RESUMEN
The bromodomain and extra-terminal (BET) family of proteins, consisting of the bromodomains containing protein 2 (BRD2), BRD3, BRD4, and the testis-specific BRDT, are key epigenetic regulators of gene transcription and has emerged as an attractive target for anticancer therapy. Herein, we describe the discovery of a novel potent BET bromodomain inhibitor, using a systematic structure-based approach focused on improving potency, metabolic stability, and permeability. The optimized dimethylisoxazole aryl-benzimidazole inhibitor exhibited high potency towards BRD4 and related BET proteins in biochemical and cell-based assays and inhibited tumor growth in two proof-of-concept preclinical animal models.
Asunto(s)
Bencimidazoles/farmacología , Descubrimiento de Drogas , Isoxazoles/farmacología , Mieloma Múltiple/tratamiento farmacológico , Factores de Transcripción/antagonistas & inhibidores , Administración Oral , Animales , Bencimidazoles/química , Bencimidazoles/metabolismo , Disponibilidad Biológica , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Isoxazoles/administración & dosificación , Isoxazoles/química , Isoxazoles/metabolismo , Ratones , Estructura Molecular , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Dominios Proteicos/efectos de los fármacos , Relación Estructura-Actividad , Factores de Transcripción/metabolismoRESUMEN
Bromodomain and extraterminal domain protein inhibitors (BETi) hold great promise as a novel class of cancer therapeutics. Because acquired resistance typically limits durable responses to targeted therapies, it is important to understand mechanisms by which tumor cells adapt to BETi. Here, through pooled shRNA screening of colorectal cancer cells, we identified tripartite motif-containing protein 33 (TRIM33) as a factor promoting sensitivity to BETi. We demonstrate that loss of TRIM33 reprograms cancer cells to a more resistant state through at least two mechanisms. TRIM33 silencing attenuates down-regulation of MYC in response to BETi. Moreover, loss of TRIM33 enhances TGF-ß receptor expression and signaling, and blocking TGF-ß receptor activity potentiates the antiproliferative effect of BETi. These results describe a mechanism for BETi resistance and suggest that combining inhibition of TGF-ß signaling with BET bromodomain inhibition may offer new therapeutic benefits.
Asunto(s)
Azepinas/farmacología , Proteínas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Triazoles/farmacología , Azepinas/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Resistencia a Medicamentos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Células HEK293 , Humanos , Estructura Molecular , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Interferencia de ARN , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/genética , Triazoles/químicaRESUMEN
Immunoglobulin heavy chain (IgH) variable region exons are assembled from V(H), D and J(H) gene segments in developing B lymphocytes. Within the 2.7-megabase mouse Igh locus, V(D)J recombination is regulated to ensure specific and diverse antibody repertoires. Here we report in mice a key Igh V(D)J recombination regulatory region, termed intergenic control region 1 (IGCR1), which lies between the V(H) and D clusters. Functionally, IGCR1 uses CTCF looping/insulator factor-binding elements and, correspondingly, mediates Igh loops containing distant enhancers. IGCR1 promotes normal B-cell development and balances antibody repertoires by inhibiting transcription and rearrangement of D(H)-proximal V(H) gene segments and promoting rearrangement of distal V(H) segments. IGCR1 maintains ordered and lineage-specific V(H)(D)J(H) recombination by suppressing V(H) joining to D segments not joined to J(H) segments, and V(H) to DJ(H) joins in thymocytes, respectively. IGCR1 is also required for feedback regulation and allelic exclusion of proximal V(H)-to-DJ(H) recombination. Our studies elucidate a long-sought Igh V(D)J recombination control region and indicate a new role for the generally expressed CTCF protein.
Asunto(s)
ADN Intergénico/genética , Reordenamiento Génico de Cadena Pesada de Linfocito B/genética , Recombinación Genética/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas Represoras/metabolismo , Exones VDJ/genética , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Factor de Unión a CCCTC , Linaje de la Célula/genética , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , Elementos de Facilitación Genéticos/genética , Retroalimentación Fisiológica , Células Germinativas/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones , Mutación/genética , Timo/citología , Transcripción Genética/genéticaRESUMEN
Regulated expression of miRNAs influences development in a wide variety of contexts. We report here that miR290-5p (100049710) and miR292-5p (100049711) are induced at the pre-B stage of murine B cell development and that they influence assembly of the Igκ light chain gene (243469) by contributing to the activation of germline Igκ transcription (κGT). We found that upon forced over-expression of miR290-5p/292-5p in Abelson Murine Leukemia Virus (AMuLV) transformed pro-B cells, two known activators of κGT, E2A (21423) and NF-κB (19697), show increased chromosomal binding to the kappa intronic enhancer. Conversely, knockdown of miR290-5p/292-5p in AMuLV pro-B cells blunts drug-induced activation of κGT. Furthermore, miR290-5p/292-5p knockdown also diminishes κGT activation, but not Rag1/2 (19373, 19374) expression, in an IL-7 dependent primary pro-B cell culture system. In addition, we identified a deficiency in κGT induction in miR290 cluster knockout mice. We hypothesize that increased expression of miR290-5p and miR292-5p contributes to the induction of κGT at the pre-B stage of B cell development through increased binding of NF-κB and E2A to kappa locus regulatory sequences.
Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Sitios Genéticos/genética , Cadenas kappa de Inmunoglobulina/genética , MicroARNs/metabolismo , Virus de la Leucemia Murina de Abelson/fisiología , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/virología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Benzamidas , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Transformación Celular Viral/efectos de los fármacos , Transformación Celular Viral/genética , ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Mesilato de Imatinib , Intrones/genética , Ratones , MicroARNs/genética , FN-kappa B/metabolismo , Piperazinas/farmacología , Pirimidinas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genéticaRESUMEN
To what extent might the regulation of translation contribute to differentiation programs, or to the molecular pathogenesis of cancer? Pre-B cells transformed with the viral oncogene v-Abl are suspended in an immortalized, cycling state that mimics leukemias with a BCR-ABL1 translocation, such as Chronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL). Inhibition of the oncogenic Abl kinase with imatinib reverses transformation, allowing progression to the next stage of B cell development. We employed a genome-wide polysome profiling assay called Gradient Encoding to investigate the extent and potential contribution of translational regulation to transformation and differentiation in v-Abl-transformed pre-B cells. Over half of the significantly translationally regulated genes did not change significantly at the level of mRNA abundance, revealing biology that might have been missed by measuring changes in transcript abundance alone. We found extensive, gene-specific changes in translation affecting genes with known roles in B cell signaling and differentiation, cancerous transformation, and cytoskeletal reorganization potentially affecting adhesion. These results highlight a major role for gene-specific translational regulation in remodeling the gene expression program in differentiation and malignant transformation.