RESUMO
Advances in the understanding of the tumor microenvironment have led to development of immunotherapeutic strategies, such as chimeric antigen receptor T cells (CAR-Ts). However, despite success in blood malignancies, CAR-T therapies in solid tumors have been hampered by their restricted infiltration. Here, we used our understanding of early cytotoxic lymphocyte infiltration of human lymphocytes in solid tumors in vivo to investigate the receptors in normal, adjacent, and tumor tissues of primary non-small-cell lung cancer specimens. We found that CX3CL1-CX3CR1 reduction restricts cytotoxic cells from the solid-tumor bed, contributing to tumor escape. Based on this, we designed a CAR-T construct using the well-established natural killer group 2, member D (NKG2D) CAR-T expression together with overexpression of CX3CR1 to promote their infiltration. These CAR-Ts infiltrate tumors at higher rates than control-activated T cells or IL-15-overexpressing NKG2D CAR-Ts. This construct also had similar functionality in a liver-cancer model, demonstrating potential efficacy in other solid malignancies.
RESUMO
Myelodysplastic Syndromes (MDSs) are bone marrow (BM) failure malignancies characterized by constitutive innate immune activation, including NLRP3 inflammasome driven pyroptotic cell death. We recently reported that the danger-associated molecular pattern (DAMP) oxidized mitochondrial DNA (ox-mtDNA) is diagnostically increased in MDS plasma although the functional consequences remain poorly defined. We hypothesized that ox-mtDNA is released into the cytosol, upon NLRP3 inflammasome pyroptotic lysis, where it propagates and further enhances the inflammatory cell death feed-forward loop onto healthy tissues. This activation can be mediated via ox-mtDNA engagement of Toll-like receptor 9 (TLR9), an endosomal DNA sensing pattern recognition receptor known to prime and activate the inflammasome propagating the IFN-induced inflammatory response in neighboring healthy hematopoietic stem and progenitor cells (HSPCs), which presents a potentially targetable axis for the reduction in inflammasome activation in MDS. We found that extracellular ox-mtDNA activates the TLR9-MyD88-inflammasome pathway, demonstrated by increased lysosome formation, IRF7 translocation, and interferon-stimulated gene (ISG) production. Extracellular ox-mtDNA also induces TLR9 redistribution in MDS HSPCs to the cell surface. The effects on NLRP3 inflammasome activation were validated by blocking TLR9 activation via chemical inhibition and CRISPR knockout, demonstrating that TLR9 was necessary for ox-mtDNA-mediated inflammasome activation. Conversely, lentiviral overexpression of TLR9 sensitized cells to ox-mtDNA. Lastly, inhibiting TLR9 restored hematopoietic colony formation in MDS BM. We conclude that MDS HSPCs are primed for inflammasome activation via ox-mtDNA released by pyroptotic cells. Blocking the TLR9/ox-mtDNA axis may prove to be a novel therapeutic strategy for MDS.
Assuntos
DNA Mitocondrial , Inflamassomos , Síndromes Mielodisplásicas , Receptor Toll-Like 9 , Humanos , DNA Mitocondrial/metabolismo , Inflamassomos/metabolismo , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transdução de Sinais/fisiologia , Receptor Toll-Like 9/metabolismoRESUMO
We have reported previously that CD33hi myeloid-derived suppressor cells (MDSCs) play a direct role in the pathogenesis of myelodysplastic syndromes (MDSs) and that their sustained activation contributes to hematopoietic and immune impairment, including modulation of PD1/PDL1. MDSCs can also limit the clinical activity of immune checkpoint inhibition in solid malignancies. We hypothesized that depletion of MDSCs may ameliorate resistance to checkpoint inhibitors and, hence, targeted them with AMV564 combined with anti-PD1 in MDS bone marrow (BM) mononuclear cells (MNCs) enhanced activation of cytotoxic T cells. AMV564 was active in vivo in a leukemia xenograft model when co-administered with healthy donor peripheral blood MNCs (PBMCs). Our findings provide a strong rationale for clinical investigation of AMV564 as a single agent or in combination with an anti-PD1 antibody and in particular for treatment of cancers resistant to checkpoint inhibitors.
