a WWE domain which are binding modules for mono-ADP-ribose and poly-ADP-ribose, respectively, in addition to two RNA recognition motifs. Catalytic inhibitors of PARP14 have been shown to reverse IL-4 driven pro-tumor gene expression in macrophages, however it is not clear what roles the non-enzymatic biomolecular recognition motifs play in PARP14-driven immunology and inflammation. To further understand this, we have discovered a heterobifunctional small molecule designed based on a catalytic inhibitor of PARP14 that binds in the enzyme's NAD+ -binding site and recruits cereblon to ubiquitinate it and selectively target it for degradation.


Assuntos
Poli(ADP-Ribose) Polimerases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química
2.
Lancet Oncol ; 19(5): 649-659, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29650362

RESUMO

BACKGROUND: Activating enhancer of zeste homolog 2 (EZH2) mutations or aberrations of the switch/sucrose non-fermentable (SWI/SNF) complex (eg, mutations or deletions of the subunits INI1 or SMARCA4) can lead to aberrant histone methylation, oncogenic transformation, and a proliferative dependency on EZH2 activity. In this first-in-human study, we aimed to investigate the safety, clinical activity, pharmacokinetics, and pharmacodynamics of tazemetostat, a first-in-class selective inhibitor of EZH2. METHODS: We did an open-label, multicentre, dose-escalation, phase 1 study using a 3 + 3 design with planned cohort expansion at the two highest doses below the maximally tolerated dose. The study was done at two centres in France: Institut Gustave Roussy (Villejuif, Val de Marne) and Institut Bergonié (Bordeaux, Gironde). Eligible patients had relapsed or refractory B-cell non-Hodgkin lymphoma or an advanced solid tumour and were older than 18 years, with Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate end-organ function. Tazemetostat was administered orally from 100 mg twice daily to 1600 mg twice daily in 28-day cycles. The primary endpoint was to establish the maximum tolerated dose or recommended phase 2 dose of tazemetostat, as determined by dose-limiting toxicities, laboratory values, and other safety or pharmacokinetic measures in cycle one according to local investigator assessment. Safety was assessed in patients who received at least one dose of tazemetostat; antitumour activity was assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01897571. The phase 1 part of the study is complete, and phase 2 is ongoing. FINDINGS: Between June 13, 2013, and Sept 21, 2016, 64 patients (21 with B-cell non-Hodgkin lymphoma, and 43 with advanced solid tumours) received doses of tazemetostat. The most common treatment-related adverse events, regardless of attribution, were asthenia (21 [33%] of 64 treatment-related events), anaemia (nine [14%]), anorexia (four [6%]), muscle spasms (nine [14%]), nausea (13 [20%]), and vomiting (six [9%]), usually grade 1 or 2 in severity. A single dose-limiting toxicity of grade 4 thrombocytopenia was identified at the highest dose of 1600 mg twice daily. No treatment-related deaths occurred; seven (11%) patients had non-treatment-related deaths (one at 200 mg twice daily, four at 400 mg twice daily, and two at 1600 mg twice daily). The recommended phase 2 dose was determined to be 800 mg twice daily. Durable objective responses, including complete responses, were observed in eight (38%) of 21 patients with B-cell non-Hodgkin lymphoma and two (5%) of 43 patients with solid tumours. INTERPRETATION: Tazemetostat showed a favourable safety profile and antitumour activity in patients with refractory B-cell non-Hodgkin lymphoma and advanced solid tumours, including epithelioid sarcoma. Further clinical investigation of tazemetostat monotherapy is ongoing in phase 2 studies in adults and a phase 1 study for children, which are currently enrolling patients who have B-cell non-Hodgkin lymphoma and INI1-negative or SMARCA4-negative tumours. FUNDING: Epizyme and Eisai.


