RESUMO
BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.
Assuntos
Benzodiazepinas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzodiazepinas/química , Benzodiazepinas/toxicidade , Proteínas de Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Análise por Conglomerados , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Cinética , Camundongos , Modelos Moleculares , Conformação Molecular , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Epigenetic mechanisms of gene regulation have a profound role in normal development and disease processes. An integral part of this mechanism occurs through lysine acetylation of histone tails which are recognized by bromodomains. While the biological and structural characterization of many bromodomain containing proteins has advanced considerably, the therapeutic tractability of this protein family is only now becoming understood. This paper describes the discovery and molecular characterization of potent (nM) small molecule inhibitors that disrupt the function of the BET family of bromodomains (Brd2, Brd3, and Brd4). By using a combination of phenotypic screening, chemoproteomics, and biophysical studies, we have discovered that the protein-protein interactions between bromodomains and acetylated histones can be antagonized by selective small molecules that bind at the acetylated lysine recognition pocket. X-ray crystal structures of compounds bound into bromodomains of Brd2 and Brd4 elucidate the molecular interactions of binding and explain the precisely defined stereochemistry required for activity.