Assuntos
Anticorpos Biespecíficos , Antineoplásicos , Melanoma , Síndromes Mielodisplásicas , Células Supressoras Mieloides , Animais , Anticorpos Biespecíficos/farmacologia , Antineoplásicos/farmacologia , Humanos , Melanoma/tratamento farmacológico , Síndromes Mielodisplásicas/tratamento farmacológico , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Linfócitos TRESUMO
The clinical utility of histone/protein deacetylase (HDAC) inhibitors in combinatorial regimens with proteasome inhibitors for patients with relapsed and refractory multiple myeloma (MM) is often limited by excessive toxicity due to HDAC inhibitor promiscuity with multiple HDACs. Therefore, more selective inhibition minimizing off-target toxicity may increase the clinical effectiveness of HDAC inhibitors. We demonstrated that plasma cell development and survival are dependent upon HDAC11, suggesting this enzyme is a promising therapeutic target in MM. Mice lacking HDAC11 exhibited markedly decreased plasma cell numbers. Accordingly, in vitro plasma cell differentiation was arrested in B cells lacking functional HDAC11. Mechanistically, we showed that HDAC11 is involved in the deacetylation of IRF4 at lysine103. Further, targeting HDAC11 led to IRF4 hyperacetylation, resulting in impaired IRF4 nuclear localization and target promoter binding. Importantly, transient HDAC11 knockdown or treatment with elevenostat, an HDAC11-selective inhibitor, induced cell death in MM cell lines. Elevenostat produced similar anti-MM activity in vivo, improving survival among mice inoculated with 5TGM1 MM cells. Elevenostat demonstrated nanomolar ex vivo activity in 34 MM patient specimens and synergistic activity when combined with bortezomib. Collectively, our data indicated that HDAC11 regulates an essential pathway in plasma cell biology establishing its potential as an emerging theraputic vulnerability in MM.
Assuntos
Inibidores de Histona Desacetilases/uso terapêutico , Histonas/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Plasmócitos/metabolismo , Animais , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Mieloma Múltiplo/fisiopatologiaRESUMO
Dendritic cells (DCs) are professional antigen presenting cells with a great capacity for cross-presentation of exogenous antigens from which robust anti-tumor immune responses ensue. However, this function is not always available and requires DCs to first be primed to induce their maturation. In particular, in the field of DC vaccine design, currently available methodologies have been limited in eliciting a sustained anti-tumor immune response. Mechanistically, part of the maturation response is influenced by the presence of stimulatory receptors relying on ITAM-containing activating adaptor molecules like DAP12, that modulates their function. We hypothesize that activating DAP12 in DC could force their maturation and enhance their potential anti-tumor activity for therapeutic intervention. For this purpose, we developed constitutively active DAP12 mutants that can promote activation of monocyte-derived DC. Here we demonstrate its ability to induce the maturation and activation of monocyte-derived DCs which enhances migration, and T cell stimulation in vitro using primary human cells. Moreover, constitutively active DAP12 stimulates a strong immune response in a murine melanoma model leading to a reduction of tumor burden. This provides proof-of-concept for investigating the pre-activation of antigen presenting cells to enhance the effectiveness of anti-tumor immunotherapies.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Células Dendríticas/imunologia , Imunidade Celular/imunologia , Melanoma Experimental/imunologia , Proteínas de Membrana/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Vacinas Anticâncer/imunologia , Movimento Celular/genética , Proliferação de Células/genética , Humanos , Imunidade Celular/genética , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Proteínas de Membrana/imunologia , Camundongos , Monócitos/imunologia , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Carga Tumoral/imunologiaRESUMO
Traditionally, basic scientists have not been as engaged in the translational continuum when it comes to engagement with patients, caregivers, and other community stakeholders. In order to address this discrepancy, a multi-disciplinary team at Moffitt Cancer Center conceived of and enacted the Patient-Researcher Forum (PRF) to promote a community-engaged research approach through communication, compassion, and bi-directional research insight for both patients/caregivers and researchers. We outline the structure and implementation of the PRF, its participants, and qualitative and quantitative results across 14 sessions. PRF sessions were conducted between July 2018 and October 2019 and included 29 patients/caregivers and close to 200 researcher/staff participants; post participation survey response rates assessing the PRF experience were 27.6% (patients/caregivers) and 60.3% (researchers) on average. Research staff overwhelmingly reported that the PRF was beneficial, citing that it helped them gain new patient-centered perspectives and helped them practice communicating research to lay audiences. Patients/caregivers also reported that the PRF was valuable, indicating that they gained a better understanding of research and that they developed a personal connection with researchers. Our PRF model may provide a strategy for improving basic scientist communication, ethics, and understanding of research impacts on the populations they wish to serve. This innovative model provides a much-needed direct connection between basic scientists and patients/caregivers which creates a 2-way learning platform that fosters understanding and research ideas in the spirit of community-engaged research.
