RESUMO
Diffuse large B-cell lymphomas (DLBCLs) contain 2 major molecular subtypes; namely, the germinal center B-cell-like (GCB) and the activated B-cell-like (ABC) DLBCLs. It is well documented that ABC-DLBCL cases have a significantly poorer survival response than GCB-DLBCLs in both the CHOP (cyclophosphamide, vincristine, doxorubicin, and prednisone) and the rituximab (R)-CHOP eras. However, the underlying cause of this subtype disparity is poorly understood. Nevertheless, these clinical observations raise the possibility for an ABC-DLBCL-specific resistance mechanism that is directed toward 1 of the CHOP components and is inadequately addressed by rituximab. Here, we report that the main cytotoxic ingredient in CHOP, doxorubicin (Dox), has subtype-specific mechanisms of cytotoxicity in DLBCLs resulting from differences in the subcellular distribution pattern. Specifically, in cell line models of ABC-DLBCL, Dox is often enriched in the cytoplasm away from the nuclear DNA. As a result, Dox-induced cytotoxicity in ABC-DLBCLs is often dependent on oxidative stress, rather than DNA damage response. These findings are corroborated by gene signature analysis, which demonstrates that basal oxidative stress status predicts treatment outcome among patients with ABC-DLBCL, but not patients with GCB-DLBCL. In terms of redox-related resistance mechanism, our results suggest that STAT3 confers Dox resistance in ABC-DLBCLs by reinforcing an antioxidant program featuring upregulation of the SOD2 gene. Furthermore, a small-molecule STAT3 inhibitor synergizes with CHOP to trigger oxidative stress and kill ABC-DLBCL cells in preclinical models. These results provide a mechanistic basis for development of novel therapies that target either STAT3 or redox homeostasis to improve treatment outcomes for ABC-DLBCLs.
Assuntos
Linfócitos B/patologia , Doxorrubicina/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologia , Estresse Oxidativo/efeitos dos fármacos , Antioxidantes/metabolismo , Linfócitos B/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Compostos Clorados/farmacologia , Dano ao DNA , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Centro Germinativo/efeitos dos fármacos , Centro Germinativo/patologia , Humanos , Compostos de Platina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Resultado do TratamentoRESUMO
After undergoing Ig somatic hypermutation and Ag selection, germinal center (GC) B cells terminally differentiate into either memory or plasma cells (PCs). It is known that the CD40L and IL-21/STAT3 signaling pathways play critical roles in this process, yet it is unclear how the B cell transcription program interprets and integrates these two types of T cell-derived signals. In this study, we characterized the role of STAT3 in the GC-associated PC differentiation using purified human tonsillar GC B cells and a GC B cell-like cell line. When primary GC B cells were cultured under PC differentiation condition, STAT3 inhibition by AG490 prevented the transition from GC centrocytes to preplasmablast, suggesting that STAT3 is required for the initiation of PC development. In a GC B cell-like human B cell line, although IL-21 alone can induce low-level Blimp-1 expression, maximum Blimp-1 upregulation and optimal PC differentiation required both IL-21 and CD40L. CD40L, although having no effect on Blimp-1 as a single agent, greatly augmented the amplitude and duration of IL-21-triggered Jak-STAT3 signaling. In the human PRDM1 locus, CD40L treatment enhanced the ability of STAT3 to upregulate Blimp-1 by removing BCL6, a potent inhibitor of Blimp-1 expression, from a shared BCL6/STAT3 site in intron 3. Thus, IL-21 and CD40L collaborate through at least two distinct mechanisms to synergistically promote Blimp-1 activation and PC differentiation.
Assuntos
Adjuvantes Imunológicos/fisiologia , Subpopulações de Linfócitos B/imunologia , Ligante de CD40/fisiologia , Diferenciação Celular/imunologia , Interleucinas/fisiologia , Plasmócitos/imunologia , Proteínas Repressoras/biossíntese , Regulação para Cima/imunologia , Subpopulações de Linfócitos B/enzimologia , Subpopulações de Linfócitos B/metabolismo , Linhagem Celular Tumoral , Humanos , Janus Quinases/fisiologia , Dados de Sequência Molecular , Técnicas de Cultura de Órgãos , Tonsila Palatina/enzimologia , Tonsila Palatina/imunologia , Tonsila Palatina/metabolismo , Plasmócitos/enzimologia , Plasmócitos/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Repressoras/fisiologia , Fator de Transcrição STAT3/fisiologiaRESUMO
Identifying the multiple dysregulated oncoproteins that contribute to tumorigenesis in a given patient is crucial for developing personalized treatment plans. However, accurate inference of aberrant protein activity in biological samples is still challenging as genetic alterations are only partially predictive and direct measurements of protein activity are generally not feasible. To address this problem we introduce and experimentally validate a new algorithm, virtual inference of protein activity by enriched regulon analysis (VIPER), for accurate assessment of protein activity from gene expression data. We used VIPER to evaluate the functional relevance of genetic alterations in regulatory proteins across all samples in The Cancer Genome Atlas (TCGA). In addition to accurately infer aberrant protein activity induced by established mutations, we also identified a fraction of tumors with aberrant activity of druggable oncoproteins despite a lack of mutations, and vice versa. In vitro assays confirmed that VIPER-inferred protein activity outperformed mutational analysis in predicting sensitivity to targeted inhibitors.
