RESUMEN
B cells infected by Epstein-Barr virus (EBV), a transforming virus endemic in humans, are rapidly cleared by the immune system, but some cells harboring the virus persist for life. Under conditions of immunosuppression, EBV can spread from these cells and cause life-threatening pathologies. We have generated mice expressing the transforming EBV latent membrane protein 1 (LMP1), mimicking a constitutively active CD40 coreceptor, specifically in B cells. Like human EBV-infected cells, LMP1+ B cells were efficiently eliminated by T cells, and breaking immune surveillance resulted in rapid, fatal lymphoproliferation and lymphomagenesis. The lymphoma cells expressed ligands for a natural killer (NK) cell receptor, NKG2D, and could be targeted by an NKG2D-Fc fusion protein. These experiments indicate a central role for LMP1 in the surveillance and transformation of EBV-infected B cells in vivo, establish a preclinical model for B cell lymphomagenesis in immunosuppressed patients, and validate a new therapeutic approach.
Asunto(s)
Modelos Animales de Enfermedad , Herpesvirus Humano 4 , Vigilancia Inmunológica , Linfoma/inmunología , Linfoma/terapia , Proteínas de la Matriz Viral/metabolismo , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Humanos , Inmunoterapia , Linfoma/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Linfocitos T/inmunología , Linfocitos T/patología , Proteínas de la Matriz Viral/genéticaRESUMEN
The role of autophagy in plasma cells is unknown. Here we found notable autophagic activity in both differentiating and long-lived plasma cells and investigated its function through the use of mice with conditional deficiency in the essential autophagic molecule Atg5 in B cells. Atg5(-/-) differentiating plasma cells had a larger endoplasmic reticulum (ER) and more ER stress signaling than did their wild-type counterparts, which led to higher expression of the transcriptional repressor Blimp-1 and immunoglobulins and more antibody secretion. The enhanced immunoglobulin synthesis was associated with less intracellular ATP and more death of mutant plasma cells, which identified an unsuspected autophagy-dependent cytoprotective trade-off between immunoglobulin synthesis and viability. In vivo, mice with conditional deficiency in Atg5 in B cells had defective antibody responses, complete selection in the bone marrow for plasma cells that escaped Atg5 deletion and fewer antigen-specific long-lived bone marrow plasma cells than did wild-type mice, despite having normal germinal center responses. Thus, autophagy is specifically required for plasma cell homeostasis and long-lived humoral immunity.
Asunto(s)
Autofagia , Linfocitos B/metabolismo , Inmunoglobulinas/biosíntesis , Proteínas Asociadas a Microtúbulos/genética , Células Plasmáticas/inmunología , Adenosina Trifosfato , Animales , Formación de Anticuerpos , Proteína 5 Relacionada con la Autofagia , Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Diferenciación Celular , Retículo Endoplásmico/genética , Estrés del Retículo Endoplásmico/genética , Centro Germinal/inmunología , Homeostasis , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/deficiencia , Células Plasmáticas/citología , Células Plasmáticas/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Factores de Transcripción/biosíntesisRESUMEN
Similar to resting mature B cells, where the B-cell antigen receptor (BCR) controls cellular survival, surface BCR expression is conserved in most mature B-cell lymphomas. The identification of activating BCR mutations and the growth disadvantage upon BCR knockdown of cells of certain lymphoma entities has led to the view that BCR signalling is required for tumour cell survival. Consequently, the BCR signalling machinery has become an established target in the therapy of B-cell malignancies. Here we study the effects of BCR ablation on MYC-driven mouse B-cell lymphomas and compare them with observations in human Burkitt lymphoma. Whereas BCR ablation does not, per se, significantly affect lymphoma growth, BCR-negative (BCR-) tumour cells rapidly disappear in the presence of their BCR-expressing (BCR+) counterparts in vitro and in vivo. This requires neither cellular contact nor factors released by BCR+ tumour cells. Instead, BCR loss induces the rewiring of central carbon metabolism, increasing the sensitivity of receptor-less lymphoma cells to nutrient restriction. The BCR attenuates glycogen synthase kinase 3 beta (GSK3ß) activity to support MYC-controlled gene expression. BCR- tumour cells exhibit increased GSK3ß activity and are rescued from their competitive growth disadvantage by GSK3ß inhibition. BCR- lymphoma variants that restore competitive fitness normalize GSK3ß activity after constitutive activation of the MAPK pathway, commonly through Ras mutations. Similarly, in Burkitt lymphoma, activating RAS mutations may propagate immunoglobulin-crippled tumour cells, which usually represent a minority of the tumour bulk. Thus, while BCR expression enhances lymphoma cell fitness, BCR-targeted therapies may profit from combinations with drugs targeting BCR- tumour cells.
