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1.
Annu Rev Immunol ; 30: 565-610, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22224767

RESUMO

The mechanisms that drive normal B cell differentiation and activation are frequently subverted by B cell lymphomas for their unlimited growth and survival. B cells are particularly prone to malignant transformation because the machinery used for antibody diversification can cause chromosomal translocations and oncogenic mutations. The advent of functional and structural genomics has greatly accelerated our understanding of oncogenic mechanisms in lymphomagenesis. The signaling pathways that normal B cells utilize to sense antigens are frequently derailed in B cell malignancies, leading to constitutive activation of prosurvival pathways. These malignancies co-opt transcriptional regulatory systems that characterize their normal B cell counterparts and frequently alter epigenetic regulators of chromatin structure and gene expression. These mechanistic insights are ushering in an era of targeted therapies for these cancers based on the principles of pathogenesis.


Assuntos
Linfoma de Células B/etiologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Epigênese Genética , Humanos , Evasão da Resposta Imune , Linfoma de Células B/tratamento farmacológico , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
2.
Nature ; 560(7718): 387-391, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29925955

RESUMO

B cell receptor (BCR) signalling has emerged as a therapeutic target in B cell lymphomas, but inhibiting this pathway in diffuse large B cell lymphoma (DLBCL) has benefited only a subset of patients1. Gene expression profiling identified two major subtypes of DLBCL, known as germinal centre B cell-like and activated B cell-like (ABC)2,3, that show poor outcomes after immunochemotherapy in ABC. Autoantigens drive BCR-dependent activation of NF-κB in ABC DLBCL through a kinase signalling cascade of SYK, BTK and PKCß to promote the assembly of the CARD11-BCL10-MALT1 adaptor complex, which recruits and activates IκB kinase4-6. Genome sequencing revealed gain-of-function mutations that target the CD79A and CD79B BCR subunits and the Toll-like receptor signalling adaptor MYD885,7, with MYD88(L265P) being the most prevalent isoform. In a clinical trial, the BTK inhibitor ibrutinib produced responses in 37% of cases of ABC1. The most striking response rate (80%) was observed in tumours with both CD79B and MYD88(L265P) mutations, but how these mutations cooperate to promote dependence on BCR signalling remains unclear. Here we used genome-wide CRISPR-Cas9 screening and functional proteomics to determine the molecular basis of exceptional clinical responses to ibrutinib. We discovered a new mode of oncogenic BCR signalling in ibrutinib-responsive cell lines and biopsies, coordinated by a multiprotein supercomplex formed by MYD88, TLR9 and the BCR (hereafter termed the My-T-BCR supercomplex). The My-T-BCR supercomplex co-localizes with mTOR on endolysosomes, where it drives pro-survival NF-κB and mTOR signalling. Inhibitors of BCR and mTOR signalling cooperatively decreased the formation and function of the My-T-BCR supercomplex, providing mechanistic insight into their synergistic toxicity for My-T-BCR+ DLBCL cells. My-T-BCR supercomplexes characterized ibrutinib-responsive malignancies and distinguished ibrutinib responders from non-responders. Our data provide a framework for the rational design of oncogenic signalling inhibitors in molecularly defined subsets of DLBCL.


Assuntos
Carcinogênese , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Complexos Multiproteicos/metabolismo , Transdução de Sinais , Adenina/análogos & derivados , Animais , Biópsia , Sistemas CRISPR-Cas/genética , Carcinogênese/genética , Desenho de Fármacos , Feminino , Humanos , Linfoma Difuso de Grandes Células B/genética , Camundongos , Complexos Multiproteicos/química , Mutação , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Piperidinas , Proteômica , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Receptores de Antígenos de Linfócitos B/antagonistas & inibidores , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Proc Natl Acad Sci U S A ; 117(11): 6092-6102, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32127472

