RESUMO
Lack or loss of tumor antigenicity represents one of the key mechanisms of immune escape and resistance to T cell-based immunotherapies. Evidence suggests that activation of stimulator of interferon genes (STING) signaling in tumor cells can augment their antigenicity by triggering a type I IFN-mediated sequence of autocrine and paracrine events. Although suppression of this pathway in melanoma and other tumor types has been consistently reported, the mechanistic basis remains unclear. In this study, we asked whether this suppression is, in part, epigenetically regulated and whether it is indeed a driver of melanoma resistance to T cell-based immunotherapies. Using genome-wide DNA methylation profiling, we show that promoter hypermethylation of cGAS and STING genes mediates their coordinated transcriptional silencing and contributes to the widespread impairment of the STING signaling function in clinically-relevant human melanomas and melanoma cell lines. This suppression is reversible through pharmacologic inhibition of DNA methylation, which can reinstate functional STING signaling in at least half of the examined cell lines. Using a series of T cell recognition assays with HLA-matched human melanoma tumor-infiltrating lymphocytes (TIL), we further show that demethylation-mediated restoration of STING signaling in STING-defective melanoma cell lines can improve their antigenicity through the up-regulation of MHC class I molecules and thereby enhance their recognition and killing by cytotoxic T cells. These findings not only elucidate the contribution of epigenetic processes and specifically DNA methylation in melanoma-intrinsic STING signaling impairment but also highlight their functional significance in mediating tumor-immune evasion and resistance to T cell-based immunotherapies.
Assuntos
Metilação de DNA , Epigênese Genética , Melanoma/genética , Proteínas de Membrana/genética , Linfócitos T/imunologia , Linhagem Celular Tumoral , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismoRESUMO
APCs are critical in T cell activation and in the induction of T cell tolerance. Epigenetic modifications of specific genes in the APC play a key role in this process, and among them histone deacetylases (HDACs) have emerged as key participants. HDAC6, one of the members of this family of enzymes, has been shown to be involved in regulation of inflammatory and immune responses. In this study, to our knowledge we show for the first time that genetic or pharmacologic disruption of HDAC6 in macrophages and dendritic cells results in diminished production of the immunosuppressive cytokine IL-10 and induction of inflammatory APCs that effectively activate Ag-specific naive T cells and restore the responsiveness of anergic CD4(+) T cells. Mechanistically, we have found that HDAC6 forms a previously unknown molecular complex with STAT3, association that was detected in both the cytoplasmic and nuclear compartments of the APC. By using HDAC6 recombinant mutants we identified the domain comprising amino acids 503-840 as being required for HDAC6 interaction with STAT3. Furthermore, by re-chromatin immunoprecipitation we confirmed that HDAC6 and STAT3 are both recruited to the same DNA sequence within the Il10 gene promoter. Of note, disruption of this complex by knocking down HDAC6 resulted in decreased STAT3 phosphorylation--but no changes in STAT3 acetylation--as well as diminished recruitment of STAT3 to the Il10 gene promoter region. The additional demonstration that a selective HDAC6 inhibitor disrupts this STAT3/IL-10 tolerogenic axis points to HDAC6 as a novel molecular target in APCs to overcome immune tolerance and tips the balance toward T cell immunity.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Histona Desacetilases/imunologia , Interleucina-10/imunologia , Fator de Transcrição STAT3/imunologia , Acetilação/efeitos dos fármacos , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Expressão Gênica , Regulação da Expressão Gênica , Desacetilase 6 de Histona , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Histona Desacetilases/genética , Tolerância Imunológica , Inflamação/imunologia , Interleucina-10/biossíntese , Ativação Linfocitária/imunologia , Macrófagos/enzimologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Oligopeptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno , Proteínas Recombinantes/genética , Fator de Transcrição STAT3/química , Transcrição GênicaRESUMO
While STING-activating agents have shown limited efficacy in early-phase clinical trials, multiple lines of evidence suggest the importance of tumor cell-intrinsic STING function in mediating antitumor immune responses. Although STING signaling is impaired in human melanoma, its restoration through epigenetic reprogramming can augment its antigenicity and T cell recognition. In this study, we show that reversal of methylation silencing of STING in murine melanoma cell lines using a clinically available DNA methylation inhibitor can improve agonist-induced STING activation and type-I IFN induction, which, in tumor-bearing mice, can induce tumor regression through a CD8+ T cell-dependent immune response. These findings not only provide mechanistic insight into how STING signaling dysfunction in tumor cells can contribute to impaired responses to STING agonist therapy, but also suggest that pharmacological restoration of STING signaling through epigenetic reprogramming might improve the therapeutic efficacy of STING agonists.