Assuntos
Cuidadores , Família , Modelos Organizacionais , Assistência Centrada no Paciente , Institutos de Câncer/organização & administração , Humanos , PesquisaRESUMO
Mantle Cell Lymphoma (MCL) is a non-Hodgkin lymphoma with a median survival rate of five years. Standard treatment with high-dose chemotherapy plus rituximab (anti-CD20 antibody) has extended overall survival although, the disease remains incurable. Histone deacetylases (HDAC) are a family of enzymes that regulate multiple proteins and cellular pathways through post-translational modification. Broad spectrum HDAC inhibitors have shown some therapeutic promise, inducing cell cycle inhibition and apoptosis in leukemia and non-Hodgkin's lymphoma. However, the therapeutic effects of these broad-spectrum HDAC inhibitors can detrimentally dampen Natural Killer (NK) cell cytotoxicity, reduce NK viability, and downregulate activation receptors important for NK mediated anti-tumor responses. Impairment of NK function in MCL patients during therapy potentially limits therapeutic activity of rituximab. Thus, there is an unmet need to decipher specific roles of individual HDACs in order to preserve and/or enhance NK function, while, directly impairing MCL viability. We investigated the impact of HDAC8 in MCL cell lines. Inhibition or genetic loss of HDAC8 caused MCL cells to undergo apoptosis. In contrast, exposure of primary human NK cells to an HDAC8 inhibitor does not alter viability, receptor expression, or antibody dependent cellular cytotoxicity (ADCC). However, an increase in effector cytokine interferon-gamma (IFNγ) producing NK cells was observed in response to HDAC8 inhibition. Taken together these data suggest that selective HDAC8 inhibitors may simultaneously preserve NK functional activity, while impairing MCL tumor growth, establishing a rationale for future clinical evaluation.
Assuntos
Células Matadoras Naturais/efeitos dos fármacos , Linfoma de Célula do Manto/tratamento farmacológico , Proteínas Repressoras/antagonistas & inibidores , Antineoplásicos Imunológicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Linfoma de Célula do Manto/imunologia , Linfoma de Célula do Manto/patologia , Proteínas Repressoras/genética , Rituximab/farmacologiaRESUMO
The Janus kinase 2 (JAK2)-driven myeloproliferative neoplasms (MPNs) are associated with clonal myelopoiesis, elevated risk of death due to thrombotic complications, and transformation to acute myeloid leukemia (AML). JAK2 inhibitors improve the quality of life for MPN patients, but these approved therapeutics do not readily reduce the natural course of disease or antagonize the neoplastic clone. An understanding of the molecular and cellular changes requisite for MPN development and progression are needed to develop improved therapies. Recently, murine MPN models were demonstrated to exhibit metabolic vulnerabilities due to a high dependence on glucose. Neoplastic hematopoietic progenitor cells in these mice express elevated levels of glycolytic enzymes and exhibit enhanced levels of glycolysis and oxidative phosphorylation, and the disease phenotype of these MPN model mice is antagonized by glycolytic inhibition. While all MPN-driving mutations lead to aberrant JAK2 activation, these mutations often co-exist with mutations in genes that encode epigenetic regulators, including loss of function mutations known to enhance MPN progression. In this perspective we discuss how altered activity of epigenetic regulators (e.g., methylation and acetylation) in MPN-driving stem and progenitor cells may alter cellular metabolism and contribute to the MPN phenotype and progression of disease. Specific metabolic changes associated with epigenetic deregulation may identify patient populations that exhibit specific metabolic vulnerabilities that are absent in normal hematopoietic cells, and thus provide a potential basis for the development of more effective personalized therapeutic approaches.