RESUMO

The KLHL14 gene acquires frequent inactivating mutations in mature B cell malignancies, especially in the MYD88L265P, CD79B mutant (MCD) genetic subtype of diffuse large B cell lymphoma (DLBCL), which relies on B cell receptor (BCR) signaling for survival. However, the pathogenic role of KLHL14 in DLBCL and its molecular function are largely unknown. Here, we report that KLHL14 is in close proximity to the BCR in the endoplasmic reticulum of MCD cell line models and promotes the turnover of immature glycoforms of BCR subunits, reducing total cellular BCR levels. Loss of KLHL14 confers relative resistance to the Bruton tyrosine kinase (BTK) inhibitor ibrutinib and promotes assembly of the MYD88-TLR9-BCR (My-T-BCR) supercomplex, which initiates prosurvival NF-κB activation. Consequently, KLHL14 inactivation allows MCD cells to maintain NF-κB signaling in the presence of ibrutinib. These findings reinforce the central role of My-T-BCR-dependent NF-κB signaling in MCD DLBCL and suggest that the genetic status of KLHL14 should be considered in clinical trials testing inhibitors of BTK and BCR signaling mediators in DLBCL.


Assuntos
Proteínas de Transporte/genética , Genes Supressores de Tumor , Linfoma Difuso de Grandes Células B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Adenina/análogos & derivados , Antígenos CD79/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Linfoma Difuso de Grandes Células B/patologia , Mutagênese Sítio-Dirigida , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Piperidinas , Proteólise , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
4.
Proc Natl Acad Sci U S A ; 117(42): 26318-26327, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33020271

RESUMO

Epstein-Barr virus (EBV) infects human B cells and reprograms them to allow virus replication and persistence. One key viral factor in this process is latent membrane protein 2A (LMP2A), which has been described as a B cell receptor (BCR) mimic promoting malignant transformation. However, how LMP2A signaling contributes to tumorigenesis remains elusive. By comparing LMP2A and BCR signaling in primary human B cells using phosphoproteomics and transcriptome profiling, we identified molecular mechanisms through which LMP2A affects B cell biology. Consistent with the literature, we found that LMP2A mimics a subset of BCR signaling events, including tyrosine phosphorylation of the kinase SYK, the calcium initiation complex consisting of BLNK, BTK, and PLCγ2, and its downstream transcription factor NFAT. However, the majority of LMP2A-induced signaling events markedly differed from those induced by BCR stimulation. These included differential phosphorylation of kinases, phosphatases, adaptor proteins, transcription factors such as nuclear factor κB (NF-κB) and TCF3, as well as widespread changes in the transcriptional output of LMP2A-expressing B cells. LMP2A affected apoptosis and cell-cycle checkpoints by dysregulating the expression of apoptosis regulators such as BCl-xL and the tumor suppressor retinoblastoma-associated protein 1 (RB1). LMP2A cooperated with MYC and mutant cyclin D3, two oncogenic drivers of Burkitt lymphoma, to promote proliferation and survival of primary human B cells by counteracting MYC-induced apoptosis and by inhibiting RB1 function, thereby promoting cell-cycle progression. Our results indicate that LMP2A is not a pure BCR mimic but rather rewires intracellular signaling in EBV-infected B cells that optimizes cell survival and proliferation, setting the stage for oncogenic transformation.


Assuntos
Herpesvirus Humano 4/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Proteínas da Matriz Viral/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/fisiologia , Linfócitos B/metabolismo , Humanos , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Fosforilação , Transdução de Sinais , Quinase Syk/metabolismo
5.
N Engl J Med ; 378(15): 1396-1407, 2018 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-29641966

