RESUMO
The interaction of lymphoma cells with their microenvironment has an important role in disease pathogenesis and is being actively pursued therapeutically using immunomodulatory drugs, including immune checkpoint inhibitors. Diffuse large B-cell lymphoma (DLBCL) is an aggressive high-grade disease that remains incurable in ~40% of patients treated with R-CHOP immunochemotherapy. The FOXP1 transcription factor is abundantly expressed in such high-risk DLBCL and we recently identified its regulation of immune response signatures, in particular, its suppression of the cell surface expression of major histocompatibility class II (MHC-II), which has a critical role in antigen presentation to T cells. Using CRISPR/Cas9 genome editing we have depleted Foxp1 expression in the aggressive murine A20 lymphoma cell line. When grown in BALB/c mice, this cell line provides a high-fidelity immunocompetent disseminated lymphoma model that displays many characteristics of human DLBCL. Transient Foxp1-depletion using siRNA, and stable depletion using CRISPR (generated by independently targeting Foxp1 exon six or seven) upregulated cell surface I-Ab (MHC-II) expression without impairing cell viability in vitro. RNA sequencing of Foxp1-depleted A20 clones identified commonly deregulated genes, such as the B-cell marker Cd19, and hallmark DLBCL signatures such as MYC-targets and oxidative phosphorylation. Immunocompetent animals bearing Foxp1-depleted A20 lymphomas showed significantly-improved survival, and 20% did not develop tumors; consistent with modulating immune surveillance, this was not observed in immunodeficient NOD SCIDγ mice. The A20 Foxp1 CRISPR model will help to further characterize the contribution of Foxp1 to lymphoma immune evasion and the potential for Foxp1 targeting to synergize with other immunotherapies.
RESUMO
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of mature B-cell lymphoma. While the majority of patients are cured with immunochemotherapy incorporating the anti-CD20 monoclonal antibody rituximab (R-CHOP), relapsed and refractory patients still have a dismal prognosis. DLBCL subtypes including an aggressive activated B-cell-like (ABC) and a more favorable prognosis germinal center-like (GCB) DLBCL have been identified by gene expression profiling and are characterized by distinct genetic abnormalities and oncogenic pathways. This identification of novel molecular targets is now enabling clinical trials to evaluate more effective personalized approaches to DLBCL therapy. The forkhead transcription factor FOXP1 is highly expressed in the ABC-DLBCL gene signature and has been extensively studied within the context of DLBCL for more than a decade. Here, we review the significance of FOXP1 in the pathogenesis of DLBCL, summarizing data supporting its utility as a prognostic and subtyping marker, its targeting by genetic aberrations, the importance of specific isoforms, and emerging data demonstrating a functional role in lymphoma biology. FOXP1 is one of the critical transcription factors whose deregulated expression makes important contributions to DLBCL pathogenesis. Thus, FOXP1 warrants further study as a potential theranostic in ABC-DLBCL.
Assuntos
Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Animais , Biomarcadores Tumorais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica , Variação Genética , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/mortalidade , MicroRNAs/genética , Terapia de Alvo Molecular , Família Multigênica , Prognóstico , Isoformas de Proteínas , Interferência de RNA , Transdução de Sinais , Translocação GenéticaRESUMO
Plasmablastic B-cell malignancies include plasmablastic lymphoma and subsets of multiple myeloma and diffuse large B-cell lymphomaDLBCL. These diseases can be difficult to diagnose and treat, and they lack well-characterized cell line models. Here, immunophenotyping and FOXP1 expression profiling identified plasmablastic characteristics in DLBCL cell lines HLY-1 and SU-DHL-9, associated with CTNNAL1, HPGD, RORA, IGF1, and/or vitamin D receptor (VDR) transcription. We demonstrated VDR protein expression in primary plasmablastic tumor cells and confirmed in cell lines expression of both VDR and the metabolic enzyme CYP27B1, which catalyzes active vitamin D3 production. Although Vdr and Cyp27b1 transcription in normal B cells were activated by interleukin 4 (IL-4) and CD40 signaling, respectively, unstimulated malignant plasmablastic cells lacking IL-4 expressed both VDR and CYP27B1. Positive autoregulation evidenced intact VDR function in all plasmablastic lines, and inhibition of growth by active vitamin D3 was both dependent on MYC protein inhibition and could be enhanced by cotreatment with a synthetic ROR ligand SR-1078. Furthermore, a VDR polymorphism, FOK1, was associated with greater vitamin D3-dependent growth inhibition. In summary, HLY-1 provides an important model of strongly plasmablastic lymphoma, and disruption of VDR pathway activity may be of therapeutic benefit in both plasmablastic lymphoma and myeloma.