Assuntos
Antineoplásicos/administração & dosagem , Benzamidas/administração & dosagem , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Inibidores Enzimáticos/administração & dosagem , Linfoma de Células B/tratamento farmacológico , Piridonas/administração & dosagem , Idoso , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Benzamidas/efeitos adversos , Benzamidas/farmacocinética , Compostos de Bifenilo , Relação Dose-Resposta a Droga , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/farmacocinética , Feminino , França , Humanos , Linfoma de Células B/enzimologia , Linfoma de Células B/patologia , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Morfolinas , Piridonas/efeitos adversos , Piridonas/farmacocinética , Fatores de Tempo , Resultado do Tratamento
3.
Pediatr Blood Cancer ; 64(3)2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27555605

RESUMO

BACKGROUND: Tazemetostat (EPZ-6438) is a selective inhibitor of the histone methyltransferase EZH2 and currently in clinical development for non-Hodgkin lymphoma and genetically defined tumors. PROCEDURES: Tazemetostat was tested against the Pediatric Preclinical Testing Program (PPTP) solid tumor xenografts using a dose of 400 mg/kg administered twice daily by oral gavage for 28 days. H3K27me3:H3 ratios were determined in control and treated tumors. RESULTS: Tazemetostat induced significant differences in event-free survival (EFS) distribution compared with control in nine of 30 (30%) of the xenografts studied. Significant differences in EFS distribution were observed in five of seven (71%) rhabdoid tumor xenograft lines compared with four of 23 (17%) nonrhabdoid xenograft lines (chi-square [χ2 ] test P = 0.006). Tazemetostat induced tumor growth inhibition meeting criteria for intermediate and high EFS treated-to-control (T/C) activity in two of 25 (8%) and one of 25 (4%) xenografts, respectively. Intermediate and high activity for the EFS T/C metric was observed exclusively among rhabdoid tumor xenografts (three of five rhabdoid tumor vs 0 of 22 nonrhabdoid tumors (χ² test P < 0.001). One rhabdoid tumor xenograft (G401) showed stable disease. For one rhabdoid tumor (G401), delayed tumor regression to tazemetostat was noted following 1 week of tumor growth. Tazemetostat induced significant reduction of H3K27me3 levels in the majority of tumors compared with controls. CONCLUSIONS: Tazemetostat demonstrated significant antitumor activity in rhabdoid tumor models but showed no consistent activity against any other histology. Tazemetostat reduced H3K27me3 levels irrespective of tumor response. Further preclinical testing to evaluate tazemetostat in combination with other anticancer agents is warranted.


Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Neoplasias Experimentais/tratamento farmacológico , Piridonas/farmacologia , Animais , Compostos de Bifenilo , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Camundongos , Camundongos SCID , Morfolinas , Neoplasias Experimentais/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Biochemistry ; 55(11): 1600-14, 2016 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-26836503

RESUMO

The dynamic structure of histones and DNA, also known as chromatin, is regulated by two classes of enzymes: those that mediate covalent modifications on either histone proteins or DNA and those that use the energy generated by ATP hydrolysis to mechanically alter chromatic structure. Both classes of enzymes are often found in large protein complexes. In this review, we describe two such complexes: polycomb repressive complex 2 (PRC2), with the protein methyltransferase EZH2 as its catalytic subunit, and the ATP-dependent chromatin remodeler switch/sucrose non-fermentable (SWI/SNF). EZH2 catalyzes the methylation of lysine 27 on histone H3, a covalent chromatin modification that is associated with repressed heterochromatin. The catalytic activity of SWI/SNF, in contrast, leads to a state of open chromatin associated with active transcription. In this review, we discuss the biochemical properties of both complexes, outline the principles of their regulation, and describe their opposing roles in normal development, which can be perturbed in disease settings such as cancer.


Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/farmacologia , Neoplasias/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Fatores de Transcrição/metabolismo , Animais , DNA de Neoplasias/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Heterocromatina/metabolismo , Heterocromatina/patologia , Histonas/metabolismo , Humanos , Neoplasias/patologia , Processamento de Proteína Pós-Traducional
5.
Proc Natl Acad Sci U S A ; 110(19): 7922-7, 2013 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-23620515

RESUMO

Inactivation of the switch/sucrose nonfermentable complex component SMARCB1 is extremely prevalent in pediatric malignant rhabdoid tumors (MRTs) or atypical teratoid rhabdoid tumors. This alteration is hypothesized to confer oncogenic dependency on EZH2 in these cancers. We report the discovery of a potent, selective, and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity, (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide). The compound induces apoptosis and differentiation specifically in SMARCB1-deleted MRT cells. Treatment of xenograft-bearing mice with (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide) leads to dose-dependent regression of MRTs with correlative diminution of intratumoral trimethylation levels of lysine 27 on histone H3, and prevention of tumor regrowth after dosing cessation. These data demonstrate the dependency of SMARCB1 mutant MRTs on EZH2 enzymatic activity and portend the utility of EZH2-targeted drugs for the treatment of these genetically defined cancers.


Assuntos
Apoptose , Neoplasias/terapia , Complexo Repressor Polycomb 2/antagonistas & inibidores , Tumor Rabdoide/enzimologia , Tumor Rabdoide/genética , Animais , Antineoplásicos/farmacologia , Compostos de Bifenilo/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Desenho de Fármacos , Proteína Potenciadora do Homólogo 2 de Zeste , Epigênese Genética , Perfilação da Expressão Gênica , Células HEK293 , Histonas/metabolismo , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Piridinas/farmacologia
6.
Nat Chem Biol ; 8(11): 890-6, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23023262

RESUMO

EZH2 catalyzes trimethylation of histone H3 lysine 27 (H3K27). Point mutations of EZH2 at Tyr641 and Ala677 occur in subpopulations of non-Hodgkin's lymphoma, where they drive H3K27 hypertrimethylation. Here we report the discovery of EPZ005687, a potent inhibitor of EZH2 (K(i) of 24 nM). EPZ005687 has greater than 500-fold selectivity against 15 other protein methyltransferases and has 50-fold selectivity against the closely related enzyme EZH1. The compound reduces H3K27 methylation in various lymphoma cells; this translates into apoptotic cell killing in heterozygous Tyr641 or Ala677 mutant cells, with minimal effects on the proliferation of wild-type cells. These data suggest that genetic alteration of EZH2 (for example, mutations at Tyr641 or Ala677) results in a critical dependency on enzymatic activity for proliferation (that is, the equivalent of oncogene addiction), thus portending the clinical use of EZH2 inhibitors for cancers in which EZH2 is genetically altered.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Histonas/metabolismo , Indazóis/farmacologia , Linfoma/tratamento farmacológico , Linfoma/patologia , Complexo Repressor Polycomb 2/antagonistas & inibidores , Piridonas/farmacologia , Antineoplásicos/química , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Proteína Potenciadora do Homólogo 2 de Zeste , Inibidores Enzimáticos/química , Histonas/química , Humanos , Indazóis/química , Linfoma/enzimologia , Linfoma/genética , Lisina/metabolismo , Metilação/efeitos dos fármacos , Estrutura Molecular , Mutação Puntual , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Piridonas/química , Relação Estrutura-Atividade
7.
Cancer Cell ; 7(2): 179-91, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15710330

RESUMO

The SH2-containing tyrosine phosphatase Shp2 (PTPN11) is required for growth factor and cytokine signaling. Germline Shp2 mutations cause Noonan Syndrome (NS), which is associated with increased risk of juvenile myelomonocytic leukemia (JMML). Somatic Shp2 mutations occur in sporadic JMML and other leukemias. We found that Shp2 mutants associated with sporadic leukemias transform murine bone marrow cells, whereas NS mutants are less potent in this assay. Transformation requires multiple domains within Shp2 and the Shp2 binding protein Gab2, and is associated with hyperactivation of the Erk, Akt, and Stat5 pathways. Mutant Shp2-transduced BM causes a fatal JMML-like disorder or, less commonly, lymphoproliferation. Shp2 mutants also cause myeloproliferation in Drosophila. Mek or Tor inhibitors potently inhibit transformation, suggesting new approaches to JMML therapy.