RESUMO

BACKGROUND: Diffuse large B-cell lymphomas (DLBCLs) are phenotypically and genetically heterogeneous. Gene-expression profiling has identified subgroups of DLBCL (activated B-cell-like [ABC], germinal-center B-cell-like [GCB], and unclassified) according to cell of origin that are associated with a differential response to chemotherapy and targeted agents. We sought to extend these findings by identifying genetic subtypes of DLBCL based on shared genomic abnormalities and to uncover therapeutic vulnerabilities based on tumor genetics. METHODS: We studied 574 DLBCL biopsy samples using exome and transcriptome sequencing, array-based DNA copy-number analysis, and targeted amplicon resequencing of 372 genes to identify genes with recurrent aberrations. We developed and implemented an algorithm to discover genetic subtypes based on the co-occurrence of genetic alterations. RESULTS: We identified four prominent genetic subtypes in DLBCL, termed MCD (based on the co-occurrence of MYD88L265P and CD79B mutations), BN2 (based on BCL6 fusions and NOTCH2 mutations), N1 (based on NOTCH1 mutations), and EZB (based on EZH2 mutations and BCL2 translocations). Genetic aberrations in multiple genes distinguished each genetic subtype from other DLBCLs. These subtypes differed phenotypically, as judged by differences in gene-expression signatures and responses to immunochemotherapy, with favorable survival in the BN2 and EZB subtypes and inferior outcomes in the MCD and N1 subtypes. Analysis of genetic pathways suggested that MCD and BN2 DLBCLs rely on "chronic active" B-cell receptor signaling that is amenable to therapeutic inhibition. CONCLUSIONS: We uncovered genetic subtypes of DLBCL with distinct genotypic, epigenetic, and clinical characteristics, providing a potential nosology for precision-medicine strategies in DLBCL. (Funded by the Intramural Research Program of the National Institutes of Health and others.).


Assuntos
Perfilação da Expressão Gênica , Heterogeneidade Genética , Linfoma Difuso de Grandes Células B/genética , Mutação , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biópsia , Epigênese Genética , Exoma , Genótipo , Humanos , Estimativa de Kaplan-Meier , Linfoma Difuso de Grandes Células B/classificação , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/mortalidade , Prognóstico , Análise de Sequência de DNA , Transcriptoma
6.
Immunity ; 36(4): 668-79, 2012 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-22464170

RESUMO

T helper 17 (Th17) cells play an important role in mucosal host defense through production of the signature cytokines IL-17 and IL-22. Prostaglandin E2 (PGE2) has been shown to enhance IL-17 production by mature Th17 cells. However, when present during Th17 cell differentiation, we found that PGE2 inhibited the transcription factor IRF4 and suppressed production of IL-17 but not IL-22. We show that IRF4 was required for IL-17 expression but inhibited IL-22 expression, highlighting the potential for discordant regulation of these two cytokines in Th17 cells. The pathogenic fungus Cryptococcus neoformans produces PGE2, and we found that it uses PGE2- and IRF4-dependent mechanisms to specifically inhibit induction of IL-17 during Th17 cell differentiation. Blockade of host PGE2 during infection led to increased IL-17 production from CD4(+) T cells and increased survival of mice. These findings suggest that host- or pathogen-derived PGE2 can act directly on Th17 cells during differentiation to inhibit IL-17-dependent antimicrobial responses.


Assuntos
Cryptococcus neoformans/metabolismo , Dinoprostona/metabolismo , Fatores Reguladores de Interferon/antagonistas & inibidores , Interleucina-17/biossíntese , Células Th17/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Criptococose/imunologia , Cryptococcus neoformans/patogenicidade , Fatores Reguladores de Interferon/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucinas/biossíntese , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Células Th17/metabolismo , Interleucina 22
8.
Proc Natl Acad Sci U S A ; 113(5): E577-86, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26787899

RESUMO

High expression of the forkhead box P1 (FOXP1) transcription factor distinguishes the aggressive activated B cell (ABC) diffuse large B-cell lymphoma (DLBCL) subtype from the better prognosis germinal center B-cell (GCB)-DLBCL subtype and is highly correlated with poor outcomes. A genetic or functional role for FOXP1 in lymphomagenesis, however, remains unknown. Here, we report that sustained FOXP1 expression is vital for ABC-DLBCL cell-line survival. Genome-wide analyses revealed direct and indirect FOXP1 transcriptional enforcement of ABC-DLBCL hallmarks, including the classical NF-κB and MYD88 (myeloid differentiation primary response gene 88) pathways. FOXP1 promoted gene expression underlying transition of the GCB cell to the plasmablast--the transient B-cell stage targeted in ABC-DLBCL transformation--by antagonizing pathways distinctive of GCB-DLBCL, including that of the GCB "master regulator," BCL6 (B-cell lymphoma 6). Cell-line derived FOXP1 target genes that were highly correlated with FOXP1 expression in primary DLBCL accurately segregated the corresponding clinical subtypes of a large cohort of primary DLBCL isolates and identified conserved pathways associated with ABC-DLBCL pathology.