Assuntos
Algoritmos , Biologia Computacional/métodos , Mutação/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Antineoplásicos/farmacologia , Bases de Dados de Produtos Farmacêuticos , Bases de Dados de Proteínas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Humanos , Neoplasias/tratamento farmacológico , Mapas de Interação de Proteínas/efeitos dos fármacos , Estrutura Terciária de ProteínaRESUMO
PURPOSE: We previously reported that constitutive STAT3 activation is a prominent feature of the activated B-cell subtype of diffuse large B-cell lymphomas (ABC-DLBCL). In this study, we investigated whether STAT3 activation can risk stratify patients with DLBCL. PATIENTS AND METHODS: By an immunohistochemical method, we investigated phosphotyrosine STAT3 (PY-STAT3) expression from 185 patients with DLBCL treated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone). Cell line-based siRNA experiments were also performed to generate an 11-gene, PY-STAT3 activation signature, which was used to study a previously published cohort of 222 patients with DLBCL. The STAT3 activation status determined by these two methods and by STAT3 mRNA levels were then correlated with survival. RESULTS: PY-STAT3 was detected in 37% of DLBCL and enriched in ABC-DLBCL cases (P = .03). PY-STAT3 positivity significantly correlated with poor overall survival (OS; P = .01) and event-free survival (EFS; P = .006). Similar observations were made for high levels of STAT3 mRNA. In multivariable analysis, PY-STAT3 status (P = .02), International Prognostic Index (P = .02), and BCL2 expression (P = .046) were independent prognosticators of OS in this cohort. Among the cell-of-origin subgroups, PY-STAT3 was associated with poor EFS among non-germinal center B-cell DLBCL cases only (P = .027). Similarly, the 11-gene STAT3 activation signature correlated with poor survival in the entire DLBCL cohort (OS, P < .001; EFS, P < .001) as well as the ABC-DLBCL subgroup (OS, P = .029; EFS, P = .025). CONCLUSION: STAT3 activation correlated with poor survival in patients with DLBCL treated with R-CHOP, especially those with tumors of the ABC-DLBCL subtype.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/mortalidade , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adulto , Idoso , Análise de Variância , Anticorpos Monoclonais Murinos/administração & dosagem , Antígenos CD20/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Ciclofosfamida/administração & dosagem , Intervalo Livre de Doença , Doxorrubicina/administração & dosagem , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Linfoma Difuso de Grandes Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prednisona/administração & dosagem , Prognóstico , Modelos de Riscos Proporcionais , RNA Mensageiro/metabolismo , Estudos Retrospectivos , Rituximab , Fator de Transcrição STAT3/efeitos dos fármacos , Fator de Transcrição STAT3/genética , Análise Serial de Tecidos , Resultado do Tratamento , Vincristina/administração & dosagemRESUMO
Germinal centers (GC) of secondary lymphoid tissues are critical to mounting a high-affinity humoral immune response. B cells within the GC undergo rapid clonal expansion and selection while diversifying their antibody genes. Although it is generally believed that GC B cells employ a unique proliferative program to accommodate these processes, little is known about how the GC-associated cell cycle is orchestrated. The D-type cyclins constitute an important component of the cell cycle engine that enables the cells to respond to physiological changes. Cell type- and developmental stage-specific roles of D-type cyclins have been described but the cyclin D requirement during GC reaction has not been addressed. In this study, we report that cyclin D3 is largely dispensable for proliferation and Ig class switching of in vitro activated B cells. In contrast, GC development in Ccnd3(-/-) mice is markedly impaired, as is the T cell-dependent antibody response. Within the GC, although both switched and unswitched B cells are affected by cyclin D3 inactivation, the IgM(-) pool is more severely reduced. Interestingly, despite a compensatory increase in cyclin D2 expression, a significant number of Ccnd3(-/-) GC B cells accumulate in quiescent G0 state. Lastly, although cyclin D3 inactivation did not disrupt BCL6 expression in GC B cells, it completely blocked the GC promoting effect of BCL6 overexpression, suggesting that cyclin D3 acts downstream of BCL6 to regulate GC formation. This is the first demonstration that cyclin D3 plays an important and unique role at the GC stage of B cell development.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Ciclina D3/fisiologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Animais , Linfócitos B/citologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Ciclina D3/deficiência , Ciclina D3/genética , Centro Germinativo/citologia , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Tecido Linfoide/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The transcription repressor BCL6 plays an essential role in the formation and function of germinal centers (GCs). While normal B cells promptly shut off BCL6 when they exit the GC, many GC-derived B-cell lymphomas sustain BCL6 expression through chromosomal translocations and activating mutations. We have previously shown that a common effect of lymphoma-associated BCL6 gene alterations is to bypass a negative autoregulatory loop that controls its transcription. In this study, we report that BCL6 autoregulation is independent of several known corepressor complexes including silencing mediator for retinoid and thyroid hormone receptors, nuclear receptor coreceptor, BCL6 corepressor, and MTA3/NuRD. Furthermore, we show that BCL6 can interact with the CtBP (C-terminal binding protein) corepressor both in vitro and in vivo and that CtBP is recruited by BCL6 to its 5' regulatory region. In lymphoma cell lines carrying BCL6 translocations, small interfering RNA-mediated CtBP knock-down selectively relieved the previously silenced wild-type BCL6 allele but not the translocated alleles, which are driven by heterologous promoters. These results demonstrate that CtBP is a novel BCL6 corepressor and suggest that a unique corepressor requirement for BCL6 autoregulation may allow GC B cells to differentially control the expression of BCL6 and other BCL6 target genes in response to environmental stimuli during the GC stage of B cell development.