Assuntos
Leucemia/genética , Mutação , Proteínas Tirosina Fosfatases/genética , Alelos , Animais , Animais Geneticamente Modificados , Células da Medula Óssea/citologia , Proliferação de Células , DNA Complementar/metabolismo , Modelos Animais de Doenças , Drosophila , Drosophila melanogaster , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Interleucina-3/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia/metabolismo , Camundongos , Modelos Genéticos , Neoplasias Experimentais , Estrutura Terciária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Retroviridae/genética , Transdução de Sinais , Fatores de Tempo
8.
J Biol Chem ; 286(11): 9225-35, 2011 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-21216966

RESUMO

Protein-tyrosine phosphatase 1B (PTP1B) and T cell protein-tyrosine phosphatase (TCPTP) are closely related intracellular phosphatases implicated in the control of glucose homeostasis. PTP1B and TCPTP can function coordinately to regulate protein tyrosine kinase signaling, and PTP1B has been implicated previously in the regulation of endoplasmic reticulum (ER) stress. In this study, we assessed the roles of PTP1B and TCPTP in regulating ER stress in the endocrine pancreas. PTP1B and TCPTP expression was determined in pancreases from chow and high fat fed mice and the impact of PTP1B and TCPTP over- or underexpression on palmitate- or tunicamycin-induced ER stress signaling assessed in MIN6 insulinoma ß cells. PTP1B expression was increased, and TCPTP expression decreased in pancreases of mice fed a high fat diet, as well as in MIN6 cells treated with palmitate. PTP1B overexpression or TCPTP knockdown in MIN6 cells mitigated palmitate- or tunicamycin-induced PERK/eIF2α ER stress signaling, whereas PTP1B deficiency enhanced ER stress. Moreover, PTP1B deficiency increased ER stress-induced cell death, whereas TCPTP deficiency protected MIN6 cells from ER stress-induced death. ER stress coincided with the inhibition of Src family kinases (SFKs), which was exacerbated by PTP1B overexpression and largely prevented by TCPTP knockdown. Pharmacological inhibition of SFKs ameliorated the protective effect of TCPTP deficiency on ER stress-induced cell death. These results demonstrate that PTP1B and TCPTP play nonredundant roles in modulating ER stress in pancreatic ß cells and suggest that changes in PTP1B and TCPTP expression may serve as an adaptive response for the mitigation of chronic ER stress.


Assuntos
Retículo Endoplasmático/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Células Secretoras de Insulina/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Animais , Morte Celular , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Ácido Palmítico/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos
9.
J Biol Chem ; 286(8): 6433-48, 2011 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-21118801

RESUMO

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.


Assuntos
Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Animais , Domínio Catalítico/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Cães , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil
10.
Immunohorizons ; 6(7): 432-446, 2022 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-35817532

RESUMO

The type 2 cytokines IL-4 and IL-13, which share use of an IL-4 receptor α-chain and its nuclear induction of the transcription factor STAT6, are crucial in elicitation and maintenance of allergic conditions including asthma. STAT6 binds poly(ADP-ribose) polymerase (PARP)14, an ADP-ribosyl monotransferase. Elimination of PARP14 by gene targeting led to attenuation of OVA-specific allergic lung inflammation. However, PARP14 has multiple functional domains apart from the portion that catalyzes ADP-ribosylation, and it is not clear whether inhibition of the catalytic function has any biological consequence. Using BALB/c mice sensitized to the allergen Alternaria alternata, we show that peroral administration of RBN012759, a highly selective inhibitor of ADP-ribosylation by PARP14 with negligible impact on other members of the PARP gene family, achieved biologically active plasma concentrations and altered several responses to the Ag. Specifically, the pharmaceutical compound decreased mucus after allergen challenge, blunted the induced increases in circulating IgE, and prevented suppression of IgG2a. We conclude that PARP14 catalytic activity can contribute to pathogenesis in allergic or atopic processes and propose that other biological endpoints dependent on ADP-ribosylation by PARP14 can be targeted using selective inhibition.