Asunto(s)
Linfocitos B/metabolismo , Genes myc , Aptitud Genética , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Linfoma/genética , Linfoma/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Linfoma de Burkitt/genética , Linfoma de Burkitt/inmunología , Linfoma de Burkitt/patología , Carbono/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Genes ras/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Linfoma/enzimología , Linfoma/patología , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Mutación , Receptores de Antígenos de Linfocitos B/deficiencia , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Células Tumorales CultivadasRESUMEN
Surface expression of a functional B cell antigen receptor (BCR) is essential for the survival and proliferation of mature B cells. Most types of B-cell lymphoproliferative disorders retain surface BCR expression, including B-cell non-Hodgkin lymphomas (B-NHL) and chronic lymphocytic leukemia (CLL). Targeting BCR effectors in B-NHL cell lines in vitro has indicated that this signaling axis is crucial for malignant B cell growth. This has led to the development of inhibitors of BCR signaling, which are currently used for the treatment of CLL and several B-NHL subtypes. Recent studies based on conditional BCR inactivation in a MYC-driven mouse B-cell lymphoma model have revisited the role of the BCR in MYC-expressing tumor B cells. Indeed, lymphoma cells losing BCR expression continue to grow unless subjected to competition with their BCR-expressing counterparts, which causes their elimination. Here, we discuss the molecular nature of the fitness signal delivered by the BCR to MYC-expressing malignant B cells, ensuring their preferential persistence within a rapidly expanding tumor population. We also review growing evidence of Ig-negative cases belonging to several B-NHL subtypes and CLL, and discuss the clinical implications of these findings in relation to an emerging picture of clinical resistances to anti-BCR therapies.
Asunto(s)
Linfocitos B/inmunología , Neoplasias Hematológicas/inmunología , Trastornos Linfoproliferativos/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Microambiente Tumoral/inmunología , Animales , Humanos , Ratones , Fenotipo , Receptores de Antígenos de Linfocitos B/genética , Transducción de SeñalRESUMEN
The mTOR inhibitor everolimus is effective against advanced pancreatic neuroendocrine tumors (pNETs). However, it can cause metabolic adverse events, such as hyperglycemia, hypertriglyceridemia and hypercholesterolemia. In this work we aimed at evaluating the impact of systemic and tumor lipid metabolism on everolimus efficacy. We carried out a monocentric, retrospective study to correlate plasma triglyceride and cholesterol levels with the progression free survival (PFS) of advanced pNET patients treated with everolimus. In formalin fixed, paraffin embedded (FFPE) tumor specimens, we also assessed by mRNA quantification and immunohistochemistry the expression of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FASN), two enzymes crucially involved in fatty acid biosynthesis, and we analyzed their impact on PFS. We evaluated 58 consecutive pNET patients who started everolimus between December 2006 and January 2015. Patients with higher plasma triglycerides during the first 3 months of treatment had an increased risk of disease progression (aHR 3.08, 95% CIs 1.15-8.21; p = 0.025). In 23 FFPE tumor specimens amenable for IHC evaluations, we found a positive correlation between ACC1 and FASN at both mRNA (r = 0.87, p = 0.00045) and protein (r = 0.68, p = 0.0004) level. Patients with higher ACC1 protein expression in metastatic lesions had significantly lower PFS when compared to patients with lower ACC1 levels (5.5 vs. 36 months; aHR 4.49, 95% CIs 1.08-18.72; p = 0.039). In conclusion, systemic and tumor lipid metabolism are associated with the PFS of everolimus-treated patients with advanced pNETs; based on these findings, dietary and pharmacological interventions targeting lipid metabolism could improve everolimus efficacy in this patient population.