Assuntos
Oxirredutases do Álcool/fisiologia , Linfócitos B/citologia , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Repressoras/fisiologia , Oxirredutases do Álcool/química , Linhagem Celular/metabolismo , Quimiocina CCL3/biossíntese , Quimiocina CCL3/genética , Proteínas de Ligação a DNA/química , Regulação da Expressão Gênica/genética , Humanos , Macrófagos/metabolismo , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/fisiologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Repressoras/química , Proteínas Repressoras/genéticaRESUMO
Diffuse large B-cell lymphoma (DLBCL) consists of at least 2 phenotypic subtypes; that is, the germinal center B-cell-like (GCB-DLBCL) and the activated B-cell-like (ABC-DLBCL) groups. It has been shown that GCB-DLBCL responds favorably to chemotherapy and expresses high levels of BCL6, a transcription repressor known to play a causative role in lymphomagenesis. In comparison, ABC-DLBCL has lower levels of BCL6, constitutively activated nuclear factor-kappaB, and tends to be refractory to chemotherapy. Here, we report that the STAT3 gene is a transcriptional target of BCL6. As a result, high-level STAT3 expression and activation are preferentially detected in ABC-DLBCL and BCL6-negative normal germinal center B cells. Most importantly, inactivating STAT3 by either AG490 or small interference RNA in ABC-DLBCL cells inhibits cell proliferation and triggers apoptosis. These phenotypes are accompanied by decreased expression of several known STAT3 target genes, including c-Myc, JunB, and Mcl-1, and increased expression of the cell- cycle inhibitor p27. In addition to identifying STAT3 as a novel BCL6 target gene, our results define a second oncogenic pathway, STAT3 activation, which operates in ABC-DLBCL, suggesting that STAT3 may be a new therapeutic target in these aggressive lymphomas.
Assuntos
Ativação Linfocitária/imunologia , Linfoma Difuso de Grandes Células B/imunologia , Linfoma Difuso de Grandes Células B/metabolismo , Fator de Transcrição STAT3/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Centro Germinativo/imunologia , Humanos , Linfoma Difuso de Grandes Células B/classificação , Linfoma Difuso de Grandes Células B/patologia , Mutação/genética , Proteínas Proto-Oncogênicas c-bcl-6 , Interferência de RNA , RNA Mensageiro/genética , Fator de Transcrição STAT3/genética , Transcrição Gênica/genética , Tirfostinas/farmacologiaRESUMO
BCL-6 is a transcription repressor frequently deregulated in non-Hodgkin's B cell lymphomas. Its activity is also critical to germinal center development and balanced Th1/Th2 differentiation. Previous studies have suggested that NF-kappaB activity is suppressed in germinal center and lymphoma B cells that express high levels of BCL-6, and yet the reason for this is unknown. We report in this study that BCL-6 can bind to three sequence motifs in the 5' regulatory region of NF-kappaB1 in vitro and in vivo, and repress NF-kappaB1 transcription both in reporter assays and in lymphoma B cell lines. BCL-6(-/-) mice further confirm the biological relevance of BCL-6-dependent regulation of NF-kappaB1 because BCL-6 inactivation caused notable increase in p105/p50 proteins in several cell types. Among these, BCL-6(-/-) macrophage cell lines displayed a hyperproliferation phenotype that can be reversed by NF-kappaB inhibitors, e.g., N-tosyl-l-phenylalanine chloromethyl ketone and SN50, a result that is consistent with increased nuclear kappaB-binding activity of p50 homodimer and p50/p65 heterodimer. Our results demonstrate that BCL-6 can negatively regulate NF-kappaB1 expression, thereby inhibiting NF-kappaB-mediated cellular functions.