Assuntos
Alérgenos , Asma , Animais , Asma/tratamento farmacológico , Modelos Animais de Doenças , Imunoglobulina E , Camundongos , Muco/metabolismo , Preparações Farmacêuticas/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/uso terapêutico
11.
Cell Chem Biol ; 28(8): 1158-1168.e13, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-33705687

RESUMO

PARP14 has been implicated by genetic knockout studies to promote protumor macrophage polarization and suppress the antitumor inflammatory response due to its role in modulating interleukin-4 (IL-4) and interferon-γ signaling pathways. Here, we describe structure-based design efforts leading to the discovery of a potent and highly selective PARP14 chemical probe. RBN012759 inhibits PARP14 with a biochemical half-maximal inhibitory concentration of 0.003 µM, exhibits >300-fold selectivity over all PARP family members, and its profile enables further study of PARP14 biology and disease association both in vitro and in vivo. Inhibition of PARP14 with RBN012759 reverses IL-4-driven protumor gene expression in macrophages and induces an inflammatory mRNA signature similar to that induced by immune checkpoint inhibitor therapy in primary human tumor explants. These data support an immune suppressive role of PARP14 in tumors and suggest potential utility of PARP14 inhibitors in the treatment of cancer.


Assuntos
Antineoplásicos/farmacologia , Inflamação/tratamento farmacológico , Interleucina-4/antagonistas & inibidores , Neoplasias Renais/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Inflamação/genética , Inflamação/metabolismo , Interleucina-4/genética , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Estrutura Molecular , Poli(ADP-Ribose) Polimerases/genética , Células RAW 264.7 , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
12.
Cancer Cell ; 39(9): 1214-1226.e10, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34375612

RESUMO

PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. Oral dosing of the PARP7 small-molecule inhibitor, RBN-2397, results in complete tumor regression in a lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model, dependent on inducing type I IFN signaling in tumor cells. PARP7 is a therapeutic target whose inhibition induces both cancer cell-autonomous and immune stimulatory effects via enhanced IFN signaling. These data support the targeting of a monoPARP in cancer and introduce a potent and selective PARP7 inhibitor to enter clinical development.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Interferon Tipo I/metabolismo , Neoplasias/tratamento farmacológico , Proteínas de Transporte de Nucleosídeos/genética , Proteínas de Transporte de Nucleosídeos/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Imunidade Adaptativa/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Evasão Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Cell Chem Biol ; 27(7): 877-887.e14, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32679093

RESUMO

Poly(ADP-ribose) polymerase (PARP) enzymes use nicotinamide adenine dinucleotide (NAD+) to modify up to seven different amino acids with a single mono(ADP-ribose) unit (MARylation deposited by PARP monoenzymes) or branched poly(ADP-ribose) polymers (PARylation deposited by PARP polyenzymes). To enable the development of tool compounds for PARP monoenzymes and polyenzymes, we have developed active site probes for use in in vitro and cellular biophysical assays to characterize active site-directed inhibitors that compete for NAD+ binding. These assays are agnostic of the protein substrate for each PARP, overcoming a general lack of knowledge around the substrates for these enzymes. The in vitro assays use less enzyme than previously described activity assays, enabling discrimination of inhibitor potencies in the single-digit nanomolar range, and the cell-based assays can differentiate compounds with sub-nanomolar potencies and measure inhibitor residence time in live cells.