Assuntos
Biomarcadores Tumorais/genética , Metabolismo Energético , Epigênese Genética , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/genética , Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Animais , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/genética , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Inibidores de Janus Quinases/uso terapêutico , Terapia de Alvo Molecular , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/metabolismo , Neoplasias/metabolismo , FenótipoRESUMO
Protein acetylation is an important contributor to cancer initiation. Histone deacetylase 6 (HDAC6) controls JAK2 translation and protein stability and has been implicated in JAK2-driven diseases best exemplified by myeloproliferative neoplasms (MPNs). By using novel classes of highly selective HDAC inhibitors and genetically deficient mouse models, we discovered that HDAC11 rather than HDAC6 is necessary for the proliferation and survival of oncogenic JAK2-driven MPN cells and patient samples. Notably, HDAC11 is variably expressed in primitive stem cells and is expressed largely upon lineage commitment. Although Hdac11is dispensable for normal homeostatic hematopoietic stem and progenitor cell differentiation based on chimeric bone marrow reconstitution, Hdac11 deficiency significantly reduced the abnormal megakaryocyte population, improved splenic architecture, reduced fibrosis, and increased survival in the MPLW515L-MPN mouse model during primary and secondary transplantation. Therefore, inhibitors of HDAC11 are an attractive therapy for treating patients with MPN. Although JAK2 inhibitor therapy provides substantial clinical benefit in MPN patients, the identification of alternative therapeutic targets is needed to reverse MPN pathogenesis and control malignant hematopoiesis. This study establishes HDAC11 as a unique type of target molecule that has therapeutic potential in MPN.
Assuntos
Hematopoese , Histona Desacetilases/fisiologia , Mutação , Transtornos Mieloproliferativos/patologia , Oncogenes , Animais , Apoptose , Ciclo Celular , Proliferação de Células , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Humanos , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Células Tumorais CultivadasRESUMO
The data presented here are related to the research article entitled "Selective expression of the transcription elongation factor ELL3 in B cells prior to ELL2 drives proliferation and survival" (Alexander et al., 2017) [1]. The cited research article characterizes Eleven-nineteen Lysine-rich Leukemia 3 (ELL3) expression in the B cell compartment and functional dependence in B lymphoma cell lines. This data report describes the mRNA expression pattern in a panel of cell lines representing the B cell compartment, supplementing the protein expression data presented in the associated research report. In addition, a reanalysis is presented of publicly available mRNA expression data from primary murine B cells to reveal dynamic regulation of the ELL family members post LPS stimulation (Barwick et al., 2016) [2]. The effect of ELL3 depletion on cell morphology, latent Epstein Barr Virus (EBV) lytic replication and differentiation markers in a Burkitt's lymphoma (BL) cell line cells are presented.
RESUMO
B cell activation is dependent on a large increase in transcriptional output followed by focused expression on secreted immunoglobulin as the cell transitions to an antibody producing plasma cell. The rapid transcriptional induction is facilitated by the release of poised RNA pol II into productive elongation through assembly of the super elongation complex (SEC). We report that a SEC component, the Eleven -nineteen Lysine-rich leukemia (ELL) family member 3 (ELL3) is dynamically up-regulated in mature and activated human B cells followed by suppression as B cells transition to plasma cells in part mediated by the transcription repressor PRDM1. Burkitt's lymphoma and a sub-set of Diffuse Large B cell lymphoma cell lines abundantly express ELL3. Depletion of ELL3 in the germinal center derived lymphomas results in severe disruption of DNA replication and cell division along with increased DNA damage and cell death. This restricted utilization and survival dependence reveal a key step in B cell activation and indicate a potential therapeutic target against B cell lymphoma's with a germinal center origin.