Assuntos
Antineoplásicos , Interferon Tipo I , Melanoma , Animais , Camundongos , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Imunidade , Interferon Tipo I/metabolismo , Epigênese GenéticaRESUMO
BACKGROUND: The temporal response to checkpoint blockade (CB) is incompletely understood. Here, we profiled the tumor infiltrating lymphocyte (TIL) landscape in response to combination checkpoint blockade at two distinct timepoints of solid tumor growth. METHODS: C57BL/6 mice bearing subcutaneous MC38 tumors were treated with anti-PD-1 and/or anti-CTLA-4 antibodies. At 11 or 21 days, TIL phenotype and effector function were analyzed in excised tumor digests using high parameter flow cytometry. The contributions of major TIL populations toward overall response were then assessed using ex vivo cytotoxicity and in vivo tumor growth assays. RESULTS: The distribution and effector function among 37 distinct TIL populations shifted dramatically between early and late MC38 growth. At 11 days, the immune response was dominated by Tumor necrosis factor alpha (TNFα)-producing NKT, representing over half of all TIL. These were accompanied by modest frequencies of natural killer (NK), CD4+, or CD8+ T cells, producing low levels of IFN-γ. At 21 days, NKT populations were reduced to a combined 20% of TIL, giving way to increased NK, CD4+, and CD8+ T cells, with increased IFN-γ production. Treatment with CB accelerated this switch. At day 11, CB reduced NKT to less than 20% of all TIL, downregulated TNFα across NKT and CD4+ T cell populations, increased CD4+ and CD8+ TIL frequencies, and significantly upregulated IFN-γ production. Degranulation was largely associated with NK and NKT TIL. Blockade of H-2kb and/or CD1d during ex vivo cytotoxicity assays revealed NKT has limited direct cytotoxicity against parent MC38. However, forced CD1d overexpression in MC38 cells significantly diminished tumor growth, suggesting NKT TIL exerts indirect control over MC38 growth. CONCLUSIONS: Despite an indirect benefit of early NKT activity, CB accelerates a switch from TNFα, NKT-driven immune response toward an IFN-γ driven CD4+/CD8+ T cell response in MC38 tumors. These results uncover a novel NKT/T cell switch that may be a key feature of CB response in CD1d+ tumors.
Assuntos
Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Interferon gama/metabolismo , Linfócitos T/metabolismo , Microambiente Tumoral/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Feminino , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , CamundongosRESUMO
STING (stimulator of IFN genes) signaling is an innate immune pathway for induction of a spontaneous antitumor T-cell response against certain immunogenic tumors. Although antigen-presenting cells are known to be involved in this process, insight into the participation of tumor cell-intrinsic STING signaling remains weak. In this study, we find diversity in the regulation of STING signaling across a panel of human melanoma cell lines. We show that intact activation of STING signaling in a subset of human melanoma cell lines enhances both their antigenicity and susceptibility to lysis by human melanoma tumor-infiltrating lymphocytes (TIL) through the augmentation of MHC class I expression. Conversely, defects in the STING signaling pathway protect melanoma cells from increased immune recognition by TILs and limit their sensitivity to TIL lysis. Based on these findings, we propose that defects in tumor cell-intrinsic STING signaling can mediate not only tumor immune evasion but also resistance to TIL-based immunotherapies.