Assuntos
Corantes Fluorescentes/química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Competitiva , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , NAD/química , NAD/metabolismo , Nanopartículas/química , Inibidores de Poli(ADP-Ribose) Polimerases/síntese química , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície
14.
Biochem Pharmacol ; 167: 97-106, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31075269

RESUMO

Poly-ADP-ribose polymerases (PARPs) are a family of enzymes responsible for transferring individual or chains of ADP-ribose subunits to substrate targets as a type of post-translational modification. PARPs regulate a wide variety of important cellular processes, ranging from DNA damage repair to antiviral response. However, most research to date has focused primarily on the polyPARPs, which catalyze the formation of ADP-ribose polymer chains, while the monoPARPs, which transfer individual ADP-ribose monomers, have not been studied as thoroughly. This is partially due to the lack of robust assays to measure mono-ADP-ribosylation in the cell. In this study, the recently developed MAR/PAR antibody has been shown to detect mono-ADP-ribosylation in cells, enabling the field to investigate the function and therapeutic potential of monoPARPs. In this study, the antibody was used in conjunction with engineered cell lines that overexpress various PARPs to establish a panel of assays to evaluate the potencies of literature-reported PARP inhibitors. These assays should be generally applicable to other PARP family members for future compound screening efforts. A convenient and generalizable workflow to identify and validate PARP substrates has been established. As an initial demonstration, aryl hydrocarbon receptor was verified as a direct PARP7 substrate and other novel substrates for this enzyme were also identified and validated. This workflow takes advantage of commercially available detection reagents and conventional mass spectrometry instrumentation and methods. Ultimately, these assays and methods will help drive research in the PARP field and benefit future therapeutics development.


Assuntos
ADP-Ribosilação/fisiologia , Descoberta de Drogas/métodos , Inibidores de Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , ADP-Ribosilação/efeitos dos fármacos , Descoberta de Drogas/tendências , Células HeLa , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/química
15.
J Steroid Biochem Mol Biol ; 109(3-5): 207-11, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18424034

RESUMO

Gene expression studies have been widely used in an effort to identify signatures that can predict clinical progression of cancer. In this study we focused instead on identifying gene expression differences between breast tumors and adjacent normal tissue, and between different subtypes of tumor classified by clinical marker status. We have collected a set of 20 breast cancer tissues, matched with the adjacent pathologically normal tissue from the same patient. The cancer samples representing each subtype of breast cancer identified by estrogen receptor ER(+/-) and Her2(+/-) status and divided into four subgroups (ER+/Her2+, ER+/Her2-, ER-/Her2+, and ER-/Her2-) were hybridized on Affymetrix HG-133 Plus 2.0 microarrays. By comparing cancer samples with their matched normal controls we have identified 3537 overall differentially expressed genes using data analysis methods from Bioconductor. When we looked at the genes in common of the four subgroups, we found 151 regulated genes, some of them encoding known targets for breast cancer treatment. Unique genes in the four subgroups instead suggested gene regulation dependent on the ER/Her2 markers selection. In conclusion, the results indicate that microarray studies using robust analysis of matched tumor and normal samples from the same patients can be used to identify genes differentially expressed in breast cancer tumor subtypes even when small numbers of samples are considered and can further elucidate molecular features of breast cancer.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Saúde , Neoplasias da Mama/classificação , Neoplasias da Mama/patologia , Proliferação de Células , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Receptor ErbB-2/genética , Receptores de Estrogênio/genética
16.
Methods Mol Biol ; 1608: 321-336, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28695519

RESUMO

Cell proliferation assays are an important component of small molecule inhibitor screens for cancer therapies. An important but often overlooked variable involves the timing and timeframe of inhibitor treatment. Whereas many traditional chemotherapeutics kill or inhibit proliferation on the timeframe of hours or in a few days of treatment, more targeted therapies that affect other cancer-relevant pathways, including differentiation or cell stress responses, can take longer, often several days to weeks to impact cellular growth and survival. Many poly(ADP-ribose) polymerases (PARPs) are involved in cellular stress pathways; therefore, phenotypic effects of PARP inhibition are often only observed with long-term inhibitor treatment. Here we summarize several assays for analyzing long-term proliferation of both adherent and suspension cells, relying either on growth in two-dimensional tissue culture or on systems than enable growth in 3D.


