RESUMEN
We report germline loss-of-function mutations in SPRED1 in a newly identified autosomal dominant human disorder. SPRED1 is a member of the SPROUTY/SPRED family of proteins that act as negative regulators of RAS->RAF interaction and mitogen-activated protein kinase (MAPK) signaling. The clinical features of the reported disorder resemble those of neurofibromatosis type 1 and consist of multiple café-au-lait spots, axillary freckling and macrocephaly. Melanocytes from a café-au-lait spot showed, in addition to the germline SPRED1 mutation, an acquired somatic mutation in the wild-type SPRED1 allele, indicating that complete SPRED1 inactivation is needed to generate a café-au-lait spot in this syndrome. This disorder is yet another member of the recently characterized group of phenotypically overlapping syndromes caused by mutations in the genes encoding key components of the RAS-MAPK pathway. To our knowledge, this is the first report of mutations in the SPRY (SPROUTY)/SPRED family of genes in human disease.
Asunto(s)
Mutación de Línea Germinal , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Neurofibromatosis 1/genética , Neurofibromatosis 1/patología , Proteínas Adaptadoras Transductoras de Señales , Adolescente , Adulto , Anciano , Análisis de Varianza , Línea Celular , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Humanos , Immunoblotting , Lactante , Péptidos y Proteínas de Señalización Intracelular/fisiología , Masculino , Proteínas de la Membrana/fisiología , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neurofibromatosis 1/metabolismo , Linaje , Fenotipo , Transducción de Señal/genética , Transducción de Señal/fisiología , Proteínas ras/metabolismoRESUMEN
Fusions of the TET-proteins (TLS/FUS, EWSR1, and TAF15/RBP56) to different transcription factors are involved in various malignancies including Ewing's sarcoma, primitive neuroectodermal tumors, and acute myeloid leukemia. These are thought to arise through transcriptional deregulation, with the transcription factor defining the tumor phenotype. We show that, as result of a t(12;17)(p13;q11) or its variant t(12;22)(p13;q12), the transcription factor gene CIZ/NMP4 is recurrently involved in acute leukemia through fusion with either EWSR1 or TAF15. The fusions possess transforming properties in NIH3T3 cells but do not affect the expression of CIZ target genes, suggesting a contribution to oncogenesis that is independent of the transactivating properties of the fusion protein. These results also extend the involvement of TET-protein fusions to acute lymphoblastic leukemia and suggest a role for CIZ/NMP4 in lymphoid and myeloid development.
Asunto(s)
Leucemia/genética , Proteínas de Fusión Oncogénica/genética , Sarcoma de Ewing/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Transactivadores/genética , Factores de Transcripción/genética , Células 3T3 , Enfermedad Aguda , Secuencia de Aminoácidos , Animales , Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 13/genética , Cromosomas Humanos Par 17/genética , Cromosomas Humanos Par 22/genética , Reordenamiento Génico , Humanos , Ratones , Datos de Secuencia Molecular , Activación Transcripcional , Translocación GenéticaRESUMEN
Mucosa-associated lymphoid tissue 1 (MALT1) controls antigen receptor-mediated signalling to nuclear factor κB (NF-κB) through both its adaptor and protease function. Upon antigen stimulation, MALT1 forms a complex with BCL10 and CARMA1, which is essential for initial IκBα phosphorylation and NF-κB nuclear translocation. Parallel induction of MALT1 protease activity serves to inactivate negative regulators of NF-κB signalling, such as A20 and RELB. Here we demonstrate a key role for auto-proteolytic MALT1 cleavage in B- and T-cell receptor signalling. MALT1 cleavage occurred after Arginine 149, between the N-terminal death domain and the first immunoglobulin-like region, and did not affect its proteolytic activity. Jurkat T cells expressing an un-cleavable MALT1-R149A mutant showed unaltered initial IκBα phosphorylation and normal nuclear accumulation of NF-κB subunits. Nevertheless, MALT1 cleavage was required for optimal activation of NF-κB reporter genes and expression of the NF-κB targets IL-2 and CSF2. Transcriptome analysis confirmed that MALT1 cleavage after R149 was required to induce NF-κB transcriptional activity in Jurkat T cells. Collectively, these data demonstrate that auto-proteolytic MALT1 cleavage controls antigen receptor-induced expression of NF-κB target genes downstream of nuclear NF-κB accumulation.