Asunto(s)
Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Antineoplásicos/administración & dosificación , Everolimus/administración & dosificación , Tumores Neuroendocrinos/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/farmacología , Colesterol/sangre , Progresión de la Enfermedad , Everolimus/farmacología , Acido Graso Sintasa Tipo I/genética , Acido Graso Sintasa Tipo I/metabolismo , Femenino , Humanos , Metabolismo de los Lípidos , Masculino , Persona de Mediana Edad , Tumores Neuroendocrinos/sangre , Tumores Neuroendocrinos/metabolismo , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/metabolismo , Estudios Retrospectivos , Análisis de Supervivencia , Resultado del Tratamiento , Triglicéridos/sangre , Regulación hacia Arriba , Adulto JovenRESUMEN
PurposeMonoallelic germ-line mutations in the BRCA1/FANCS, BRCA2/FANCD1 and PALB2/FANCN genes confer high risk of breast cancer. Biallelic mutations in these genes cause Fanconi anemia (FA), characterized by malformations, bone marrow failure, chromosome fragility, and cancer predisposition (BRCA2/FANCD1 and PALB2/FANCN), or an FA-like disease presenting a phenotype similar to FA but without bone marrow failure (BRCA1/FANCS). FANCM monoallelic mutations have been reported as moderate risk factors for breast cancer, but there are no reports of any clinical phenotype observed in carriers of biallelic mutations.MethodsBreast cancer probands were subjected to mutation analysis by sequencing gene panels or testing DNA damage response genes.ResultsFive cases homozygous for FANCM loss-of-function mutations were identified. They show a heterogeneous phenotype including cancer predisposition, toxicity to chemotherapy, early menopause, and possibly chromosome fragility. Phenotype severity might correlate with mutation position in the gene.ConclusionOur data indicate that biallelic FANCM mutations do not cause classical FA, providing proof that FANCM is not a canonical FA gene. Moreover, our observations support previous findings suggesting that FANCM is a breast cancer-predisposing gene. Mutation testing of FANCM might be considered for individuals with the above-described clinical features.
Asunto(s)
Alelos , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Fragilidad Cromosómica , ADN Helicasas/genética , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Predisposición Genética a la Enfermedad , Mutación , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Consanguinidad , Resistencia a Antineoplásicos/genética , Femenino , Estudios de Asociación Genética , Genotipo , Mutación de Línea Germinal , Humanos , Masculino , Linaje , Fenotipo , Medición de Riesgo , Factores de RiesgoRESUMEN
The RAG1 and RAG2 proteins are the only lymphoid-specific factors required to perform the first step of V(D)J recombination, DNA cleavage. While the catalytic domain of RAG1, the core region, has been well characterized, the role of the noncore region in modulating chromosomal V(D)J recombination efficiency remains ill defined. Recent studies have highlighted the role of chromatin structure in regulation of V(D)J recombination. Here we show that RAG1 itself, through a RING domain within its N-terminal noncore region, preferentially interacts directly with and promotes monoubiquitylation of histone H3. Mutations affecting the RAG1 RING domain reduce histone H3 monoubiquitylation activity, decrease V(D)J recombination activity in vivo, reduce formation of both signal-joint and coding-joint products on episomal substrates, and decrease efficiency of V(D)J recombination at the endogenous IgH locus in lymphoid cells. The results reveal that RAG1-mediated histone monoubiquitylation activity plays a role in regulating the joining phase of chromosomal V(D)J recombination.
