RESUMEN
Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma with poor long-term overall survival. Currently, MCL research and development of potential cures is hampered by the lack of good in vivo models. MCL is characterized by recurrent translocations of CCND1 or CCND2, resulting in overexpression of the cell cycle regulators cyclin D1 or D2, respectively. Here, we show, for the first time, that hematopoiesis-specific activation of cyclin D2 is sufficient to drive murine MCL-like lymphoma development. Furthermore, we demonstrate that cyclin D2 overexpression can synergize with loss of p53 to form aggressive and transplantable MCL-like lymphomas. Strikingly, cyclin D2-driven lymphomas display transcriptional, immunophenotypic, and functional similarities with B1a B cells. These MCL-like lymphomas have B1a-specific B cell receptors (BCRs), show elevated BCR and NF-κB pathway activation, and display increased MALT1 protease activity. Finally, we provide preclinical evidence that inhibition of MALT1 protease activity, which is essential for the development of early life-derived B1a cells, can be an effective therapeutic strategy to treat MCL.
Asunto(s)
Ciclina D2/genética , Linfoma de Células del Manto/genética , Linfoma de Células del Manto/patología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/antagonistas & inhibidores , Aloinjertos , Animales , Linfocitos B/metabolismo , Linfocitos B/patología , Ciclina D2/metabolismo , Regulación Neoplásica de la Expresión Génica , Linfoma de Células del Manto/tratamiento farmacológico , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Células Neoplásicas Circulantes , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antigen receptor-dependent (AgR-dependent) stimulation of the NF-κB transcription factor in lymphocytes is a required event during adaptive immune response, but dysregulated activation of this signaling pathway can lead to lymphoma. AgR stimulation promotes assembly of the CARMA1-BCL10-MALT1 complex, wherein MALT1 acts as (a) a scaffold to recruit components of the canonical NF-κB machinery and (b) a protease to cleave and inactivate specific substrates, including negative regulators of NF-κB. In multiple lymphoma subtypes, malignant B cells hijack AgR signaling pathways to promote their own growth and survival, and inhibiting MALT1 reduces the viability and growth of these tumors. As such, MALT1 has emerged as a potential pharmaceutical target. Here, we identified G protein-coupled receptor kinase 2 (GRK2) as a new MALT1-interacting protein. We demonstrated that GRK2 binds the death domain of MALT1 and inhibits MALT1 scaffolding and proteolytic activities. We found that lower GRK2 levels in activated B cell-type diffuse large B cell lymphoma (ABC-DLBCL) are associated with reduced survival, and that GRK2 knockdown enhances ABC-DLBCL tumor growth in vitro and in vivo. Together, our findings suggest that GRK2 can function as a tumor suppressor by inhibiting MALT1 and provide a roadmap for developing new strategies to inhibit MALT1-dependent lymphomagenesis.
Asunto(s)
Carcinogénesis/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Linfoma de Células B Grandes Difuso/metabolismo , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Proteínas Oncogénicas/metabolismo , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Femenino , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Humanos , Células Jurkat , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Ratones , Ratones Endogámicos NOD , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Proteínas Oncogénicas/genéticaRESUMEN
MALT1 is a central signaling component in innate and adaptive immunity by regulating NF-κB and other key signaling pathways in different cell types. Activities of MALT1 are mediated by its scaffold and protease functions. Because of its role in lymphocyte activation and proliferation, inhibition of MALT1 proteolytic activity is of high interest for therapeutic targeting in autoimmunity and certain lymphomas. However, recent studies showing that Malt1 protease-dead knock-in (Malt1-PD) mice suffer from autoimmune disease have somewhat tempered the initial enthusiasm. Although it has been proposed that an imbalance between immune suppressive regulatory T cells (Tregs) and activated effector CD4+ T cells plays a key role in the autoimmune phenotype of Malt1-PD mice, the specific contribution of MALT1 proteolytic activity in T cells remains unclear. Using T cell-conditional Malt1 protease-dead knock-in (Malt1-PDT) mice, we here demonstrate that MALT1 has a T cell-intrinsic role in regulating the homeostasis and function of thymic and peripheral T cells. T cell-specific ablation of MALT1 proteolytic activity phenocopies mice in which MALT1 proteolytic activity has been genetically inactivated in all cell types. The Malt1-PDT mice have a reduced number of Tregs in the thymus and periphery, although the effect in the periphery is less pronounced compared to full-body Malt1-PD mice, indicating that also other cell types may promote Treg induction in a MALT1 protease-dependent manner. Despite the difference in peripheral Treg number, both T cell-specific and full-body Malt1-PD mice develop ataxia and multi-organ inflammation to a similar extent. Furthermore, reconstitution of the full-body Malt1-PD mice with T cell-specific expression of wild-type human MALT1 eliminated all signs of autoimmunity. Together, these findings establish an important T cell-intrinsic role of MALT1 proteolytic activity in the suppression of autoimmune responses.