Assuntos
Linfócitos B/imunologia , Divisão Celular/imunologia , Regulação da Expressão Gênica/imunologia , Fatores de Elongação da Transcrição/imunologia , Divisão Celular/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Replicação do DNA/genética , Replicação do DNA/imunologia , Humanos , Células Jurkat , RNA Polimerase II/genética , RNA Polimerase II/imunologia , Fatores de Elongação da Transcrição/genéticaRESUMO
UNLABELLED: Epstein-Barr virus (EBV) maintains a lifelong latent infection within a subset of its host's memory B cells, while lytic EBV replication takes place in plasma cells and differentiated epithelial cells. Therefore, cellular transcription factors, such as BLIMP1, that are key mediators of differentiation likely contribute to the EBV latent-to-lytic switch. Previous reports showed that ectopic BLIMP1 expression induces reactivation in some EBV-positive (EBV(+)) B-cell lines and transcription from Zp, with all Z(+) cells in oral hairy leukoplakia being BLIMP1(+). Here, we examined BLIMP1's role in inducing EBV lytic gene expression in numerous EBV(+) epithelial and B-cell lines and activating transcription from Rp. BLIMP1 addition was sufficient to induce reactivation in latently infected epithelial cells derived from gastric cancers, nasopharyngeal carcinomas, and normal oral keratinocytes (NOK) as well as some, but not all B-cell lines. BLIMP1 strongly induced transcription from Rp as well as Zp, with there being three or more synergistically acting BLIMP1-responsive elements (BRE) within Rp. BLIMP1's DNA-binding domain was required for reactivation, but BLIMP1 did not directly bind the nucleotide (nt) -660 Rp BRE. siRNA knockdown of BLIMP1 inhibited 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced lytic reactivation in NOK-Akata cells, cells that can be reactivated by R, but not Z. Thus, we conclude that BLIMP1 expression is both necessary and sufficient to induce EBV lytic replication in many (possibly all) EBV(+) epithelial-cell types, but in only a subset of EBV(+) B-cell types; it does so, at least in part, by strongly activating expression of both EBV immediately early genes, BZLF1 and BRLF1. IMPORTANCE: This study is the first one to show that the cellular transcription factor BLIMP1, a key player in both epithelial and B-cell differentiation, induces reactivation of the oncogenic herpesvirus Epstein-Barr virus (EBV) out of latency into lytic replication in a variety of cancerous epithelial cell types as well as in some, but not all, B-cell types that contain this virus in a dormant state. The mechanism by which BLIMP1 does so involves strongly turning on expression of both of the immediate early genes of the virus, probably by directly acting upon the promoters as part of protein complexes or indirectly by altering the expression or activities of some cellular transcription factors and signaling pathways. The fact that EBV(+) cancers usually contain mostly undifferentiated cells may be due in part to these cells dying from lytic EBV infection when they differentiate and express wild-type BLIMP1.
Assuntos
Linfócitos B/virologia , Células Epiteliais/virologia , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Ativação Viral , Linhagem Celular , Herpesvirus Humano 4/genética , Humanos , Proteínas Imediatamente Precoces/biossíntese , Fator 1 de Ligação ao Domínio I Regulador Positivo , Transativadores/biossíntese , Transcrição Gênica , Latência ViralRESUMO
Barriers persist in the development and delivery of effective cancer therapies to under-represented minority populations. In Puerto Rico, cancer is the second leading cause of death, yet cancer research awareness and training opportunities remain somewhat limited on the island. These limitations hinder progress toward decreasing the cancer health disparities that exist within the Puerto Rican population. The predominantly Hispanic population of Puerto Rico is the focus of a partnership between the Ponce Health Sciences University-Medical School and Ponce Research Institute (PHSU) in Ponce, Puerto Rico and the H. Lee Moffitt Cancer Center in Tampa, Florida. The Partnership goals are to reduce these barriers through an integrated, multipronged approach of training and education alongside outreach and research components. This report describes the approaches, successes and challenges of enhancing clinical cancer research capacity on the island and the unique challenges of a partnership between two institutes physically separated by long distances. Once fully developed this model may be exportable to other Latin American countries where the need is even greater.