Assuntos
Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Proteínas de Membrana/metabolismo , Transdução de Sinais/imunologia , Citotoxicidade Imunológica , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Linfócitos do Interstício Tumoral/metabolismo , Melanoma/metabolismo , Proteínas de Membrana/agonistas , Nucleotidiltransferases/metabolismo , Células Tumorais Cultivadas , Evasão Tumoral/imunologia , Regulação para CimaRESUMO
Tertiary lymphoid structures (TLSs) associate with better prognosis in certain cancer types, but their underlying formation and immunological benefit remain to be determined. We established a mouse model of TLSs to study their contribution to antitumor immunity. Because the stroma in lymph nodes (sLN) participates in architectural support, lymphogenesis, and lymphocyte recruitment, we hypothesized that TLSs can be created by sLN. We selected a sLN line with fibroblast morphology that expressed sLN surface markers and lymphoid chemokines. The subcutaneous injection of the sLN line successfully induced TLSs that attracted infiltration of host immune cell subsets. Injection of MC38 tumor lysate-pulsed dendritic cells activated TLS-residing lymphocytes to demonstrate specific cytotoxicity. The presence of TLSs suppressed MC38 tumor growth in vivo by improving antitumor activity of tumor-infiltrating lymphocytes with downregulated immune checkpoint proteins (PD-1 and Tim-3). Future engineering of sLN lines may allow for further enhancements of TLS functions and immune cell compositions.
RESUMO
Epigenetic changes in chromatin structure have been recently associated with the deregulated expression of critical genes in normal and malignant processes. HDAC11, the newest member of the HDAC family of enzymes, functions as a negative regulator of IL-10 expression in APCs, as previously described by our lab. However, at the present time, its role in other hematopoietic cells, specifically in neutrophils, has not been fully explored. In this report, for the first time, we present a novel physiologic role for HDAC11 as a multifaceted regulator of neutrophils. Thus far, we have been able to demonstrate a lineage-restricted overexpression of HDAC11 in neutrophils and committed neutrophil precursors (promyelocytes). Additionally, we show that HDAC11 appears to associate with the transcription machinery, possibly regulating the expression of inflammatory and migratory genes in neutrophils. Given the prevalence of neutrophils in the peripheral circulation and their central role in the first line of defense, our results highlight a unique and novel role for HDAC11. With the consideration of the emergence of new, selective HDAC11 inhibitors, we believe that our findings will have significant implications in a wide range of diseases spanning malignancies, autoimmunity, and inflammation.
Assuntos
Regulação da Expressão Gênica/imunologia , Hematopoese/imunologia , Histona Desacetilases/imunologia , Neutrófilos/enzimologia , Animais , Imunoprecipitação da Cromatina , Epigênese Genética , Citometria de Fluxo , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Reação em Cadeia da PolimeraseRESUMO
Histone deacetylase 10 (HDAC10) belongs to the class IIb HDAC family and its biological role remains mostly unidentified. A decreased HDAC10 expression has been reported in patients with aggressive solid tumors (Osada et al. Int J Cancer 112: 26-32, 2004; Jin et al. Int J Clin Exp Pathol 7: 5872-5879, 2014), suggesting that loss of HDAC10 expression might confer a survival advantage to malignant cells. Consequently, results from our lab suggests that overexpression of HDAC10 in aggressive mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) Z138c and MEC1 cells, respectively, resulted in a rapid induction of cell death in vitro with only 5 % of cells being alive at 48 h, cell cycle arrest, and up-regulation of co-stimulatory molecules. Here we present several standard methods to study the function of HDAC10 in B cell malignancies.
Assuntos
Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/metabolismo , Animais , Western Blotting , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The physiological role of histone deacetylase 11 (HDAC11), the newest member of the HDAC family, remained largely unknown until the discovery of its regulatory function in immune cells. Among them, the regulation of cytokine production by antigen-presenting cells and the modulation of the suppressive ability of myeloid-derived suppressor cells (MDSCs) (Sahakian et al. Mol Immunol 63: 579-585, 2015; Wang et al. J Immunol 186: 3986-3996, 2011; Villagra et al. Nat Immunol 10: 92-100, 2009). Our earlier data has demonstrated that HDAC11, by interacting at the chromatin level with the IL-10 promoter, downregulates il-10 transcription in both murine and human APCs in vitro and ex vivo models (Villagra et al. Nat Immunol 10: 92-100, 2009). However the role of HDAC11 in other cell types still remains unknown. Here we present several methods that can potentially be used to identify the functional role of HDAC11, assigning special attention to the evaluation of immunological parameters.