Assuntos
Bioensaio/métodos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos
17.
Mol Cancer Ther ; 16(5): 850-860, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28292935

RESUMO

The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival. Mol Cancer Ther; 16(5); 850-60. ©2017 AACR.


Assuntos
Carcinoma de Células Pequenas/tratamento farmacológico , DNA Helicases/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteínas Nucleares/genética , Neoplasias Ovarianas/tratamento farmacológico , Tumor Rabdoide/tratamento farmacológico , Fatores de Transcrição/genética , Animais , Carcinoma de Células Pequenas/diagnóstico , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Diagnóstico Diferencial , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Humanos , Hipercalcemia/diagnóstico , Hipercalcemia/tratamento farmacológico , Hipercalcemia/genética , Hipercalcemia/patologia , Camundongos , Mutação , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Tumor Rabdoide/diagnóstico , Tumor Rabdoide/genética , Tumor Rabdoide/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Mol Cancer Ther ; 16(11): 2586-2597, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28835384

RESUMO

The EZH2 small-molecule inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase II clinical trials for the treatment of non-Hodgkin lymphoma (NHL). We have previously shown that EZH2 inhibitors display an antiproliferative effect in multiple preclinical models of NHL, and that models bearing gain-of-function mutations in EZH2 were consistently more sensitive to EZH2 inhibition than lymphomas with wild-type (WT) EZH2 Here, we demonstrate that cell lines bearing EZH2 mutations show a cytotoxic response, while cell lines with WT-EZH2 show a cytostatic response and only tumor growth inhibition without regression in a xenograft model. Previous work has demonstrated that cotreatment with tazemetostat and glucocorticoid receptor agonists lead to a synergistic antiproliferative effect in both mutant and wild-type backgrounds, which may provide clues to the mechanism of action of EZH2 inhibition in WT-EZH2 models. Multiple agents that inhibit the B-cell receptor pathway (e.g., ibrutinib) were found to have synergistic benefit when combined with tazemetostat in both mutant and WT-EZH2 backgrounds of diffuse large B-cell lymphomas (DLBCL). The relationship between B-cell activation and EZH2 inhibition is consistent with the proposed role of EZH2 in B-cell maturation. To further support this, we observe that cell lines treated with tazemetostat show an increase in the B-cell maturation regulator, PRDM1/BLIMP1, and gene signatures corresponding to more advanced stages of maturation. These findings suggest that EZH2 inhibition in both mutant and wild-type backgrounds leads to increased B-cell maturation and a greater dependence on B-cell activation signaling. Mol Cancer Ther; 16(11); 2586-97. ©2017 AACR.


Assuntos
Benzamidas/administração & dosagem , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Pirazóis/administração & dosagem , Piridonas/administração & dosagem , Pirimidinas/administração & dosagem , Adenina/análogos & derivados , Animais , Linfócitos B/efeitos dos fármacos , Compostos de Bifenilo , Proliferação de Células/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Sinergismo Farmacológico , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Morfolinas , Mutação , Piperidinas , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Cancer Res ; 77(23): 6651-6666, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28978636

RESUMO

Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represents a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers, including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft model that is intrinsically resistant to the RTKi sunitinib, but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its antiangiogenic and antimetastatic activity but lost its direct antitumor effects due to kinome reprogramming, which resulted in suppression of proapoptotic and cell-cycle-regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTKs, restoring the antitumor effects of sunitinib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease. Cancer Res; 77(23); 6651-66. ©2017 AACR.


Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Indóis/farmacologia , Neoplasias Renais/tratamento farmacológico , Pirróis/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Animais , Bevacizumab/farmacologia , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Feminino , Humanos , Neoplasias Renais/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Neovascularização Patológica/tratamento farmacológico , Fosforilação , Receptores Proteína Tirosina Quinases/metabolismo , Sunitinibe , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
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