Asunto(s)
Cromatina/metabolismo , Histonas/metabolismo , Proteínas de Homeodominio/fisiología , Dominios RING Finger/fisiología , Sitios de Unión , Línea Celular , Proteínas de Homeodominio/química , Humanos , Mutagénesis Sitio-Dirigida , Recombinación Genética , UbiquitinaciónRESUMEN
In mammals, VDJ recombination is responsible for the establishment of a highly diversified preimmune antibody repertoire. Acquisition of a functional Ig heavy (H) chain variable (V) gene rearrangement is thought to prevent further recombination at the IgH locus. Here, we describe VHQ52(NT); Vκgr32(NT) Ig monoclonal mice reprogrammed from the nucleus of an intestinal IgA(+) plasma cell. In VHQ52(NT) mice, IgA replaced IgM to drive early B-cell development and peripheral B-cell maturation. In VHQ52(NT) animals, over 20% of mature B cells disrupted the single productive, nonautoimmune IgH rearrangement through VH replacement and exchanged it with a highly diversified pool of IgH specificities. VH replacement occurred in early pro-B cells, was independent of pre-B-cell receptor signaling, and involved predominantly one adjacent VH germ-line gene. VH replacement was also identified in 5% of peripheral B cells of mice inheriting a different productive VH rearrangement expressed in the form of an IgM H chain. In summary, editing of a productive IgH rearrangement through VH replacement can account for up to 20% of the IgH repertoire expressed by mature B cells.
Asunto(s)
Clonación de Organismos , Inmunoglobulina A/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Animales , Secuencia de Bases , Ratones , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Transducción de SeñalRESUMEN
Fas is highly expressed in activated and germinal center (GC) B cells but can potentially be inactivated by misguided somatic hypermutation. We employed conditional Fas-deficient mice to investigate the physiological functions of Fas in various B cell subsets. B cell-specific Fas-deficient mice developed fatal lymphoproliferation due to activation of B cells and T cells. Ablation of Fas specifically in GC B cells reproduced the phenotype, indicating that the lymphoproliferation initiates in the GC environment. B cell-specific Fas-deficient mice also showed an accumulation of IgG1(+) memory B cells expressing high amounts of CD80 and the expansion of CD28-expressing CD4(+) Th cells. Blocking T cell-B cell interaction and GC formation completely prevented the fatal lymphoproliferation. Thus, Fas-mediated selection of GC B cells and the resulting memory B cell compartment is essential for maintaining the homeostasis of both T and B lymphocytes.
Asunto(s)
Linfocitos B/inmunología , Centro Germinal/inmunología , Linfocitos T/inmunología , Receptor fas/metabolismo , Animales , Antígenos CD/inmunología , Antígenos CD/metabolismo , Linfocitos B/metabolismo , Antígeno B7-1/inmunología , Antígeno B7-1/metabolismo , Antígenos CD28/inmunología , Antígenos CD28/metabolismo , Antígenos CD40/inmunología , Antígenos CD40/metabolismo , Antígeno CTLA-4 , Comunicación Celular , Diferenciación Celular , Proliferación Celular , Citocinas/sangre , Centro Germinal/metabolismo , Homeostasis , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Linfocitos T/metabolismo , Receptor fas/deficiencia , Receptor fas/inmunologíaRESUMEN
Transcription factor (TF)-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSC) is associated with genome-wide changes in chromatin modifications. Polycomb-mediated histone H3 lysine-27 trimethylation (H3K27me3) has been proposed as a defining mark that distinguishes the somatic from the iPSC epigenome. Here, we dissected the functional role of H3K27me3 in TF-induced reprogramming through the inactivation of the H3K27 methylase EZH2 at the onset of reprogramming. Our results demonstrate that surprisingly the establishment of functional iPSC proceeds despite global loss of H3K27me3. iPSC lacking EZH2 efficiently silenced the somatic transcriptome and differentiated into tissues derived from the three germ layers. Remarkably, the genome-wide analysis of H3K27me3 in Ezh2 mutant iPSC cells revealed the retention of this mark on a highly selected group of Polycomb targets enriched for developmental regulators controlling the expression of lineage specific genes. Erasure of H3K27me3 from these targets led to a striking impairment in TF-induced reprogramming. These results indicate that PRC2-mediated H3K27 trimethylation is required on a highly selective core of Polycomb targets whose repression enables TF-dependent cell reprogramming.