Assuntos
Pesquisa Biomédica/estatística & dados numéricos , Educação Médica/métodos , Hispânico ou Latino , Oncologia/educação , Neoplasias/etnologia , Humanos , Neoplasias/prevenção & controle , Porto Rico/etnologia , Estados Unidos/epidemiologiaRESUMO
Cancer-associated protein tyrosine kinase (PTK) mutations usually are gain-of-function (GOF) mutations that drive tumor growth and metastasis. We have found 50 JAK1 truncating mutations in 36 of 635 gynecologic tumors in the Total Cancer Care® (TCC®) tumor bank. Among cancer cell lines containing JAK1 truncating mutations in the Cancer Cell Line Encyclopedia databank, 68% are gynecologic cancer cells. Within JAK1 the K142, P430, and K860 frame-shift mutations were identified as hot spot mutation sites. Sanger sequencing of cancer cell lines, primary tumors, and matched normal tissues confirmed the JAK1 mutations and showed that these mutations are somatic. JAK1 mediates interferon (IFN)-γ-regulated tumor immune surveillance. Functional assays show that JAK1 deficient cancer cells are defective in IFN-γ-induced LMP2 and TAP1 expression, loss of which inhibits presentation of tumor antigens. These findings identify recurrent JAK1 truncating mutations that could contribute to tumor immune evasion in gynecologic cancers, especially in endometrial cancer.
Assuntos
Neoplasias dos Genitais Femininos/genética , Janus Quinase 1/genética , Mutação , Proteínas Tirosina Quinases/genética , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Apresentação de Antígeno/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Análise Mutacional de DNA , Feminino , Neoplasias dos Genitais Femininos/metabolismo , Antígenos HLA/metabolismo , Humanos , Imunofenotipagem , Interferon gama/farmacologia , Janus Quinase 1/metabolismo , Regiões Promotoras Genéticas , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
Dendritic cell (DC) maturation is characterized by upregulation of cell-surface MHC class II (MHC-II) and costimulatory molecules, and production of a variety of cytokines that can shape both innate and adaptive immunity. Paradoxically, transcription of the MHC-II genes, as well as its activator, CIITA, is rapidly silenced during DC maturation. The mechanisms that control CIITA/MHC-II expression and silencing have not been fully understood. We report in this article that the tumor suppressor tuberous sclerosis complex 1 (TSC1) is a critical regulator of DC function for both innate and adaptive immunity. Its deficiency in DCs results in increased mammalian target of rapamycin (mTOR) complex 1 but decreased mTORC2 signaling, altered cytokine production, impaired CIITA/MHC-II expression, and defective Ag presentation to CD4 T cells after TLR4 stimulation. We demonstrate further that IFN regulatory factor 4 can directly bind to CIITA promoters, and decreased IFN regulatory factor 4 expression is partially responsible for decreased CIITA/MHC-II expression in TSC1-deficient DCs. Moreover, we identify that CIITA/MHC-II silencing during DC maturation requires mTOR complex 1 activity. Together, our data reveal unexpected roles of TSC1/mTOR that control multifaceted functions of DCs.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Fatores Reguladores de Interferon/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Apresentação de Antígeno/imunologia , Células da Medula Óssea/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Células Cultivadas , Antígenos de Histocompatibilidade Classe II/imunologia , Ativação Linfocitária , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/metabolismo , Receptor 4 Toll-Like/metabolismo , Transativadores/genética , Proteína 1 do Complexo Esclerose TuberosaRESUMO
We investigated the transcriptional and epigenetic repression of miR-29 by MYC, HDAC3, and EZH2 in mantle cell lymphoma and other MYC-associated lymphomas. We demonstrate that miR-29 is repressed by MYC through a corepressor complex with HDAC3 and EZH2. MYC contributes to EZH2 upregulation via repression of the EZH2 targeting miR-26a, and EZH2 induces MYC via inhibition of the MYC targeting miR-494 to create positive feedback. Combined inhibition of HDAC3 and EZH2 cooperatively disrupted the MYC-EZH2-miR-29 axis, resulting in restoration of miR-29 expression, downregulation of miR-29-targeted genes, and lymphoma growth suppression in vitro and in vivo. These findings define a MYC-mediated miRNA repression mechanism, shed light on MYC lymphomagenesis mechanisms, and reveal promising therapeutic targets for aggressive B-cell malignancies.