Asunto(s)
Autoinmunidad/inmunología , Activación de Linfocitos/inmunología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/inmunología , Linfocitos T/inmunología , Animales , Autoinmunidad/genética , Homeostasis/genética , Homeostasis/inmunología , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Activación de Linfocitos/genética , Ratones Noqueados , Ratones Transgénicos , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Proteolisis , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Timo/citología , Timo/inmunología , Timo/metabolismoRESUMEN
Mucosa-associated lymphoid tissue 1 (Malt1) regulates immune cell function by mediating the activation of nuclear factor κB (NF-κB) signaling through both its adaptor and proteolytic function. Malt1 is also a target of its own protease activity and this self-cleavage further contributes to NF-κB activity. Until now, the functional distinction between Malt1 self-cleavage and its general protease function in regulating NF-κB signaling and immune activation remained unclear. Here we demonstrate, using a new mouse model, the importance of Malt1 self-cleavage in regulating expression of NF-κB target genes and subsequent T cell activation. Significantly, we further establish that Treg homeostasis is critically linked to Malt1 function via a Treg intrinsic and extrinsic mechanism. TCR-mediated Malt1 proteolytic activity and self-cleavage was found to drive Il2 expression in conventional CD4+ T cells, thereby regulating Il2 availability for Treg homeostasis. Remarkably, the loss of Malt1-mediated self-cleavage alone was sufficient to cause a significant Treg deficit resulting in increased anti-tumor immune reactivity without associated autoimmunity complications. These results establish for the first time that inhibition of MALT1 proteolytic activity could be a viable therapeutic strategy to augment anti-tumor immunity.
Asunto(s)
Activación de Linfocitos , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/inmunología , Neoplasias/inmunología , Linfocitos T Reguladores/inmunología , Animales , Regulación de la Expresión Génica , Homeostasis , Interleucina-2/inmunología , Ratones , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , FN-kappa B/genética , Proteínas de Neoplasias/inmunología , Proteolisis , Transducción de Señal/inmunologíaRESUMEN
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
The genetic background of mature B-cell neoplasms with villous lymphocytes is poorly understood. We identified a novel breakpoint region at 14q32.13 that was rearranged together with IGH/14q32.33 in four cases of BRAF/V600E-negative leukemia/lymphoma with villous lymphocytes carrying either t(14;14)(q32.13;q32.33) (three patients) or del(14)(q32.13q32.33) (one patient). The 14q32.13 breakpoints were mapped by fluorescence in situ hybridization (FISH) in the region harboring the TCL1A/TCL1B/TCL6 genes, known to be affected by TCRA/D-mediated t(14;14)(q11;q32)/inv(14)(q11q32) occurring in T-cell leukemia/lymphoma. To identify the target of t(14;14)(q32.13; q32.33) and del(14)(q32.13q32.33), quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 25 candidate genes located centromerically and telomerically to the 14q32.13 breakpoint was performed. Any of the analyzed genes was commonly overexpressed in the presented cases. Of note, up-regulated transcription of TCL1A was observed in two cases. In summary, we provide evidence that IGH-mediated chromosomal aberrations affecting the 14q32.13/TCL1A-TCL6 region are recurrent in mature B-cell neoplasms with villous lymphocytes. Despite extensive qRT-PCR studies, molecular consequences of these novel aberrations remain elusive.