Asunto(s)
Caspasas/fisiología , Linfocitos/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/fisiología , Transducción de Señal/fisiología , Transcripción Genética/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteína 10 de la LLC-Linfoma de Células B , Secuencia de Bases , Western Blotting , Caspasas/genética , Caspasas/metabolismo , Membrana Celular/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Células Jurkat , Datos de Secuencia Molecular , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Mutación Missense/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteolisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Antígenos/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal/genética , Transcripción Genética/fisiologíaRESUMEN
BACKGROUND: The translocation t(11;18)(q21;q21) is the most frequent chromosomal aberration associated with MALT lymphoma and results in constitutive NF-kappaB activity via the expression of an API2-MALT1 fusion protein. The properties of the reciprocal MALT1-API2 were never investigated as it was reported to be rarely transcribed. PRINCIPAL FINDINGS: Our data indicate the presence of MALT1-API2 transcripts in the majority of t(11;18)(q21;q21)-positive MALT lymphomas. Based on the breakpoints in the MALT1 and API2 gene, the MALT1-API2 protein contains the death domain and one or both immunoglobulin-like domains of MALT1 (approximately 90% of cases)--mediating the possible interaction with BCL10--fused to the RING domain of API2. Here we show that this RING domain enables MALT1-API2 to function as an E3 ubiquitin ligase for BCL10, inducing its ubiquitination and proteasomal degradation in vitro. Expression of MALT1-API2 transcripts in t(11;18)(q21;q21)-positive MALT lymphomas was however not associated with a reduction of BCL10 protein levels. CONCLUSION: As we observed MALT1-API2 to be an efficient target of its own E3 ubiquitin ligase activity, our data suggest that this inherent instability of MALT1-API2 prevents its accumulation and renders a potential effect on MALT lymphoma development via destabilization of BCL10 unlikely.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 18 , Linfoma de Células B de la Zona Marginal/genética , Proteínas de Fusión Oncogénica/metabolismo , Translocación Genética , Proteína 10 de la LLC-Linfoma de Células B , Línea Celular , Núcleo Celular/metabolismo , Humanos , Hidrólisis , Complejo de la Endopetidasa Proteasomal/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , UbiquitinaciónRESUMEN
Molecular cloning of the translocations t(12;22)(p13;q12) and t(12;17)(p13;q11) in acute leukaemia showed that either EWSR1 or its homologue TAF15 are fused to the transcription factor CIZ. EWSR1 and TAF15 belong to the TET family (TLS/FUS, EWSR1 and TAF15) of proteins. TET fusions have been identified in both solid tumours and acute myeloid leukaemia. The novel 12p translocations directly implicated TET fusions in acute lymphoblastic leukaemia as well, and demonstrated the involvement of CIZ in haematopoietic malignancies. In addition, a new fusion E2A-CIZ was recently cloned as a result of a t(12;19)(p13;p13) in a patient with acute lymphoblastic leukaemia. NIH3T3 cells stably expressing TET-CIZ fusions display a transformed phenotype in a focus formation assay. We show here that E2A-CIZ also transforms 3T3 fibroblasts, suggesting that the addition of a transactivation domain to the CIZ protein is involved in this phenotype. An artificial VP16-CIZ construct reveals similar transforming properties, supporting this. We have then analysed the domains within TAF15-CIZ that are necessary for 3T3 fibroblast transformation. Deletion of the zinc fingers of CIZ resulted in loss of both DNA-binding and transforming properties of TAF15-CIZ, whereas deletion of the other functional domains of CIZ had no effect. Fusion of a transactivation domain to CIZ is suggestive for a transactivating function in transformation. Luciferase experiments indeed showed that E2A-CIZ as well as VP16-CIZ transactivates the MMP7 promoter. Taken together, our results reported here suggest that transformation of 3T3 fibroblasts by CIZ fusions is dependent on DNA-binding and might involve transactivation of CIZ target genes.