Assuntos
Linfócitos B/imunologia , Fatores de Transcrição Forkhead/fisiologia , Linfoma Difuso de Grandes Células B/imunologia , Proteínas Repressoras/fisiologia , Diferenciação Celular , Linhagem Celular Tumoral , Humanos , Ativação Linfocitária , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Transcrição Gênica
9.
Proc Natl Acad Sci U S A ; 113(46): E7260-E7267, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27799566

RESUMO

Janus kinases (JAKs) classically signal by activating STAT transcription factors but can also regulate gene expression by epigenetically phosphorylating histone H3 on tyrosine 41 (H3Y41-P). In diffuse large B-cell lymphomas (DLBCLs), JAK signaling is a feature of the activated B-cell (ABC) subtype and is triggered by autocrine production of IL-6 and IL-10. Whether this signaling involves STAT activation, epigenetic modification of chromatin, or both mechanisms is unknown. Here we use genetic and pharmacological inhibition to show that JAK1 signaling sustains the survival of ABC DLBCL cells. Whereas STAT3 contributed to the survival of ABC DLBCL cell lines, forced STAT3 activity could not protect these cells from death following JAK1 inhibition, suggesting epigenetic JAK1 action. JAK1 regulated the expression of nearly 3,000 genes in ABC DLBCL cells, and the chromatin surrounding many of these genes was modified by H3Y41-P marks that were diminished by JAK1 inhibition. These JAK1 epigenetic target genes encode important regulators of ABC DLBCL proliferation and survival, including IRF4, MYD88, and MYC. A small molecule JAK1 inhibitor cooperated with the BTK inhibitor ibrutinib in reducing IRF4 levels and acted synergistically to kill ABC DLBCL cells, suggesting that this combination should be evaluated in clinical trials.


Assuntos
Janus Quinase 1/genética , Linfoma Difuso de Grandes Células B/genética , Apoptose , Linhagem Celular Tumoral , Epigênese Genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Janus Quinase 1/antagonistas & inibidores , Fator de Transcrição STAT3/genética
10.
Proc Natl Acad Sci U S A ; 113(14): E2039-46, 2016 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-26993806

RESUMO

The requirement for the B-cell transcription factor OCT2 (octamer-binding protein 2, encoded by Pou2f2) in germinal center B cells has proved controversial. Here, we report that germinal center B cells are formed normally after depletion of OCT2 in a conditional knockout mouse, but their proliferation is reduced and in vivo differentiation to antibody-secreting plasma cells is blocked. This finding led us to examine the role of OCT2 in germinal center-derived lymphomas. shRNA knockdown showed that almost all diffuse large B-cell lymphoma (DLBCL) cell lines are addicted to the expression of OCT2 and its coactivator OCA-B. Genome-wide chromatin immunoprecipitation (ChIP) analysis and gene-expression profiling revealed the broad transcriptional program regulated by OCT2 that includes the expression of STAT3, IL-10, ELL2, XBP1, MYC, TERT, and ADA. Importantly, genetic alteration of OCT2 is not a requirement for cellular addiction in DLBCL. However, we detected amplifications of the POU2F2 locus in DLBCL tumor biopsies and a recurrent mutation of threonine 223 in the DNA-binding domain of OCT2. This neomorphic mutation subtly alters the DNA-binding preference of OCT2, leading to the transactivation of noncanonical target genes including HIF1a and FCRL3 Finally, by introducing mutations designed to disrupt the OCT2-OCA-B interface, we reveal a requirement for this protein-protein interface that ultimately might be exploited therapeutically. Our findings, combined with the predominantly B-cell-restricted expression of OCT2 and the absence of a systemic phenotype in our knockout mice, suggest that an OCT2-targeted therapeutic strategy would be efficacious in both major subtypes of DLBCL while avoiding systemic toxicity.