Asunto(s)
Linfocitos B/metabolismo , Aberraciones Cromosómicas , Cromosomas Humanos Par 14/genética , Proteínas Proto-Oncogénicas/genética , Anciano , Linfocitos B/patología , Puntos de Rotura del Cromosoma , Deleción Cromosómica , Cromosomas Humanos Par 4/genética , Resultado Fatal , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Leucemia Linfocítica Crónica de Células B/genética , Linfoma de Células B/genética , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Translocación GenéticaRESUMEN
Genetic events underlying pathogenesis of nodal and extranodal marginal zone lymphoma are not completely understood. We report here a novel t(X;14)(p11.4;q32.33) identified in 4 lymphoma cases: 2 with a mucosa-associated lymphoid tissue lymphoma, one with a nodal marginal zone lymphoma and one with gastric diffuse large B-cell lymphoma. In all cases, lymphoma evolved from a previous auto-immune disorder. Fluorescence in situ hybridization and molecular studies showed that t(X;14), which is mediated by immunoglobulin heavy chain locus, targets the GPR34 gene at Xp11.4. Upregulation of GPR34 mRNA and aberrant expression of GPR34 protein has been demonstrated in 3 presented cases by quantitative real-time polymerase chain reaction and immunohistochemistry, respectively. GPR34 belongs to the largest family of cell surface molecules involved in signal transmission that play important roles in many physiological and pathological processes, including tumorigenesis. Although functional consequences of t(X;14) have not been identified, our studies suggest that up-regulated GPR34 activate neither nuclear factor-κB nor ELK-related tyrosine kinase.
Asunto(s)
Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 14/genética , Linfoma de Células B de la Zona Marginal/genética , Receptores Lisofosfolípidos/metabolismo , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Linfoma de Células B de la Zona Marginal/metabolismo , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Masculino , Receptores Lisofosfolípidos/genética , Regulación hacia ArribaRESUMEN
The identification of mucosa-associated lymphoid tissue lymphoma translocation 1 (MALT1) as a gene that is perturbed in the B-cell neoplasm MALT lymphoma, already more than a decade ago, was the starting point for an intense area of research. The fascination with MALT1 was fueled further by the observation that it contains a domain homologous to the catalytic domain of caspases and thus, potentially, could function as a protease. Discoveries since then initially revealed that MALT1 is a key adaptor molecule in antigen receptor signaling to the transcription factor NF-κB, which is crucial for lymphocyte function. However, recent discoveries show that this function of MALT1 is not restricted to lymphocytes, witnessed by the ever-increasing list of receptors from cells within and outside of the immune system that require MALT1 for NF-κB activation. Yet, a role for MALT1 protease activity was shown only recently in immune signaling, and its importance was then further strengthened by the dependency of NF-κB-addicted B-cell lymphomas on this proteolytic activity. Therapeutic targeting of MALT1 protease activity might, therefore, become a useful approach for the treatment of these lymphomas and, additionally, an effective strategy for treating other neoplastic and inflammatory disorders associated with deregulated NF-κB signaling.