Assuntos
Histona Desacetilases/fisiologia , Histonas/metabolismo , Linfoma de Células B/etiologia , MicroRNAs/fisiologia , Complexo Repressor Polycomb 2/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste , Inativação Gênica , Humanos , Linfoma de Células B/genética , Linfoma de Células B/terapia , Metilação , Camundongos , MicroRNAs/análise , MicroRNAs/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
B-lymphocyte-induced maturation protein 1 (BLIMP1) exists as two major isoforms, α and ß, which arise from alternate promoters. Inactivation of the full length BLIMP1α isoform is thought to contribute to B cell lymphomagenesis by blocking post-germinal centre (GC) B cell differentiation. In contrast, the shorter ß isoform is functionally impaired and over-expressed in several haematological malignancies, including diffuse large B cell lymphomas (DLBCL). We have studied the influence on BLIMP1ß expression of the Epstein-Barr virus (EBV), a human herpesvirus that is implicated in the pathogenesis of several GC-derived lymphomas, including a subset of DLBCL and Hodgkin's lymphoma (HL). We show that BLIMP1ß expression is increased following the EBV infection of normal human tonsillar GC B cells. We also show that this change in expression is accompanied by hypomethylation of the BLIMP1ß-specific promoter. Furthermore, we confirmed previous reports that the BLIMP1ß promoter is hypomethylated in DLBCL cell lines and show for the first time that BLIMP1ß is hypomethylated in the Hodgkin/Reed-Sternberg (HRS) cells of HL. Our results provide evidence in support of a role for BLIMP1ß in the pathogenesis of EBV-associated B cell lymphomas.
RESUMO
Human germinal center-associated lymphoma (HGAL) and LIM domain only-2 (LMO2) are proteins highly expressed in germinal center (GC) B lymphocytes. HGAL and LMO2 are also expressed in GC-derived lymphomas and distinguish biologically distinct subgroups of diffuse large B-cell lymphomas (DLBCL) associated with improved survival. However, little is known about their regulation. PRDM1/Blimp1 is a master regulator of terminal B cell differentiation and may also function as a tumor suppressor in the pathogenesis of DLBCL, where it is frequently inactivated by mutations and deletions. We now demonstrate that both HGAL and LMO2 are directly regulated by the transcription repressor PRDM1. In vivo studies demonstrate that PRDM1 directly binds to the recognition sites within the upstream promoters of both HGAL and LMO2. PRDM1 binding suppresses endogenous protein and mRNA levels of HGAL and LMO2. In addition, promoter analysis reveals that site-specific binding of PRDM1 to the promoters is capable of repressing transcriptional activity. This inhibitory effect of PRDM1 suggests that it has a key role in the loss of HGAL and LMO2 expression upon differentiation of GC B cells to plasma cells and may also contribute to absence of HGAL and LMO2 expression in post-GC lymphoid tumors.
Assuntos
Linfócitos B/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Metaloproteínas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Genes Reporter , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas com Domínio LIM , Linfoma de Células B/metabolismo , Metaloproteínas/genética , Proteínas dos Microfilamentos , Proteínas de Neoplasias/genética , Fator 1 de Ligação ao Domínio I Regulador Positivo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
An important pathogenic event in Epstein-Barr virus (EBV)-associated lymphomas is the suppression of virus replication, which would otherwise lead to cell death. Because virus replication in B cells is intimately linked to their differentiation toward plasma cells, we asked whether the physiologic signals that drive normal B-cell differentiation are absent in EBV-transformed cells. We focused on BLIMP1α, a transcription factor that is required for plasma cell differentiation and that is inactivated in diffuse large B-cell lymphomas. We show that BLIMP1α expression is down-regulated after EBV infection of primary germinal center B cells and that the EBV oncogene, latent membrane protein-1 (LMP-1), is alone capable of inducing this down-regulation in these cells. Furthermore, the down-regulation of BLIMP1α by LMP-1 was accompanied by a partial disruption of the BLIMP1α transcriptional program, including the aberrant induction of MYC, the repression of which is required for terminal differentiation. Finally, we show that the ectopic expression of BLIMP1α in EBV-transformed cells can induce the viral lytic cycle. Our results suggest that LMP-1 expression in progenitor germinal center B cells could contribute to the pathogenesis of EBV-associated lymphomas by down-regulating BLIMP1α, in turn preventing plasma cell differentiation and induction of the viral lytic cycle.