Asunto(s)
Células Madre Pluripotentes Inducidas , Factor 3 de Transcripción de Unión a Octámeros , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Animales , Diferenciación Celular , Proliferación Celular , Metilación de ADN , Proteína Potenciadora del Homólogo Zeste 2 , Fibroblastos/citología , Fibroblastos/metabolismo , Silenciador del Gen , Histonas/genética , Histonas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismoRESUMEN
B-cell development is a multistep process sustained by a highly coordinated transcriptional network under the control of a limited set of transcription factors. Epigenetic mechanisms, including DNA methylation, histone posttranslational modifications and microRNAs act in concert with transcription factors to promote lineage commitment, define and sustain cell identity and establish heritable cell-type- and stage-specific gene expression profiles. Epigenetic modifiers have recently emerged as key regulators of B-cell development and activation. Central to B-cell-mediated immunity are germinal centers, transient structures formed in secondary lymphoid organs where antigen-specific B cells undergo intense proliferation, immunoglobulin somatic hypermutation and isotype switching, to generate ultimately long-lived memory B cells and terminally differentiated plasma cells expressing high-affinity antibodies. Deregulation of one or more epigenetic axes represents a common feature of several B-cell disorders arising from germinal center B cells, including autoimmunity and lymphoma. Moreover, the hijacking of epigenetic determinants is central to the ability of the B-lymphotropic Epstein-Barr virus (EBV) to establish, via the germinal center reaction, life-long latency and occasionally contribute to malignant B-cell transformation. In the light of recent findings, this review will discuss the relevance of epigenetic deregulation in the pathogenesis of B-cell diseases. Understanding how specific epigenetic alterations contribute to the development of lymphomas, autoimmunity and EBV-associated disorders is instrumental to develop novel therapeutic interventions for the cure of these often fatal pathologies.
Asunto(s)
Enfermedades Autoinmunes/genética , Linfocitos B/inmunología , Epigénesis Genética , Infecciones por Virus de Epstein-Barr/genética , Linfoma de Células B/genética , Animales , Enfermedades Autoinmunes/inmunología , Carcinogénesis/genética , Diferenciación Celular , Infecciones por Virus de Epstein-Barr/inmunología , Centro Germinal/virología , Humanos , Cambio de Clase de Inmunoglobulina , Memoria Inmunológica , Linfoma de Células B/inmunología , Hipermutación Somática de InmunoglobulinaRESUMEN
Th17 cells are a proinflammatory subset of effector T cells that have been implicated in the pathogenesis of asthma. Their production of the cytokine IL-17 is known to induce local recruitment of neutrophils, but the direct impact of IL-17 on the lung epithelium is poorly understood. In this study, we describe a novel mouse model of spontaneous IL-17-driven lung inflammation that exhibits many similarities to asthma in humans. We have found that STAT3 hyperactivity in T lymphocytes causes an expansion of Th17 cells, which home preferentially to the lungs. IL-17 secretion then leads to neutrophil infiltration and lung epithelial changes, in turn leading to a chronic inflammatory state with increased mucus production and decreased lung function. We used this model to investigate the effects of IL-17 activity on airway epithelium and identified CXCL5 and MIP-2 as important factors in neutrophil recruitment. The neutralization of IL-17 greatly reduces pulmonary neutrophilia, underscoring a key role for IL-17 in promoting chronic airway inflammation. These findings emphasize the role of IL-17 in mediating neutrophil-driven pulmonary inflammation and highlight a new mouse model that may be used for the development of novel therapies targeting Th17 cells in asthma and other chronic pulmonary diseases.
