RESUMEN
DICER1 plays a critical role in microRNA (miRNA) biogenesis. Recurrent somatic 'hotspot' mutations at the four metal-binding sites within the RNase IIIb domain of DICER1 were identified in ovarian sex cord-stromal tumours and have since been described in other paediatric tumours. In this study, we screened the RNase IIIb domain of DICER1 in 290 endometrial tumours and identified six cases with hotspot mutations, including two cases affected by an atypical G1809R mutation directly adjacent to a metal-binding site. Using Illumina and Sanger targeted resequencing, we observed and validated biallelic DICER1 mutations in several cases with hotspot mutations. Through in vitro DICER1 cleavage assays, small RNA deep sequencing and real-time PCR, we demonstrated that mutations adding a positively charged side chain to residue 1809 have similar detrimental effects on 5p miRNA production to mutations at the metal-binding sites. As expected, 5p miRNAs were globally reduced in tumours and cell lines with hotspot mutations. Pathway analysis of gene expression profiles indicated that genes de-repressed due to loss of 5p miRNAs are strongly associated with pathways regulating the cell cycle. Using a Dicer1-null mouse cell line model, we found that expression of DICER1 hotspot mutants promoted cell proliferation, whereas wild-type (WT) DICER1 inhibited cell proliferation. Furthermore, targets of let-7 family miRNAs are enriched among the up-regulated genes, suggesting that loss of let-7 may be impacting downstream pathways. Our results reveal that DICER1 hotspot mutations are implicated in common malignancies and may constitute a unique oncogenic pathway.
Asunto(s)
ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Neoplasias Endometriales/enzimología , Neoplasias Endometriales/genética , MicroARNs/biosíntesis , Mutación , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Transcripción Genética , Animales , Línea Celular Tumoral , Proliferación Celular , ARN Helicasas DEAD-box/deficiencia , Análisis Mutacional de ADN , Neoplasias Endometriales/patología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones Noqueados , MicroARNs/genética , Fenotipo , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Ribonucleasa III/deficiencia , Factores de Tiempo , TransfecciónRESUMEN
ABL tyrosine kinase inhibitor (TKI) therapy induces clinical remission in chronic myeloid leukemia (CML) patients but early relapses and later emergence of TKI-resistant disease remain problematic. We recently demonstrated that the AHI-1 oncogene physically interacts with BCR-ABL and JAK2 and mediates cellular resistance to TKI in CML stem/progenitor cells. We now show that deletion of the SH3 domain of AHI-1 significantly enhances apoptotic response of BCR-ABL(+) cells to TKIs compared to cells expressing full-length AHI-1. We have also discovered a novel interaction between AHI-1 and Dynamin-2, a GTPase, through the AHI-1 SH3 domain. The crystal structure of the AHI-1 SH3 domain at 1.53-Å resolution reveals that it adopts canonical SH3 folding, with the exception of an unusual C-terminal α helix. PD1R peptide, known to interact with the PI3K SH3 domain, was used to model the binding pattern between the AHI-1 SH3 domain and its ligands. These studies showed that an "Arg-Arg-Trp" stack may form within the binding interface, providing a potential target site for designing specific drugs. The crystal structure of the AHI-1 SH3 domain thus provides a valuable tool for identification of key interaction sites in regulation of drug resistance and for the development of small molecule inhibitors for CML.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Dominios Homologos src , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras del Transporte Vesicular , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Dinamina II/metabolismo , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Ligandos , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Alineación de Secuencia , Eliminación de SecuenciaRESUMEN
Myocyte enhancer factor 2B (MEF2B) is a transcription factor with mutation hotspots at K4, Y69 and D83 in diffuse large B-cell lymphoma (DLBCL). To provide insight into the regulatory network of MEF2B, in this study, we analyse global gene expression and DNA-binding patterns. We find that candidate MEF2B direct target genes include RHOB, RHOD, CDH13, ITGA5 and CAV1, and that indirect target genes of MEF2B include MYC, TGFB1, CARD11, MEF2C, NDRG1 and FN1. MEF2B overexpression increases HEK293A cell migration and epithelial-mesenchymal transition, and decreases DLBCL cell chemotaxis. K4E, Y69H and D83V MEF2B mutations decrease the capacity of MEF2B to activate transcription and decrease its' effects on cell migration. The K4E and D83V mutations decrease MEF2B DNA binding. In conclusion, our map of the MEF2B regulome connects MEF2B to drivers of oncogenesis.