Assuntos
Linfócitos B/citologia , Diferenciação Celular , Sobrevivência Celular , Linfoma Difuso de Grandes Células B/patologia , Proteínas de Transporte de Cátions Orgânicos/fisiologia , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos Knockout , Proteínas de Transporte de Cátions Orgânicos/genética , Transportador 2 de Cátion Orgânico
11.
Nature ; 490(7418): 116-20, 2012 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-22885699

RESUMO

Burkitt's lymphoma (BL) can often be cured by intensive chemotherapy, but the toxicity of such therapy precludes its use in the elderly and in patients with endemic BL in developing countries, necessitating new strategies. The normal germinal centre B cell is the presumed cell of origin for both BL and diffuse large B-cell lymphoma (DLBCL), yet gene expression analysis suggests that these malignancies may use different oncogenic pathways. BL is subdivided into a sporadic subtype that is diagnosed in developed countries, the Epstein-Barr-virus-associated endemic subtype, and an HIV-associated subtype, but it is unclear whether these subtypes use similar or divergent oncogenic mechanisms. Here we used high-throughput RNA sequencing and RNA interference screening to discover essential regulatory pathways in BL that cooperate with MYC, the defining oncogene of this cancer. In 70% of sporadic BL cases, mutations affecting the transcription factor TCF3 (E2A) or its negative regulator ID3 fostered TCF3 dependency. TCF3 activated the pro-survival phosphatidylinositol-3-OH kinase pathway in BL, in part by augmenting tonic B-cell receptor signalling. In 38% of sporadic BL cases, oncogenic CCND3 mutations produced highly stable cyclin D3 isoforms that drive cell cycle progression. These findings suggest opportunities to improve therapy for patients with BL.


Assuntos
Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/genética , Genômica , Terapia de Alvo Molecular , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Ciclo Celular , Ciclina D3/genética , Ciclina D3/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Genes myc/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Interferência de RNA , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais
12.
Blood ; 126(20): 2291-301, 2015 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-26400962

RESUMO

The serine-threonine kinase ataxia-telangiectasia mutated (ATM) plays a central role in maintaining genomic integrity. In mice, ATM deficiency is exclusively associated with T-cell lymphoma development, whereas B-cell tumors predominate in human ataxia-telangiectasia patients. We demonstrate in this study that when T cells are removed as targets for lymphomagenesis and as mediators of immune surveillance, ATM-deficient mice exclusively develop early-onset immunoglobulin M(+) B-cell lymphomas that do not transplant to immunocompetent mice and that histologically and genetically resemble the activated B cell-like (ABC) subset of human diffuse large B-cell lymphoma (DLBCL). These B-cell lymphomas show considerable chromosomal instability and a recurrent genomic amplification of a 4.48-Mb region on chromosome 18 that contains Malt1 and is orthologous to a region similarly amplified in human ABC DLBCL. Of importance, amplification of Malt1 in these lymphomas correlates with their dependence on nuclear factor (NF)-κB, MALT1, and B-cell receptor (BCR) signaling for survival, paralleling human ABC DLBCL. Further, like some human ABC DLBCLs, these mouse B-cell lymphomas also exhibit constitutive BCR-dependent NF-κB activation. This study reveals that ATM protects against development of B-cell lymphomas that model human ABC DLBCL and identifies a potential role for T cells in preventing the emergence of these tumors.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Vigilância Imunológica , Linfoma Difuso de Grandes Células B/imunologia , Proteínas Supressoras de Tumor/deficiência , Animais , Proteínas Mutadas de Ataxia Telangiectasia/imunologia , Caspases/genética , Caspases/imunologia , Linhagem Celular Tumoral , Instabilidade Cromossômica/imunologia , Loci Gênicos/imunologia , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos Knockout , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , NF-kappa B/genética , NF-kappa B/imunologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Proteínas Supressoras de Tumor/imunologia
13.
J Immunol ; 194(6): 2504-12, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25681333