Asunto(s)
Inhibidores de Caspasas , Caspasas/metabolismo , Linfoma de Células B de la Zona Marginal/enzimología , Linfoma de Células B de la Zona Marginal/terapia , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Animales , Caspasas/genética , Técnicas de Inactivación de Genes , Humanos , Linfoma de Células B de la Zona Marginal/tratamiento farmacológico , Linfoma de Células B de la Zona Marginal/genética , Terapia Molecular Dirigida/métodos , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/genética , Inhibidores de Proteasas/farmacologíaRESUMEN
AIM: To investigate how t(11;18)(q21;q21)-positive gastrointestinal MALT lymphomas relate to other marginal zone lymphomas with respect to the somatic mutation pattern of the V(H) genes and the expression of the marker CD27. METHODS: The V(H) gene of 7 t(11;18)(q21;q21)-positive gastrointestinal MALT lymphomas was amplified by PCR using family specific V(H) primers and a consensus J(H) primer. PCR products were sequenced and mutation analysis of the CDR and the FR regions was performed. All cases were immunostained for CD27. RESULTS: One case showed unmutated V(H) genes while the others showed mutated V(H) genes with mutation frequencies ranging from 1.3 to 14.7% and with evidence of antigen selection in 2 cases. These data suggest that the translocation t(11;18)(q21;q21) can target either B-cells at different stages of differentiation or naive B-cells that retain the capacity to differentiate upon antigen stimulation. All cases but one displayed weak to strong CD27 expression which did not correlate with the V(H) gene mutation status. CONCLUSION: t(11;18)(q21;q21)-positive gastrointestinal MALT lymphomas are heterogeneous with respect to the V(H) mutation status and CD27 is not a marker of somatically mutated B-cells.
RESUMEN
Proper regulation of nuclear factor κB (NF-κB) transcriptional activity is required for normal lymphocyte function, and deregulated NF-κB signaling can facilitate lymphomagenesis. We demonstrate that the API2-MALT1 fusion oncoprotein created by the recurrent t(11;18)(q21;q21) in mucosa-associated lymphoid tissue (MALT) lymphoma induces proteolytic cleavage of NF-κB-inducing kinase (NIK) at arginine 325. NIK cleavage requires the concerted actions of both fusion partners and generates a C-terminal NIK fragment that retains kinase activity and is resistant to proteasomal degradation. The resulting deregulated NIK activity is associated with constitutive noncanonical NF-κB signaling, enhanced B cell adhesion, and apoptosis resistance. Our study reveals the gain-of-function proteolytic activity of a fusion oncoprotein and highlights the importance of the noncanonical NF-κB pathway in B lymphoproliferative disease.
Asunto(s)
Linfocitos B/metabolismo , Linfoma de Células B de la Zona Marginal/metabolismo , FN-kappa B/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Apoptosis , Adhesión Celular , Línea Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Quinasa I-kappa B/metabolismo , Linfoma de Células B de la Zona Marginal/genética , Subunidad p52 de NF-kappa B/metabolismo , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Especificidad por Sustrato , Quinasa de Factor Nuclear kappa BRESUMEN
Among the genetic abnormalities reported to occur in MALT lymphomas, the translocation t(11;18)(q21;q21) is of particular interest because it is exclusively documented in MALT lymphomas, mainly with gastrointestinal location. It results in the creation of a fusion protein API2-MALT1 that activates the transcription factor NF-kappaB through enhanced IKK gamma polyubiquitination. Here, we apply the recently developed molecular technique termed comparative expressed sequence hybridization to identify differentially expressed chromosomal regions related to the pathogenesis of gastric MALT lymphomas. By comparing t(11;18)(q21;q21)-positive gastric MALT lymphomas to their t(11;18)(q21;q21)-negative counterparts, we found that the location of the MALT1 break point determines a difference in expression pattern within the t(11;18)(q21;q21)-positive group. Moreover, we could define a gastric MALT lymphoma signature, which most likely comprises the regions and genes with significance in the development of MALT lymphomas, by comparing both t(11;18)(q21;q21)-positive and -negative MALT lymphomas to normal lymphoid tissue. Finally, a significant imprint of the marginal zone signature, established by comparing microdissected, splenic B follicles with and without marginal zone, was evident in the expression profile of MALT lymphoma, further supporting a marginal zone origin for this type of B-cell non-Hodgkin's lymphoma.
