RESUMEN
USP7, which encodes a deubiquitylating enzyme, is among the most frequently mutated genes in pediatric T-ALL, with somatic heterozygous loss-of-function mutations (haploinsufficiency) predominantly affecting the subgroup that has aberrant TAL1 oncogene activation. Network analysis of > 200 T-ALL transcriptomes linked USP7 haploinsufficiency with decreased activities of E-proteins. E-proteins are also negatively regulated by TAL1, leading to concerted down-regulation of E-protein target genes involved in T-cell development. In T-ALL cell lines, we showed the physical interaction of USP7 with E-proteins and TAL1 by mass spectrometry and ChIP-seq. Haploinsufficient but not complete CRISPR knock-out of USP7 showed accelerated cell growth and validated transcriptional down-regulation of E-protein targets. Our study unveiled the synergistic effect of USP7 haploinsufficiency with aberrant TAL1 activation on T-ALL, implicating USP7 as a haploinsufficient tumor suppressor in T-ALL. Our findings caution against a universal oncogene designation for USP7 while emphasizing the dosage-dependent consequences of USP7 inhibitors currently under development as potential cancer therapeutics.
Asunto(s)
Oncogenes/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteína 1 de la Leucemia Linfocítica T Aguda/genética , Peptidasa Específica de Ubiquitina 7/genética , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Linaje de la Célula/genética , Proliferación Celular/genética , Regulación Leucémica de la Expresión Génica/genética , Haploinsuficiencia/genética , Humanos , Pediatría , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Activación Transcripcional/genéticaRESUMEN
Infant leukaemia is an embryonal disease in which the underlying MLL translocations initiate in utero. Zebrafish offer unique potential to understand how MLL impacts haematopoiesis from the earliest embryonic timepoints and how translocations cause leukaemia as an embryonal process. In this study, a zebrafish mll cDNA syntenic to human MLL spanning the 5' to 3' UTRs, was cloned from embryos, and mll expression was characterized over the zebrafish lifespan. The protein encoded by the 35-exon ORF exhibited 46·4% overall identity to human MLL and 68-100% conservation in functional domains (AT-hooks, SNL, CXXC, PHD, bromodomain, FYRN, taspase1 sites, FYRC, SET). Maternally supplied transcripts were detected at 0-2 hpf. Strong ubiquitous early zygotic expression progressed to a cephalo-caudal gradient during later embryogenesis. mll was expressed in the intermediate cell mass (ICM) where primitive erythrocytes are produced and in the kidney where definitive haematopoiesis occurs in adults. mll exhibits high cross species conservation, is developmentally regulated in haematopoietic and other tissues and is expressed from the earliest embryonic timepoints throughout the zebrafish lifespan. Haematopoietic tissue expression validates using zebrafish for MLL haematopoiesis and leukaemia models.
Asunto(s)
Sistema Hematopoyético/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Pez Cebra/metabolismo , Envejecimiento/genética , Envejecimiento/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Biología Computacional , ADN Complementario/genética , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis/fisiología , Humanos , Datos de Secuencia Molecular , Proteína de la Leucemia Mieloide-Linfoide/genética , Sistemas de Lectura Abierta , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Especificidad de la Especie , Pez Cebra/genéticaRESUMEN
Neuroblastoma, an embryonal tumour of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer. High-risk neuroblastomas are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germ line. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK complementary DNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed interleukin-3-dependent murine haematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to the small-molecule inhibitor of ALK, TAE684 (ref. 4). Furthermore, two human neuroblastoma cell lines harbouring the F1174L mutation were also sensitive to the inhibitor. Cytotoxicity was associated with increased amounts of apoptosis as measured by TdT-mediated dUTP nick end labelling (TUNEL). Short hairpin RNA (shRNA)-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumours and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.
Asunto(s)
Mutación/genética , Neuroblastoma/genética , Neuroblastoma/terapia , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Alelos , Quinasa de Linfoma Anaplásico , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Activación Enzimática/genética , Genoma Humano/genética , Humanos , Hibridación Fluorescente in Situ , Etiquetado Corte-Fin in Situ , Ratones , Neuroblastoma/enzimología , Neuroblastoma/patología , Polimorfismo de Nucleótido Simple/genética , Estructura Terciaria de Proteína/genética , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras , Análisis de Secuencia de ADNAsunto(s)
Adamantano/análogos & derivados , Proteínas Proto-Oncogénicas/genética , Piridinas/farmacología , Factores de Transcripción/genética , Proteínas de Pez Cebra/genética , Adamantano/química , Adamantano/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Ciclina B/efectos de los fármacos , Ciclina B/genética , Ciclina B1 , Evaluación Preclínica de Medicamentos/métodos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Modelos Animales , Mutación , Fenotipo , Proteínas Proto-Oncogénicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Piridinas/química , Fase S/efectos de los fármacos , Fase S/genética , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/efectos de los fármacos , Proteínas de Pez Cebra/metabolismoRESUMEN
For decades immunologists have relied heavily on the mouse model for their experimental designs. With the realization of the important role innate immunity plays in orchestrating immune responses, invertebrates such as worms and flies have been added to the repertoire. Here, we discuss the advent of the zebrafish as a powerful vertebrate model organism that promises to positively impact immunologic research.