RESUMO

Endosomal TLRs play an important role in systemic autoimmune diseases, such as systemic erythematosus lupus, in which DNA- and RNA-associated autoantigens activate autoreactive B cells through TLR9- and TLR7-dependent pathways. Nevertheless, TLR9-deficient autoimmune-prone mice develop more severe clinical disease, whereas TLR7-deficient and TLR7/9-double deficient autoimmune-prone mice develop less severe disease. To determine whether the regulatory activity of TLR9 is B cell intrinsic, we directly compared the functional properties of autoantigen-activated wild-type, TLR9-deficient, and TLR7-deficient B cells in an experimental system in which proliferation depends on BCR/TLR coengagement. In vitro, TLR9-deficient cells are less dependent on survival factors for a sustained proliferative response than are either wild-type or TLR7-deficient cells. The TLR9-deficient cells also preferentially differentiate toward the plasma cell lineage, as indicated by expression of CD138, sustained expression of IRF4, and other molecular markers of plasma cells. In vivo, autoantigen-activated TLR9-deficient cells give rise to greater numbers of autoantibody-producing cells. Our results identify distinct roles for TLR7 and TLR9 in the differentiation of autoreactive B cells that explain the capacity of TLR9 to limit, as well as TLR7 to promote, the clinical features of systemic erythematosus lupus.


Assuntos
Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptor 7 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Animais , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Autoantígenos/imunologia , Autoimunidade/genética , Autoimunidade/imunologia , Linfócitos B/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proliferação de Células/genética , Células Cultivadas , Citometria de Fluxo , Fatores Reguladores de Interferon/imunologia , Fatores Reguladores de Interferon/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Plasmócitos/imunologia , Plasmócitos/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator Reumatoide/imunologia , Sindecana-1/imunologia , Sindecana-1/metabolismo , Receptor 7 Toll-Like/deficiência , Receptor 7 Toll-Like/genética , Receptor Toll-Like 9/deficiência , Receptor Toll-Like 9/genética , Transcriptoma/imunologia
14.
Nature ; 470(7332): 115-9, 2011 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-21179087

RESUMO

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt's lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-ß. Hence, the MYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations.


Assuntos
Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Mutação/genética , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Oncogenes/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Linfoma de Burkitt/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Citocinas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Interações Hidrofóbicas e Hidrofílicas , Quinases Associadas a Receptores de Interleucina-1/biossíntese , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Janus Quinases/metabolismo , Linfoma de Zona Marginal Tipo Células B/genética , Linfoma Difuso de Grandes Células B/classificação , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fator 88 de Diferenciação Mieloide/química , NF-kappa B/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Interferência de RNA , Receptores de Interleucina-1/metabolismo , Fator de Transcrição STAT3/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Receptores Toll-Like/metabolismo
15.
Proc Natl Acad Sci U S A ; 111(11): E998-1006, 2014 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-24591644

RESUMO

The plasmacytoid dendritic cell (pDC) is vital to the coordinated action of innate and adaptive immunity. pDC development has not been unequivocally traced, nor has its transcriptional regulatory network been fully clarified. Here we confirm an essential requirement for the BCL11A transcription factor in fetal pDC development, and demonstrate this lineage-specific requirement in the adult organism. Furthermore, we identify BCL11A gene targets and provide a molecular mechanism for its action in pDC commitment. Embryonic germ-line deletion of Bcl11a revealed an absolute cellular, molecular, and functional absence of pDCs in fetal mice. In adults, deletion of Bcl11a in hematopoietic stem cells resulted in perturbed yet continued generation of progenitors, loss of downstream pDC and B-cell lineages, and persisting myeloid, conventional dendritic, and T-cell lineages. Challenge with virus resulted in a marked reduction of antiviral response in conditionally deleted adults. Genome-wide analyses of BCL11A DNA binding and expression revealed that BCL11A regulates transcription of E2-2 and other pDC differentiation modulators, including ID2 and MTG16. Our results identify BCL11A as an essential, lineage-specific factor that regulates pDC development, supporting a model wherein differentiation into pDCs represents a primed "default" pathway for common dendritic cell progenitors.