Assuntos
Imunoprecipitação da Cromatina/métodos , Citocinas/genética , Histona Desacetilases/metabolismo , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Citocinas/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica , Histona Desacetilases/genética , Camundongos , Células Supressoras Mieloides/citologia , Células Supressoras Mieloides/imunologia , Regiões Promotoras GenéticasRESUMO
The anti-inflammatory cytokine IL-10 is a key modulator of immune responses. A better understanding of the regulation of this cytokine offers the possibility of tipping the balance of the immune response toward either tolerance, or enhanced immune responses. Histone deacetylases (HDACs) have been widely described as negative regulators of transcriptional regulation, and in this context, the primarily nuclear protein HDAC11 was shown to repress il-10 gene transcriptional activity in antigen-presenting cells (APCs). Here we report that another HDAC, HDAC6, primarily a cytoplasmic protein, associates with HDAC11 and modulates the expression of IL-10 as a transcriptional activator. To our knowledge, this is the first demonstration of two different HDACs being recruited to the same gene promoter to dictate divergent transcriptional responses. This dynamic interaction results in dynamic changes in the expression of IL-10 and might help to explain the intrinsic plasticity of the APC to determine T-cell activation versus T-cell tolerance.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Histona Desacetilases/imunologia , Interleucina-10/genética , Animais , Linhagem Celular , Regulação da Expressão Gênica , Desacetilase 6 de Histona , Histona Desacetilases/genética , Tolerância Imunológica/genética , Tolerância Imunológica/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Interferência de RNA , RNA Interferente Pequeno , Linfócitos T/imunologia , Transcrição Gênica , Ativação Transcricional/imunologiaRESUMO
Melanoma is the deadliest skin cancer, and its incidence has been increasing faster than any other cancer. Although immunogenic, melanoma is not effectively cleared by host immunity. In this study, we investigate the therapeutic, antimelanoma potential of the histone deacetylase inhibitor (HDACi) panobinostat (LBH589) by assessing both its cytotoxic effects on melanoma cells as well as enhancement of immune recognition of melanoma. Utilizing murine and human melanoma cell lines, we analyzed the effects of LBH589 on proliferation and survival. In addition, we analyzed the expression of several immunologically relevant surface markers and melanoma differentiation antigens, and the ability of LBH589-treated melanoma to activate antigen-specific T cells. Finally, we assessed the in-vivo effects of LBH589 in a mouse melanoma model. Low nanomolar concentrations of LBH589 inhibit the growth of all melanoma cell lines tested, but not normal melanocytes. This inhibition is characterized by increased apoptosis as well as a G1 cell cycle arrest. In addition, LBH589 augments the expression of major histocompatibility complex and costimulatory molecules on melanoma cells leading to an increased ability to activate antigen-specific T cells. Treatment also increases expression of melanoma differentiation antigens. In vivo, LBH589 treatment of melanoma-bearing mice results in a significant increase in survival. However, in immunodeficient mice, the therapeutic effect of LBH589 is lost. Taken together, LBH589 exerts a dual effect upon melanoma cells by affecting not only growth/survival but also by increasing melanoma immunogenicity. These effects provide the framework for future evaluation of this HDAC inhibitor in melanoma treatment.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Imunidade Celular/efeitos dos fármacos , Indóis/farmacologia , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Hospedeiro Imunocomprometido , Ativação Linfocitária/efeitos dos fármacos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Melanoma/enzimologia , Melanoma/imunologia , Melanoma/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Panobinostat , Neoplasias Cutâneas/enzimologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The incidence of malignant melanoma has dramatically increased in recent years thus requiring the need for improved therapeutic strategies. In our efforts to design selective histone deactylase inhibitors (HDACI), we discovered that the aryl urea 1 is a modestly potent yet nonselective inhibitor. Structure-activity relationship studies revealed that adding substituents to the nitrogen atom of the urea so as to generate compounds bearing a branched linker group results in increased potency and selectivity for HDAC6. Compound 5 g shows low nanomolar inhibitory potency against HDAC6 and a selectivity of â¼600-fold relative to the inhibition of HDAC1. These HDACIs were evaluated for their ability to inhibit the growth of B16 melanoma cells with the most potent and selective HDAC6I being found to decrease tumor cell growth. To the best of our knowledge, this work constitutes the first report of HDAC6-selective inhibitors that possess antiproliferative effects against melanoma cells.