Asunto(s)
Cromosomas Humanos Par 11 , Cromosomas Humanos Par 18 , Linfoma de Células B de la Zona Marginal/genética , Neoplasias Gástricas/genética , Adulto , Anciano , Mapeo Cromosómico , Hibridación Genómica Comparativa , Etiquetas de Secuencia Expresada , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Linfoma de Células B de la Zona Marginal/metabolismo , Linfoma de Células B de la Zona Marginal/patología , Masculino , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Translocación GenéticaRESUMEN
The Carma1-Bcl10-Malt1 signaling module bridges TCR signaling to the canonical IkappaB kinase (IKK)/NF-kappaB pathway. Covalent attachment of regulatory ubiquitin chains to Malt1 paracaspase directs TCR signaling to IKK activation. Further, the ubiquitin-editing enzyme A20 was recently suggested to suppress T cell activation, but molecular targets for A20 remain elusive. In this paper, we show that A20 regulates the strength and duration of the IKK/NF-kappaB response upon TCR/CD28 costimulation. By catalyzing the removal of K63-linked ubiquitin chains from Malt1, A20 prevents sustained interaction between ubiquitinated Malt1 and the IKK complex and thus serves as a negative regulator of inducible IKK activity. Upon T cell stimulation, A20 is rapidly removed and paracaspase activity of Malt1 has been suggested to cleave A20. Using antagonistic peptides or reconstitution of Malt1(-/-) T cells, we show that Malt1 paracaspase activity is required for A20 cleavage and optimal IL-2 production, but dispensable for initial IKK/NF-kappaB signaling in CD4(+) T cells. However, proteasomal inhibition impairs A20 degradation and impedes TCR/CD28-induced IKK activation. Taken together, A20 functions as a Malt1 deubiquitinating enzyme and proteasomal degradation and de novo synthesis of A20 contributes to balance TCR/CD28-induced IKK/NF-kappaB signaling.
Asunto(s)
Caspasas/metabolismo , Regulación hacia Abajo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Ubiquitina/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasas/genética , Línea Celular , Proteínas de Unión al ADN , Activación Enzimática , Humanos , Quinasa I-kappa B/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , ARN Interferente Pequeño/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
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
The paracaspase MALT1 mediates T cell antigen receptor-induced signaling to the transcription factor NF-kappaB and is indispensable for T cell activation and proliferation. Enhanced expression of MALT1 or aberrant expression of a fusion protein of the apoptosis inhibitor API2 and MALT1 has been linked to mucosa-associated lymphoid tissue lymphoma. Despite the presence of a caspase-like domain, MALT1 proteolytic activity has not yet been demonstrated. Here we show that T cell antigen receptor stimulation induced recruitment of the NF-kappaB inhibitor A20 into a complex of MALT1 and the adaptor protein Bcl-10, leading to MALT1-mediated processing of A20. API2-MALT1 expression likewise resulted in cleavage of A20. MALT1 cleaved human A20 after arginine 439 and impaired its NF-kappaB-inhibitory function. Our studies identify A20 as a substrate of MALT1 and emphasize the importance of MALT1 proteolytic activity in the 'fine tuning' of T cell antigen receptor signaling.
Asunto(s)
Caspasas/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , FN-kappa B/antagonistas & inhibidores , Proteínas de Neoplasias/fisiología , Proteínas Nucleares/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Caspasas/genética , Línea Celular , Proteínas de Unión al ADN , Humanos , Immunoblotting , Células Jurkat , Activación de Linfocitos/inmunología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/genética , Péptido Hidrolasas/fisiología , Transducción de Señal/inmunología , Transfección , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
The recurrent translocation t(11;18)(q21;q21) associated with mucosa-associated lymphoid tissue (MALT) lymphoma results in the expression of an API2.MALT1 fusion protein that constitutively activates NF-kappaB. The first baculovirus IAP repeat (BIR) domain of API2 and the C terminus of MALT1, which contains its caspase-like domain, are present in all reported fusion variants and interact with TRAF2 and TRAF6, respectively, suggesting their contribution to NF-kappaB signaling by API2.MALT1. Also, the involvement of BCL10 has been suggested via binding to BIR1 of API2 and via its interaction with the immunoglobulin domains of MALT1, present in half of the fusion variants. However, conflicting reports exist concerning their roles in API2.MALT1-induced NF-kappaB signaling. In this report, streptavidin pulldowns of biotinylated API2.MALT1 fusion variants showed that none of the fusion variants interacted with endogenous BCL10; its role in NF-kappaB signaling by API2.MALT1 was further questioned by RNA interference experiments. In contrast, TRAF6 was essential for NF-kappaB activation by all fusion variants, and we identified a novel TRAF6 binding site in the second immunoglobulin domain of MALT1, which enhanced NF-kappaB activation when present in the fusion protein. Furthermore, inclusion of both immunoglobulin domains in API2.MALT1 further enhanced NF-kappaB signaling via intramolecular TRAF6 activation. Finally, binding of TRAF2 to BIR1 contributed to NF-kappaB activation by API2.MALT1, although additional mechanisms involving BIR1-mediated raft association are also important. Taken together, these data reveal distinct mechanisms of NF-kappaB activation by the different API2.MALT1 fusion variants with an essential role for TRAF6.
