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
p27Kip1 (p27) is an inhibitor of cyclin-dependent kinases. Inhibiting p27 protein degradation is an actively developing cancer therapy strategy. One focus has been to identify small molecule inhibitors to block recruitment of Thr-187-phosphorylated p27 (p27T187p) to SCF(Skp2/Cks1) ubiquitin ligase. Since phosphorylation of Thr-187 is required for this recruitment, p27T187A knockin (KI) mice were generated to determine the effects of systemically blocking interaction between p27 and Skp2/Cks1 on tumor susceptibility and other proliferation related mouse physiology. Rb1(+/-) mice develop pituitary tumors with full penetrance and the tumors are invariably Rb1(-/-), modeling tumorigenesis by two-hit loss of RB1 in humans. Immunization induced humoral immunity depends on rapid B cell proliferation and clonal selection in germinal centers (GCs) and declines with age in mice and humans. Here, we show that p27T187A KI prevented pituitary tumorigenesis in Rb1(+/-) mice and corrected decline in humoral immunity in older mice following immunization with sheep red blood cells (SRBC). These findings reveal physiological contexts that depend on p27 ubiquitination by SCF(Skp2-Cks1) ubiquitin ligase and therefore help forecast clinical potentials of Skp2/Cks1-p27T187p interaction inhibitors. We further show that GC B cells and T cells use different mechanisms to regulate their p27 protein levels, and propose a T helper cell exhaustion model resembling that of stem cell exhaustion to understand decline in T cell-dependent humoral immunity in older age.
Assuntos
Substituição de Aminoácidos , Inibidor de Quinase Dependente de Ciclina p27/genética , Imunidade Humoral/genética , Hipófise/metabolismo , Neoplasias Hipofisárias/genética , Proteína do Retinoblastoma/genética , Fatores Etários , Alanina/genética , Alanina/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Eritrócitos/imunologia , Citometria de Fluxo , Técnicas de Introdução de Genes , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Humanos , Imunidade Humoral/imunologia , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Hipófise/patologia , Neoplasias Hipofisárias/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Ovinos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Treonina/genética , Treonina/metabolismoRESUMO
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
Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR-ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Inibidores de Proteínas Quinases/farmacologia , Fator 1 de Ribosilação do ADP/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-6 , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismoRESUMO
BCL6 protects germinal center (GC) B cells against DNA damage-induced apoptosis during somatic hypermutation and class-switch recombination. Although expression of BCL6 was not found in early IL-7-dependent B cell precursors, we report that IL-7Ralpha-Stat5 signaling negatively regulates BCL6. Upon productive VH-DJH gene rearrangement and expression of a mu heavy chain, however, activation of pre-B cell receptor signaling strongly induces BCL6 expression, whereas IL-7Ralpha-Stat5 signaling is attenuated. At the transition from IL-7-dependent to -independent stages of B cell development, BCL6 is activated, reaches expression levels resembling those in GC B cells, and protects pre-B cells from DNA damage-induced apoptosis during immunoglobulin (Ig) light chain gene recombination. In the absence of BCL6, DNA breaks during Ig light chain gene rearrangement lead to excessive up-regulation of Arf and p53. As a consequence, the pool of new bone marrow immature B cells is markedly reduced in size and clonal diversity. We conclude that negative regulation of Arf by BCL6 is required for pre-B cell self-renewal and the formation of a diverse polyclonal B cell repertoire.
Assuntos
Linfócitos B/citologia , Linfócitos B/imunologia , Proteínas de Ligação a DNA/imunologia , Fatores de Ribosilação do ADP/metabolismo , Animais , Apoptose , Sequência de Bases , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Citoproteção , Dano ao DNA/genética , Regulação para Baixo/genética , Rearranjo Gênico de Cadeia Leve de Linfócito B/genética , Humanos , Interleucina-7/metabolismo , Linfopoese , Camundongos , Dados de Sequência Molecular , Receptores de Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6 , Proteínas Proto-Oncogênicas c-myc/metabolismo , Recombinação Genética/genética , Transdução de Sinais , Transcrição Gênica , Regulação para Cima/genéticaRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/metabolismo , NF-kappa B/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/imunologia , Humanos , Imunoprecipitação , Linfoma de Células B/metabolismo , Camundongos , NF-kappa B/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas c-bcl-6 , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/imunologiaRESUMO
The transcription repressor BCL-6 is known to play critical roles in B-cell lymphomagenesis, germinal center formation, and balanced Th1/Th2 differentiation. In macrophages, although BCL-6 has also been shown to regulate the expression of several chemokine genes, its function in other aspects of macrophage biology has not been studied. In addition, the precise role of BCL-6 in cell proliferation is poorly understood in general. Here we report that BCL-6(-/-) macrophages hyperproliferate due to an accelerated G(1)/S transition accompanied by increased cyclin D2 and c-myc and decreased expression of p27. Crucial to this enhanced proliferation is spontaneous interleukin 6 (IL-6) production and signal transducer and activator of transcription 3 (STAT3) activation in BCL-6(-/-) macrophages. In colony-forming assays, BCL- 6(-/-) bone marrow progenitor cells form spontaneous macrophage colonies that can be inhibited by anti-IL-6 antibodies. Gene expression studies demonstrate that BCL-6 binds to several sequence motifs scattered in the IL-6 locus and can repress IL-6 transcription both in 293T cells and in macrophages. In conclusion, our results indicate that BCL-6 negatively regulates proliferation of the monocytic/macrophage lineage by suppressing an autocrine IL-6/STAT3-mediated gene expression program. Our work also suggests that BCL-6 prevents abnormal Th2 differentiation by suppressing basal level IL-6 production in antigen-presenting cells (APCs).