Assuntos
Colecalciferol/uso terapêutico , Mieloma Múltiplo/metabolismo , Linfoma Plasmablástico/metabolismo , Receptores de Calcitriol/metabolismo , Animais , Benzamidas , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colecalciferol/farmacologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Imunofenotipagem , Camundongos Endogâmicos C57BL , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Linfoma Plasmablástico/tratamento farmacológico , Linfoma Plasmablástico/genética , Receptores de Calcitriol/genética , Proteínas Repressoras/metabolismoRESUMO
FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥ 20% nuclear tumoral FOXP2-positivity (n = 24/158) correlated with significantly inferior overall survival (OS: P = 0.0017) and progression-free survival (PFS: P = 0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P < 0.05 for both OS and PFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P = 0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P = 0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Fatores de Transcrição Forkhead/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Murinos/administração & dosagem , Linhagem Celular Tumoral , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Feminino , Fatores de Transcrição Forkhead/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Humanos , Estimativa de Kaplan-Meier , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Prednisona/administração & dosagem , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/genética , Transcriptoma/genética , Vincristina/administração & dosagem , Adulto JovemRESUMO
Strong FOXP1 protein expression is a poor risk factor in diffuse large B-cell lymphoma and has been linked to an activated B-cell-like subtype, which preferentially expresses short FOXP1 (FOXP1S) proteins. However, both short isoform generation and function are incompletely understood. Here we prove by mass spectrometry and N-terminal antibody staining that FOXP1S proteins in activated B-cell-like diffuse large B-cell lymphoma are N-terminally truncated. Furthermore, a rare strongly FOXP1-expressing population of normal germinal center B cells lacking the N-terminus of the regular long protein (FOXP1L) was identified. Exon-targeted silencing and transcript analyses identified three alternate 5' non-coding exons [FOXP1-Ex6b(s), FOXP1-Ex7b and FOXP1-Ex7c], downstream of at least two predicted promoters, giving rise to FOXP1S proteins. These were differentially controlled by B-cell activation and methylation, conserved in murine lymphoma cells, and significantly correlated with FOXP1S protein expression in primary diffuse large B-cell lymphoma samples. Alternatively spliced isoforms lacking exon 9 (e.g. isoform 3) did not encode FOXP1S, and an alternate long human FOXP1 protein (FOXP1AL) likely generated from a FOXP1-Ex6b(L) transcript was detected. The ratio of FOXP1L:FOXP1S isoforms correlated with differential expression of plasmacytic differentiation markers in U-2932 subpopulations, and altering this ratio was sufficient to modulate CD19 expression in diffuse large B-cell lymphoma cell lines. Thus, the activity of multiple alternate FOXP1 promoters to produce multiple protein isoforms is likely to regulate B-cell maturation.
Assuntos
Linfócitos B/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/genética , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Processamento Alternativo , Animais , Antígenos CD19/genética , Antígenos CD19/metabolismo , Linhagem Celular Tumoral , Éxons , Fatores de Transcrição Forkhead/química , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/químicaRESUMO
Mutations of the forkhead transcription factor FOXP2 gene have been implicated in inherited speech-and-language disorders, and specific Foxp2 expression patterns in neuronal populations and neuronal phenotypes arising from Foxp2 disruption have been described. However, molecular functions of FOXP2 are not completely understood. Here we report a requirement for FOXP2 in growth arrest of the osteosarcoma cell line 143B. We observed endogenous expression of this transcription factor both transiently in normally developing murine osteoblasts and constitutively in human SAOS-2 osteosarcoma cells blocked in early osteoblast development. Critically, we demonstrate that in 143B osteosarcoma cells with minimal endogenous expression, FOXP2 induced by growth arrest is required for up-regulation of p21WAF1/CIP1. Upon growth factor withdrawal, FOXP2 induction occurs rapidly and precedes p21WAF1/CIP1 activation. Additionally, FOXP2 expression could be induced by MAPK pathway inhibition in growth-arrested 143B cells, but not in traditional cell line models of osteoblast differentiation (MG-63, C2C12, MC3T3-E1). Our data are consistent with a model in which transient upregulation of Foxp2 in pre-osteoblast mesenchymal cells regulates a p21-dependent growth arrest checkpoint, which may have implications for normal mesenchymal and osteosarcoma biology.