Asunto(s)
Caspasas/metabolismo , Linfoma de Células B de la Zona Marginal/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Transducción de Señal , Factor 6 Asociado a Receptor de TNF/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteína 10 de la LLC-Linfoma de Células B , Sitios de Unión/genética , Caspasas/genética , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 18/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Humanos , Células Jurkat , Linfoma de Células B de la Zona Marginal/genética , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , FN-kappa B/genética , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica , Unión Proteica/genética , Estructura Terciaria de Proteína/genética , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor 6 Asociado a Receptor de TNF/genética , Translocación Genética/genéticaRESUMEN
Recently, we described a transgenic mouse model to analyze the effect of the API2-MALT1 fusion-protein in vivo. Our results showed that the expression of API2-MALT1 is not sufficient to induce the development of lymphoma masses. Here, we demonstrate that immunization with Freund's complete adjuvant led to the loss of compartmentalization of the splenic white pulp in API2-MALT1 transgenic mice, resulting in a splenic marginal zone lymphoma-like lymphoid hyperplasia of a peculiar B-cell subset that disappeared as soon as the antigenic stimulation faded away. These data indicate an effect of API2-MALT1 expression on the normal immune response.
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Adyuvante de Freund/inmunología , Linfoma/genética , Linfoma/patología , Proteínas de Fusión Oncogénica/genética , Neoplasias del Bazo/genética , Neoplasias del Bazo/patología , Animales , Adyuvante de Freund/administración & dosificación , Regulación Neoplásica de la Expresión Génica/fisiología , Ratones , Ratones Transgénicos , Proteínas de Fusión Oncogénica/biosíntesisRESUMEN
Enhanced Green Fluorescent Protein (EGFP) is the most commonly used live cell reporter despite a number of conflicting reports that it can affect cell physiology. Thus far, the precise mechanism of GFP-associated defects remained unclear. Here we demonstrate that EGFP and EGFP fusion proteins inhibit polyubiquitination, a posttranslational modification that controls a wide variety of cellular processes, like activation of kinase signalling or protein degradation by the proteasome. As a consequence, the NF-kappaB and JNK signalling pathways are less responsive to activation, and the stability of the p53 tumour suppressor is enhanced in cell lines and in vivo. In view of the emerging role of polyubiquitination in the regulation of numerous cellular processes, the use of EGFP as a live cell reporter should be carefully considered.