Assuntos
Proteínas de Transporte/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Redes Reguladoras de Genes/imunologia , Proteínas Nucleares/imunologia , Animais , Linfócitos B/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA , Células Dendríticas/citologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Perfilação da Expressão Gênica , Camundongos , Camundongos Knockout , Camundongos Mutantes , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Proc Natl Acad Sci U S A ; 111(31): 11365-70, 2014 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-25049379

RESUMO

In the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), NF-κB activity is essential for viability of the malignant cells and is sustained by constitutive activity of IκB kinase (IKK) in the cytoplasm. Here, we report an unexpected role for the bromodomain and extraterminal domain (BET) proteins BRD2 and BRD4 in maintaining oncogenic IKK activity in ABC DLBCL. IKK activity was reduced by small molecules targeting BET proteins as well as by genetic knockdown of BRD2 and BRD4 expression, thereby inhibiting downstream NF-κB-driven transcriptional programs and killing ABC DLBCL cells. Using a high-throughput platform to screen for drug-drug synergy, we observed that the BET inhibitor JQ1 combined favorably with multiple drugs targeting B-cell receptor signaling, one pathway that activates IKK in ABC DLBCL. The BTK kinase inhibitor ibrutinib, which is in clinical development for the treatment of ABC DLBCL, synergized strongly with BET inhibitors in killing ABC DLBCL cells in vitro and in a xenograft mouse model. These findings provide a mechanistic basis for the clinical development of BET protein inhibitors in ABC DLBCL, particularly in combination with other modulators of oncogenic IKK signaling.


Assuntos
Quinase I-kappa B/antagonistas & inibidores , Linfoma Difuso de Grandes Células B/enzimologia , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Adenina/análogos & derivados , Animais , Azepinas/farmacologia , Azepinas/toxicidade , Proteínas de Ciclo Celular , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular , Sinergismo Farmacológico , Humanos , Quinase I-kappa B/química , Quinase I-kappa B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos SCID , Proteínas Nucleares/metabolismo , Piperidinas , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Pirazóis/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Triazóis/farmacologia , Triazóis/toxicidade , Ensaios Antitumorais Modelo de Xenoenxerto
17.
J Immunol ; 192(11): 5265-72, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24748495

RESUMO

We have shown previously that priming of respiratory mucosa with live Lactobacillus species promotes robust and prolonged survival from an otherwise lethal infection with pneumonia virus of mice, a property known as heterologous immunity. Lactobacillus priming results in a moderate reduction in virus recovery and a dramatic reduction in virus-induced proinflammatory cytokine production; the precise mechanisms underlying these findings remain to be elucidated. Because B cells have been shown to promote heterologous immunity against respiratory virus pathogens under similar conditions, in this study we explore the role of B cells in Lactobacillus-mediated protection against acute pneumovirus infection. We found that Lactobacillus-primed mice feature elevated levels of airway Igs IgG, IgA, and IgM and lung tissues with dense, B cell (B220(+))-enriched peribronchial and perivascular infiltrates with germinal centers consistent with descriptions of BALT. No B cells were detected in lung tissue of Lactobacillus-primed B cell deficient µMT mice or Jh mice, and Lactobacillus-primed µMT mice had no characteristic infiltrates or airway Igs. Nonetheless, we observed diminished virus recovery and profound suppression of virus-induced proinflammatory cytokines CCL2, IFN-γ, and CXCL10 in both wild-type and Lactobacillus-primed µMT mice. Furthermore, Lactobacillus plantarum-primed, B cell-deficient µMT and Jh mice were fully protected from an otherwise lethal pneumonia virus of mice infection, as were their respective wild-types. We conclude that B cells are dispensable for Lactobacillus-mediated heterologous immunity and were not crucial for promoting survival in response to an otherwise lethal pneumovirus infection.


Assuntos
Linfócitos B/imunologia , Lactobacillus/imunologia , Pulmão/imunologia , Infecções por Pneumovirus/imunologia , Pneumovirus/imunologia , Mucosa Respiratória/imunologia , Animais , Anticorpos Antibacterianos/genética , Anticorpos Antibacterianos/imunologia , Citocinas/genética , Citocinas/imunologia , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Pneumovirus/genética , Infecções por Pneumovirus/genética , Infecções por Pneumovirus/patologia , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia
18.
Nature ; 463(7277): 88-92, 2010 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-20054396