Asunto(s)
Asma/inmunología , Enfermedades del Sistema Inmune/inmunología , Interleucina-17/inmunología , Trastornos Leucocíticos/inmunología , Neutrófilos/inmunología , Mucosa Respiratoria/inmunología , Animales , Asma/metabolismo , Separación Celular , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Interleucina-17/metabolismo , Ratones , Ratones Endogámicos C57BL , Neumonía/inmunología , Neumonía/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Mucosa Respiratoria/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , TransfecciónRESUMEN
B-cell terminal differentiation into antibody secreting plasma cells (PCs) features a transcriptional shift driven by the activation of plasma cell lineage determinants such as Blimp-1 and Xbp-1, together with the extinction of Pax5. Little is known about the signals inducing this change in transcriptional networks and the role of the B-cell receptor (BCR) in terminal differentiation remains especially controversial. Here, we show that tonic BCR signal strength influences PC commitment in vivo. Using immuno-globulin light chain transgenic mice expressing suboptimal surface BCR levels and latent membrane protein 2A knock-in animals with defined BCR-like signal strengths, we show that weak, antigen-independent constitutive BCR signaling facilitates spontaneous PC differentiation in vivo and in vitro in response to TLR agonists or CD40/IL-4. Conversely, increasing tonic signaling completely prevents this process that is rescued by lowering surface BCR expression or through the inhibition of Syk phosphorylation. These findings provide new insights into the role of basal BCR signaling in PC differentiation and point to the need to resolve a strong BCR signal in order to guarantee terminal differentiation.
Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Diferenciación Celular , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Animales , Diferenciación Celular/inmunología , Ratones , Ratones Noqueados , Células Plasmáticas/citología , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Sindecano-1/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Receptores Toll-Like/metabolismo , Proteínas de la Matriz Viral/metabolismoRESUMEN
The sialic-acid-binding immunoglobulin-like lectin SIGLEC-G is a negative regulator of B-cell receptor-mediated calcium signaling. Its deficiency leads to reduced turnover and increased proliferation and survival of murine B-1a cells. Siglecg(-/-) mice show a premature expansion of polyclonal CD5(+) B cells in the spleen and the peritoneal cavity. Here we studied the fate of B lymphocytes in Siglecg(-/-) mice over time. We demonstrate that in aging animals SIGLEC-G deficiency promotes progressive accumulation of monoclonal B lymphocytes and increases the susceptibility to develop B-cell lymphoproliferative disorders. Lymphoid tumors arising in aged Siglecg(-/-) mice are monoclonal and histologically heterogeneous as they include diffuse large B-cell lymphoma, follicular lymphoma, and medium-to-large B-cell monomorphic lymphoma but surprisingly not chronic lymphocytic leukemia. The tumors express high levels of BCL-2 and are transplantable. In keeping with these findings we have also observed a remarkable down-regulation of the human ortholog SIGLEC10 in human B-cell lymphoma and leukemia cell lines. Taken together, these observations indicate that the down-regulation of negative B-cell receptor regulators such as SIGLEC-G/SIGLEC10 may represent another mechanism relevant to the pathogenesis of B-cell lymphomas.
Asunto(s)
Linfocitos B/metabolismo , Predisposición Genética a la Enfermedad , Lectinas/deficiencia , Leucemia de Células B/metabolismo , Linfoma de Células B/metabolismo , Receptores de Antígenos de Linfocitos B/deficiencia , Animales , Predisposición Genética a la Enfermedad/genética , Humanos , Lectinas/genética , Leucemia de Células B/genética , Leucemia de Células B/patología , Linfoma de Células B/genética , Linfoma de Células B/patología , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/metabolismo , Trastornos Linfoproliferativos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Receptores de Antígenos de Linfocitos B/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido SiálicoRESUMEN
Enhancers are noncoding regulatory DNA regions that modulate the transcription of target genes, often over large distances along with the genomic sequence. Enhancer alterations have been associated with various pathological conditions, including cancer. However, the identification and characterization of somatic mutations in noncoding regulatory regions with a functional effect on tumorigenesis and prognosis remain a major challenge. Here, we present a strategy for detecting and characterizing enhancer mutations in a genome-wide analysis of patient cohorts, across three lung cancer subtypes. Lung tissue-specific enhancers were defined by integrating experimental data and public epigenomic profiles, and the genome-wide enhancer-target gene regulatory network of lung cells was constructed by integrating chromatin three-dimensional architecture data. Lung cancers possessed a similar mutation burden at tissue-specific enhancers and exons but with differences in their mutation signatures. Functionally relevant alterations were prioritized on the basis of the pathway-level integration of the effect of a mutation and the frequency of mutations on individual enhancers. The genes enriched for mutated enhancers converged on the regulation of key biological processes and pathways relevant to tumor biology. Recurrent mutations in individual enhancers also affected the expression of target genes, with potential relevance for patient prognosis. Together, these findings show that noncoding regulatory mutations have a potential relevance for cancer pathogenesis and can be exploited for patient classification. SIGNIFICANCE: Mapping enhancer-target gene regulatory interactions and analyzing enhancer mutations at the level of their target genes and pathways reveal convergence of recurrent enhancer mutations on biological processes involved in tumorigenesis and prognosis.