Assuntos
Apoptose , Neoplasias Ósseas/patologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Osteoblastos/citologia , Osteossarcoma/patologia , Animais , Western Blotting , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Ciclo Celular , Proliferação de Células , Células Cultivadas , Imunoprecipitação da Cromatina , Inibidor de Quinase Dependente de Ciclina p21/genética , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/genética , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteossarcoma/genética , Osteossarcoma/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaAssuntos
Fator 1 Induzível por Hipóxia/metabolismo , Mutação , Policitemia/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Análise Mutacional de DNA , Hipertensão Pulmonar Primária Familiar , Humanos , Hipertensão Pulmonar/complicações , Hipertensão Pulmonar/genética , Fator 1 Induzível por Hipóxia/fisiologia , Lactente , Masculino , Mutação/fisiologia , Policitemia/complicações , Índice de Gravidade de Doença , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/fisiologiaRESUMO
A variety of genetic lesions, including chromosomal translocations, internal tandem duplications, and mutations, have been described in acute myeloid leukemia (AML). Expression profiling has shown that chromosomal translocations, in particular, are associated with distinctive patterns of gene expression. AML exhibiting the translocation t(8;21), which fuses the AML1 and ETO genes, has such a characteristic expression profile. One gene whose expression is highly correlated with the presence of the AML1/ETO fusion is POU4F1, which encodes the POU homeodomain transcription factor BRN3A. Here we show using specific siRNA in t(8;21) cells and overexpression studies in progenitor cells that AML1/ETO promotes expression of POU4F1/BRN3A. This effect requires DNA-binding function of AML1/ETO, and accordingly, AML1/ETO is bound to the POU4F1 locus in t(8;21) cells. Functionally, whereas overexpression of Brn3a in murine hematopoietic progenitor cells induces terminal myeloid differentiation, coexpression of AML1/ETO or AML1/ETO9a blocks this effect. Furthermore, Brn3a reduction by shRNA impairs AML1/ETO-induced immortalization of murine progenitors. In summary, we identify POU4F1/BRN3A as a novel potential upregulated AML1/ETO target gene whose dramatically high expression may cooperate with AML1/ETO in t(8;21) cells.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fator de Transcrição Brn-3A/metabolismo , Adulto , Animais , Sequência de Bases , Western Blotting , Diferenciação Celular , Imunoprecipitação da Cromatina , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 8/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Ensaio de Desvio de Mobilidade Eletroforética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Feto , Imunofluorescência , Células-Tronco Hematopoéticas/fisiologia , Humanos , Técnicas Imunoenzimáticas , Leucemia Mieloide Aguda/patologia , Fígado/citologia , Fígado/metabolismo , Luciferases/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Proteína 1 Parceira de Translocação de RUNX1 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Brn-3A/antagonistas & inibidores , Fator de Transcrição Brn-3A/genética , Transfecção , Translocação Genética/genéticaRESUMO
Natural killer (NK) cells are a subset of lymphocytes crucial for innate immunity and modification of adaptive immune responses. In contrast to commitment to the T cell or B cell lineage, little is known about NK cell lineage commitment. Here we show that the basic leucine zipper (bZIP) transcription factor E4BP4 (also called NFIL3) is essential for generation of the NK cell lineage. E4BP4-deficient mice (Nfil3(-/-); called 'E4bp4(-/-)' here) had B cells, T cells and NKT cells but specifically lack NK cells and showed severely impaired NK cell-mediated cytotoxicity. Overexpression of E4bp4 was sufficient to increase NK cell production from hematopoietic progenitor cells. E4BP4 acted in a cell-intrinsic manner 'downstream' of the interleukin 15 receptor (IL-15R) and through the transcription factor Id2. E4bp4(-/-) mice may provide a model for definitive analysis of the contribution of NK cells to immune responses and pathologies.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Diferenciação Celular/imunologia , Citotoxicidade Imunológica/imunologia , Células Matadoras Naturais/citologia , Animais , Linfócitos B/imunologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Citotoxicidade Imunológica/genética , Citometria de Fluxo , Expressão Gênica/imunologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Proteína 2 Inibidora de Diferenciação/genética , Proteína 2 Inibidora de Diferenciação/imunologia , Proteína 2 Inibidora de Diferenciação/metabolismo , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Camundongos , Camundongos Knockout , Células T Matadoras Naturais/imunologia , Receptores de Interleucina-15/genética , Receptores de Interleucina-15/imunologia , Receptores de Interleucina-15/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologiaRESUMO
The Brn-3 family of POU (Pit-Oct-Unc) homeodomain transcription factors regulate differentiation of neuronal cell types. The transcriptional activator Brn-3a is expressed in Ewing's sarcomas, which also express characteristic chimaeric proteins as a consequence of fusion of the TET family gene EWS to one of several ETS genes. We have previously demonstrated a physical interaction between Brn-3a and EWS proteins, and show here that the C-terminal POU domain but not N-terminal activation domain of Brn-3a can interact in vitro with the RNA-binding domain of EWS. Likely due to POU domain homology, the related factor Brn-3b can also interact with EWS, but to a lesser extent than Brn-3a. Importantly, Brn-3a but not Brn-3b interacts in vitro with chimaeric EWS/Fli-1, EWS/ATF-1 and EWS/ERG proteins. Furthermore, overexpression of EWS/Fli-1 but not EWS or Fli-1 inhibits Brn-3a-associated growth arrest and neurite outgrowth in neuronal cells, and specifically inhibits Brn-3a-dependent activation of p21 and SNAP-25 transcription. In contrast, upregulation of Bcl-2 expression and inhibition of apoptosis by Brn-3a is antagonized more by EWS than by EWS/Fli-1. These data demonstrate that oncogenic rearrangement of EWS to produce EWS/Fli-1 may enhance the antiapoptotic effect of Brn-3a and inhibit its ability to promote neuronal differentiation.
Assuntos
Apoptose , Proteínas de Ligação a DNA/fisiologia , Neurônios/citologia , Proteínas de Fusão Oncogênica/fisiologia , Proteína EWS de Ligação a RNA/fisiologia , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Humanos , Proteínas de Membrana/análise , Proteínas do Tecido Nervoso/análise , Neuritos/fisiologia , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-fli-1 , RNA/metabolismo , Proteína 25 Associada a Sinaptossoma , Fator de Transcrição Brn-3 , Fator de Transcrição Brn-3A , Fator de Transcrição Brn-3BRESUMO
Mice carrying the bovine papillomavirus type I genome develop dermal fibrosarcomas in a multiple step process characterized by distinctive proliferative stages. Chromosomal aberrations are identified early in this tumorigenic pathway, however, the mechanism that originates them is unknown. Using a functional assay, we investigated the status of the mitotic spindle cell cycle checkpoint (MSCCC) that regulates the metaphase to anaphase transition, in cells representing different stages of fibrosarcoma progression. Loss of MSCCC activity was apparent in mild fibromatosis and completely abolished in aggressive fibromatosis and fibrosarcoma lesions. This altered MSCCC status was confirmed biochemically by deregulated expression of Cks1 protein and unscheduled cyclin B metabolism. Immunoprecipitation and sequencing analyses indicated that mutation of p53 was not required for the abrogation of the MSCCC. These results demonstrate that loss of mitotic spindle checkpoint activity predisposes to chromosomal instability at early stages of fibrosarcoma development. To our knowledge, these studies constitute the first report of a transition in MSCCC activity in a tumorigenesis model.
Assuntos
Aberrações Cromossômicas , Dano ao DNA/genética , Fibrossarcoma/genética , Genes cdc/fisiologia , Predisposição Genética para Doença/genética , Mitose/genética , Mutação/genética , Fuso Acromático/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/genética , Ciclina B/genética , Ciclina B/metabolismo , Progressão da Doença , Fibroma/genética , Camundongos , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Glucocorticoids influence many physiological processes, and in particular apoptosis, often with opposite effects depending on the cell type examined. We found that during fibrosarcoma development there is a strong increase in apoptosis at the tumor stage, which is repressed by dexamethasone to levels observed in normal fibroblasts. The anti-apoptotic Bcl-2 family protein Bcl-x(L) is induced by dexamethasone at the transcriptional level at all stages of fibrosarcoma development. The ligand-activated glucocorticoid receptor (GR) activates the Bcl-x promoter in transient transfection experiments, and GR binds to specific Bcl-x promoter sequences in vitro and in vivo. Furthermore, a GR antagonist abolishes this effect, indicating that Bcl-x(L) induction is mediated by GR. Importantly, exogenous Bcl-x(L) inhibits apoptosis and caspase-3 activity in fibrosarcoma cells to levels found in dexamethasone-treated fibrosarcoma cells. We conclude that Bcl-x(L) is a key target mediating the anti-apoptotic effects of glucocorticoids during fibrosarcoma development. These observations provide further understanding of the molecular basis of glucocorticoid regulation of cell death during tumorigenesis.