RESUMO

A role for B-cell-receptor (BCR) signalling in lymphomagenesis has been inferred by studying immunoglobulin genes in human lymphomas and by engineering mouse models, but genetic and functional evidence for its oncogenic role in human lymphomas is needed. Here we describe a form of 'chronic active' BCR signalling that is required for cell survival in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). The signalling adaptor CARD11 is required for constitutive NF-kappaB pathway activity and survival in ABC DLBCL. Roughly 10% of ABC DLBCLs have mutant CARD11 isoforms that activate NF-kappaB, but the mechanism that engages wild-type CARD11 in other ABC DLBCLs was unknown. An RNA interference genetic screen revealed that a BCR signalling component, Bruton's tyrosine kinase, is essential for the survival of ABC DLBCLs with wild-type CARD11. In addition, knockdown of proximal BCR subunits (IgM, Ig-kappa, CD79A and CD79B) killed ABC DLBCLs with wild-type CARD11 but not other lymphomas. The BCRs in these ABC DLBCLs formed prominent clusters in the plasma membrane with low diffusion, similarly to BCRs in antigen-stimulated normal B cells. Somatic mutations affecting the immunoreceptor tyrosine-based activation motif (ITAM) signalling modules of CD79B and CD79A were detected frequently in ABC DLBCL biopsy samples but rarely in other DLBCLs and never in Burkitt's lymphoma or mucosa-associated lymphoid tissue lymphoma. In 18% of ABC DLBCLs, one functionally critical residue of CD79B, the first ITAM tyrosine, was mutated. These mutations increased surface BCR expression and attenuated Lyn kinase, a feedback inhibitor of BCR signalling. These findings establish chronic active BCR signalling as a new pathogenetic mechanism in ABC DLBCL, suggesting several therapeutic strategies.


Assuntos
Linfócitos B/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Tirosina Quinase da Agamaglobulinemia , Motivos de Aminoácidos , Linfócitos B/patologia , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Antígenos CD79/química , Antígenos CD79/genética , Antígenos CD79/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Humanos , Linfoma Difuso de Grandes Células B/genética , Mutação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Interferência de RNA , Receptores de Antígenos de Linfócitos B/deficiência , Receptores de Antígenos de Linfócitos B/genética , Quinases da Família src/metabolismo
19.
Blood ; 120(5): 1095-106, 2012 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-22718837

RESUMO

Dysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G(1) arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G(1) and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G(1) block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy.


Assuntos
Apoptose/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Fatores Reguladores de Interferon/genética , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/genética , Ácidos Borônicos/administração & dosagem , Ácidos Borônicos/farmacologia , Bortezomib , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Citotoxinas/administração & dosagem , Citotoxinas/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Fatores Reguladores de Interferon/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Pirazinas/administração & dosagem , Pirazinas/farmacologia , Especificidade por Substrato , Fatores de Tempo , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Nature ; 454(7201): 226-31, 2008 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-18568025

RESUMO

The transcription factor IRF4 (interferon regulatory factor 4) is required during an immune response for lymphocyte activation and the generation of immunoglobulin-secreting plasma cells. Multiple myeloma, a malignancy of plasma cells, has a complex molecular aetiology with several subgroups defined by gene expression profiling and recurrent chromosomal translocations. Moreover, the malignant clone can sustain multiple oncogenic lesions, accumulating genetic damage as the disease progresses. Current therapies for myeloma can extend survival but are not curative. Hence, new therapeutic strategies are needed that target molecular pathways shared by all subtypes of myeloma. Here we show, using a loss-of-function, RNA-interference-based genetic screen, that IRF4 inhibition is toxic to myeloma cell lines, regardless of transforming oncogenic mechanism. Gene expression profiling and genome-wide chromatin immunoprecipitation analysis uncovered an extensive network of IRF4 target genes and identified MYC as a direct target of IRF4 in activated B cells and myeloma. Unexpectedly, IRF4 was itself a direct target of MYC transactivation, generating an autoregulatory circuit in myeloma cells. Although IRF4 is not genetically altered in most myelomas, they are nonetheless addicted to an aberrant IRF4 regulatory network that fuses the gene expression programmes of normal plasma cells and activated B cells.


Assuntos
Fatores Reguladores de Interferon/metabolismo , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Sobrevivência Celular , Transformação Celular Neoplásica/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes myc/genética , Humanos , Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/genética , Camundongos , Mieloma Múltiplo/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , Ativação Transcricional
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