Asunto(s)
Redes Reguladoras de Genes , Neoplasias Pulmonares , Humanos , Elementos de Facilitación Genéticos/genética , Neoplasias Pulmonares/genética , Mutación , Carcinogénesis/genéticaRESUMEN
Cyclic fasting-mimicking diet (FMD) is an experimental nutritional intervention with potent antitumor activity in preclinical models of solid malignancies. FMD cycles are also safe and active metabolically and immunologically in cancer patients. Here, we reported on the outcome of FMD cycles in two patients with chronic lymphocytic leukemia (CLL) and investigated the effects of fasting and FMD cycles in preclinical CLL models. Fasting-mimicking conditions in murine CLL models had mild cytotoxic effects, which resulted in apoptosis activation mediated in part by lowered insulin and IGF1 concentrations. In CLL cells, fasting conditions promoted an increase in proteasome activity that served as a starvation escape pathway. Pharmacologic inhibition of this escape mechanism with the proteasome inhibitor bortezomib resulted in a strong enhancement of the proapoptotic effects of starvation conditions in vitro. In mouse CLL models, combining cyclic fasting/FMD with bortezomib and rituximab, an anti-CD20 antibody, delayed CLL progression and resulted in significant prolongation of mouse survival. Overall, the effect of proteasome inhibition in combination with FMD cycles in promoting CLL death supports the targeting of starvation escape pathways as an effective treatment strategy that should be tested in clinical trials. SIGNIFICANCE: Chronic lymphocytic leukemia cells resist fasting-mimicking diet by inducing proteasome activation to escape starvation, which can be targeted using proteasome inhibition by bortezomib treatment to impede leukemia progression and prolong survival.
Asunto(s)
Antineoplásicos , Leucemia Linfocítica Crónica de Células B , Humanos , Animales , Ratones , Bortezomib/farmacología , Bortezomib/uso terapéutico , Rituximab/uso terapéutico , Leucemia Linfocítica Crónica de Células B/patología , Complejo de la Endopetidasa Proteasomal , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , AyunoRESUMEN
The germinal center (GC) dark zone (DZ) and light zone (LZ) regions spatially separate expansion and diversification from selection of antigen-specific B-cells to ensure antibody affinity maturation and B cell memory. The DZ and LZ differ significantly in their immune composition despite the lack of a physical barrier, yet the determinants of this polarization are poorly understood. This study provides novel insights into signals controlling asymmetric T-cell distribution between DZ and LZ regions. We identify spatially-resolved DNA damage response and chromatin compaction molecular features that underlie DZ T-cell exclusion. The DZ spatial transcriptional signature linked to T-cell immune evasion clustered aggressive Diffuse Large B-cell Lymphomas (DLBCL) for differential T cell infiltration. We reveal the dependence of the DZ transcriptional core signature on the ATR kinase and dissect its role in restraining inflammatory responses contributing to establishing an immune-repulsive imprint in DLBCL. These insights may guide ATR-focused treatment strategies bolstering immunotherapy in tumors marked by DZ transcriptional and chromatin-associated features.