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Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Animales , Línea Celular , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lisina/química , Ratones , Ratones Transgénicos , FN-kappa B/metabolismo , Proteínas de Fusión Oncogénica/química , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , UbiquitinaciónRESUMEN
The translocation t(11;18)(q21;q21) that generates an API2-MALT1 fusion protein is the most common structural abnormality among the genetic defects reported in mucosa-associated lymphoid tissue (MALT)-type lymphomas, and its presence correlates with the apparent lack of further genetic instability or chromosomal imbalances. Hence, constitutive nuclear factor-kappaB (NF-kappaB) activation induced by the API2-MALT1 fusion protein is considered essential for B-cell transformation. To examine its role in B-cell development and lymphomagenesis, Emu-API2-MALT1 transgenic mice were produced. Our data show that expression of the API2-MALT1 fusion protein alone is not sufficient for the development of lymphoma masses within 50 weeks. Nevertheless, API2-MALT1 expression affected B-cell maturation in the bone marrow and triggered the specific expansion of splenic marginal zone B cells. Polyubiquitination of IkappaB kinase gamma (IKKgamma), indicative for enhanced NF-kappaB activation, was increased in splenic lymphocytes and promoted the survival of B cells ex vivo. In addition, we show that the API2-MALT1 fusion resided in the cholesterol- and sphingolipid-enriched membrane microdomains, termed lipid rafts. We provide evidence that association of the MALT1 COOH terminal with the lipid rafts, which is mediated by the API2 portion, is sufficient to trigger NF-kappaB activation via enhanced polyubiquitination of IKKgamma. Taken together, these data support the hypothesis that the API2-MALT1 fusion protein can contribute to MALT lymphoma formation via increased NF-kappaB activation.
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Linfocitos B/metabolismo , Quinasa I-kappa B/metabolismo , Linfoma de Células B de la Zona Marginal/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Ubiquitinas/metabolismo , Animales , Linfocitos B/enzimología , Linfocitos B/patología , Supervivencia Celular/fisiología , Femenino , Humanos , Inmunohistoquímica , Linfoma de Células B de la Zona Marginal/genética , Linfoma de Células B de la Zona Marginal/patología , Masculino , Microdominios de Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas de Fusión Oncogénica/biosíntesis , Proteínas de Fusión Oncogénica/genéticaRESUMEN
The pathogenesis of mucosa-associated lymphoid tissue (MALT) lymphomas is associated with independent chromosomal translocations that lead to the upregulation of either BCL10 or MALT1 or the generation of a fusion protein, cIAP2-MALT1. While both BCL10 and MALT1 are critically involved in antigen receptor-mediated NF-kappaB activation, the role of cIAP2 is not clear. Here we show that cIAP2 is a ubiquitin ligase (E3) of BCL10 and targets it for degradation, inhibiting antigen receptor-mediated cytokine production. cIAP2-MALT1 lacks E3 activity, and concomitantly, the BCL10 protein is stabilized in MALT lymphomas harboring this fusion. Furthermore, BCL10 and cIAP2-MALT1 synergistically activate NF-kappaB. These results reveal cIAP2 as an inhibitor of antigenic signaling and implicate its dysfunction in MALT lymphomas.
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Citocinas/inmunología , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Linfoma de Células B de la Zona Marginal/enzimología , Proteínas de Neoplasias/metabolismo , Caspasas , Línea Celular , Fusión Génica , Humanos , Linfoma de Células B de la Zona Marginal/genética , Linfoma de Células B de la Zona Marginal/metabolismo , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/genética , Proteínas Recombinantes de Fusión/metabolismo , Translocación GenéticaRESUMEN
Recurrent chromosome 12p deletions are associated with distinct tumor types and suggest the presence of a tumor suppressor gene (TSG). Previously, we mapped an EST with similarity to a protein tyrosine phosphatase to the minimally deleted region for all these neoplasms. The corresponding gene, DUSP16/MKP-7, was recently shown to code for a mitogen-activated protein kinase phosphatase, suggestive for a function as tumor suppressor. Overexpression of DUSP16 in BCR-ABL-transformed Rat-1 fibroblasts reduces their transforming capacity in vitro and in vivo via downregulation of BCR-ABL-induced JNK activation. A role for DUSP16 as a regulator of JNK signaling was further demonstrated via overexpression in Ba/F3 cells, which increased their antiapoptosis. However, no inactivating mutations could be detected in leukemia patients hemizygous for DUSP16, and the effect of hemizygosity on DUSP16 expression level could not be assessed due to the variability of DUSP16 transcript levels observed in leukaemia cell lines and in patients. Taken together, the functional data point to a context-dependent role for DUSP16 on cell transformation and apoptosis, reflecting the dual role of JNK, and therefore suggest that DUSP16 might be haploinsufficient for tumor suppression.