Asunto(s)
Fusión Artificial Génica , Proteínas Asociadas a Matriz Nuclear/fisiología , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Línea Celular Transformada , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Técnica del Anticuerpo Fluorescente , Eliminación de Gen , Humanos , Ratones , Mutación , Células 3T3 NIH , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Plásmidos , Fracciones Subcelulares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The BCR-ABL1 fusion kinase is frequently associated with chronic myeloid leukemia and B-cell acute lymphoblastic leukemia but is rare in T-cell acute lymphoblastic leukemia (T-ALL). We recently identified NUP214-ABL1 as a variant ABL1 fusion gene in 6% of T-ALL patients. Here we describe the identification of another ABL1 fusion, EML1-ABL1, in a T-ALL patient with a cryptic t(9;14)(q34;q32) associated with deletion of CDKN2A (p16) and expression of TLX1 (HOX11). Echinoderm microtubule-associated protein-like 1-Abelson 1 (EML1-ABL1) is a constitutively phosphorylated tyrosine kinase that transforms Ba/F3 cells to growth factor-independent growth through activation of survival and proliferation pathways, including extracellular signal-related kinase 1/2 (Erk1/2), signal transducers and activators of transcription 5 (Stat5), and Lyn kinase. Deletion of the coiled-coil domain of EML1 abrogated the transforming properties of the fusion kinase. EML1-ABL1 and breakpoint cluster region (BCR)-ABL1 were equally sensitive to the tyrosine kinase inhibitor imatinib. These data further demonstrate the involvement of ABL1 fusions in the pathogenesis of T-ALL and identify EML1-ABL1 as a novel therapeutic target of imatinib.
Asunto(s)
Cromosomas Humanos Par 14 , Cromosomas Humanos Par 9 , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Proteínas de Fusión bcr-abl/química , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia de Células T/patología , Proteínas de Fusión Oncogénica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocación Genética , Adolescente , Secuencia de Bases , Benzamidas , Western Blotting , Línea Celular , Supervivencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , ADN Complementario/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , Eliminación de Gen , Genes abl , Humanos , Mesilato de Imatinib , Hibridación Fluorescente in Situ , Cariotipificación , Microtúbulos/metabolismo , Proteínas de la Leche/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Fenotipo , Fosforilación , Piperazinas/farmacología , Reacción en Cadena de la Polimerasa , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/metabolismo , Pirimidinas/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Retroviridae , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT5 , Transducción de Señal , Factores de Tiempo , Transactivadores/metabolismoRESUMEN
ALK-positive anaplastic large-cell lymphoma (ALCL) has been recognized as a distinct type of lymphoma in the heterogeneous group of T/Null-ALCL. While most of the ALK-positive ALCL (ALKomas) are characterized by the presence of the NPM-ALK fusion protein, the product of the t(2;5)(p23;q35), 10-20% of ALKomas contain variant ALK fusions, including ATIC-ALK, TFG-ALK, CLTC-ALK (previously designated CLTCL-ALK), TMP3-ALK, and MSN-ALK. TMP3-ALK and TMP4-ALK fusions also have been detected in inflammatory myofibroblastic tumors (IMTs), making clear that aberrations of the ALK gene are not associated exclusively with the pathogenesis of ALK-positive ALCL. Here we report results of molecular studies on two lymphoma cases and one IMT case with variant rearrangements of ALK. Our study led to the detection of the CLTC-ALK fusion in an ALCL case and to the identification of two novel fusion partners of ALK: ALO17 (KIAA1618), a gene with unknown function, which was fused to ALK in an ALCL case with a t(2;17)(p23;q25), and CARS, encoding the cysteinyl-tRNA synthetase, which was fused to ALK in an IMT case with a t(2;11;2)(p23;p15;q31). These results confirm the recurrent involvement of ALK in IMT and further demonstrate the diversity of ALK fusion partners, with the ability to homodimerize as a common characteristic.