RESUMEN
Jmjd3, a JmjC family histone demethylase, is induced by the transcription factor NF-kB in response to microbial stimuli. Jmjd3 erases H3K27me3, a histone mark associated with transcriptional repression and involved in lineage determination. However, the specific contribution of Jmjd3 induction and H3K27me3 demethylation to inflammatory gene expression remains unknown. Using chromatin immunoprecipitation-sequencing we found that Jmjd3 is preferentially recruited to transcription start sites characterized by high levels of H3K4me3, a marker of gene activity, and RNA polymerase II (Pol_II). Moreover, 70% of lipopolysaccharide (LPS)-inducible genes were found to be Jmjd3 targets. Although most Jmjd3 target genes were unaffected by its deletion, a few hundred genes, including inducible inflammatory genes, showed moderately impaired Pol_II recruitment and transcription. Importantly, most Jmjd3 target genes were not associated with detectable levels of H3K27me3, and transcriptional effects of Jmjd3 absence in the window of time analysed were uncoupled from measurable effects on this histone mark. These data show that Jmjd3 fine-tunes the transcriptional output of LPS-activated macrophages in an H3K27 demethylation-independent manner.
Asunto(s)
Regulación de la Expresión Génica , Histona Demetilasas con Dominio de Jumonji/metabolismo , Lipopolisacáridos/inmunología , Activación de Macrófagos , Macrófagos/inmunología , Animales , Células Cultivadas , Femenino , Técnicas de Inactivación de Genes , Histona Demetilasas con Dominio de Jumonji/análisis , Histona Demetilasas con Dominio de Jumonji/genética , Macrófagos/metabolismo , RatonesRESUMEN
The telomerase reverse transcriptase elongates telomeres to prevent replicative senescence. This process requires exposure of the 3'-end, which is thought to occur when two sister telomeres are generated at replication completion. Using two-dimensional agarose gel electrophoresis (2D-gels) and electron microscopy, we found that telomeric repeats are hotspots for replication fork reversal. Fork reversal generates 3' telomeric ends before replication completion. To verify whether these ends are elongated by telomerase, we probed de novo telomeric synthesis in situ and at replication intermediates by reconstituting mutant telomerase that adds a variant telomere sequence. We found variant telomeric repeats overlapping with telomeric reversed forks in 2D-gels, but not with normal forks, nontelomeric reversed forks, or telomeric reversed forks with a C-rich 3'-end. Our results define reversed telomeric forks as a substrate of telomerase during replication.
Asunto(s)
Telomerasa , Telomerasa/genética , Telomerasa/metabolismo , Telómero/genética , Telómero/metabolismo , Replicación del ADNRESUMEN
Evaluation of B-cell clonality can be challenging in the interpretation of lymphoid infiltrates on tissue sections. Clonality testing based on IG gene rearrangements analysis by PCR (IG-PCR) is the gold standard. Alternatively, B-cell clonality can be assessed by the recognition of immunoglobulin light chain (IgLC) restriction, by immunohistochemistry (IHC), chromogenic in situ hybridization (ISH) or flow cytometry (FC). IG-PCR requires molecular facilities, and FC requires cell suspensions, both not widely available in routine pathology units. This study evaluates the performance of B-cell clonality detection by IgLC-RNAscope® (RNAsc) in a group of 216 formalin-fixed, paraffin-embedded samples including 185 non-Hodgkin B-cell lymphomas, 11 Hodgkin lymphomas (HL) and 20 reactive samples. IgLC-RNAsc, performed in parallel with FC in 53 cases, demonstrated better performances (93% vs 83%), particularly in diffuse large B-cell lymphoma (98% vs 71%) and follicular lymphoma (93% vs 83%) diagnosis. IgLC-RNAsc was also superior to IHC and ISH especially in samples with limited tumor cell content, where IG-PCR was not informative. Performed for the first time on mediastinal lymphomas, IgLC-RNAsc identified monotypic IgLC transcripts in 69% of primary mediastinal large B-cell lymphoma (PMBCL) and 67% of mediastinal gray zone lymphomas (MGZL). IGK/L double-negative cells were detected in 1 PMBCL, 2 MGZL, and all classical HL, while monotypic IgLC expression appeared to be a hallmark in nodular lymphocyte-predominant HL. IgLC-RNAsc demonstrates to be a powerful tool in B-cell lymphoma diagnosis, above all in challenging cases with limited tumor cell content, ensuring in situ investigations on mechanisms of Ig regulation across lymphoma entities.