RESUMEN
To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.
Asunto(s)
Biocatálisis , Histonas/metabolismo , Oncogenes , Transcripción Genética , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Línea Celular , Cromatina/metabolismo , Proteínas Co-Represoras/metabolismo , Secuencia Conservada , Evolución Molecular , Redes Reguladoras de Genes , Genoma , Histona Desacetilasas/metabolismo , Humanos , Cinética , Metilación , Modelos Biológicos , ARN Polimerasa II/metabolismoRESUMEN
Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.
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Quinasa 9 Dependiente de la Ciclina/metabolismo , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteína Fosfatasa 2/metabolismo , Proteínas de Unión al ARN/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Línea Celular Tumoral , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Endogámicos NOD , Fosforilación , Unión Proteica , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , Especificidad por SustratoRESUMEN
Chronic stimulation of CD8+ T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells.
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Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Anergia Clonal/inmunología , Proteína 2 de la Respuesta de Crecimiento Precoz/metabolismo , Linfopoyesis/fisiología , Animales , Antígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Proteína 2 de la Respuesta de Crecimiento Precoz/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.
RESUMEN
UNLABELLED: Human immunodeficiency virus (HIV) persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs) are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi) may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART). The primary endpoint was change in cell associated unspliced (CA-US) HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065). Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90%) with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1). CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells. TRIAL REGISTRATION: ClinicalTrials.gov NCT01365065.
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Linfocitos T CD4-Positivos/virología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Inhibidores de Histona Desacetilasas/uso terapéutico , Ácidos Hidroxámicos/uso terapéutico , Activación Viral/efectos de los fármacos , Adulto , Linfocitos T CD4-Positivos/inmunología , Femenino , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , VIH-1/fisiología , Humanos , Activación de Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , ARN Viral/genética , Transcripción Genética/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , VorinostatRESUMEN
Immunohistochemistry is commonly used to show the presence of apoptotic cells in situ. In this protocol, B-cell lymphoma cells are injected into recipient mice and, on tumor formation, the mice are treated with the apoptosis inducer vorinostat (a histone deacetylase inhibitor). Tumor samples are fixed and sectioned, and fragmented DNA (a feature of apoptotic cells) is end-labeled by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Immunohistochemical methods are then used to detect the labeled DNA and identify B-cell lymphoma cells in the last stage of apoptosis. Because the assay can lead to false-positive results, it is advisable to carry out an additional assay (e.g., immunohistochemistry for active caspase-3) to confirm the presence of apoptotic cells.
Asunto(s)
Apoptosis , Inmunohistoquímica/métodos , Linfoma de Células B/patología , Animales , ADN/análisis , Fragmentación del ADN , Inhibidores de Histona Desacetilasas/administración & dosificación , Ácidos Hidroxámicos/administración & dosificación , Etiquetado Corte-Fin in Situ , Ratones , VorinostatRESUMEN
Flow cytometry assays are often used to detect apoptotic cells in in vitro cultures. Depending on the experimental model, these assays can also be useful in evaluating apoptosis in vivo. In this protocol, we describe a propidium iodide (PI) flow cytometry assay to evaluate B-cell lymphomas that have undergone apoptosis in vivo. B-cell lymphoma cells are injected into recipient mice and, on tumor formation, the mice are treated with the apoptosis inducer vorinostat (a histone deacetylase inhibitor). Tumor samples collected from the lymph nodes and/or the spleen are used to prepare a single-cell suspension that is exposed to a hypotonic solution containing the fluorochrome PI. The DNA content of the cells, now labeled with PI, is analyzed by flow cytometry. Nuclear DNA content is lost during apoptosis, resulting in a hypodiploid (or sub-G1) DNA profile during flow cytometry. In contrast, healthy cells display a sharp diploid DNA profile.
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Apoptosis , Citometría de Flujo/métodos , Linfoma de Células B/patología , Propidio/metabolismo , Coloración y Etiquetado/métodos , Animales , ADN/análisis , Inhibidores de Histona Desacetilasas/administración & dosificación , Ácidos Hidroxámicos/administración & dosificación , Ratones , VorinostatRESUMEN
Apoptosis is a mode of cell death that is essential in multicellular organisms for the removal of superfluous, damaged, or potentially dangerous cells during development, infection, or normal tissue homeostasis. To prevent inflammation, cells undergoing apoptosis produce "find-me" signals that trigger the recruitment of phagocytes, which clear the apoptotic cells on recognition of "eat-me" signals. Despite the loss of billions of cells per day by apoptosis in the human body, the number of apoptotic cells found in healthy tissue is surprisingly low and reflects the efficiency of this process. However, in certain conditions (e.g., in cancer cells responding to chemotherapy), the number of apoptotic cells is too high to be efficiently cleared by phagocytes, and apoptotic cells can be observed. In these situations, the detection of apoptosis may be helpful in monitoring disease progression as well as in predicting the responses of tumors to anticancer therapies. Here we introduce various methods for monitoring apoptotic cells in vivo using a murine model of B-cell lymphoma and a solid tumor xenograft.
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Apoptosis , Linfoma de Células B/patología , Animales , Xenoinjertos/patología , Ratones , Trasplante de NeoplasiasRESUMEN
Positron emission tomography (PET) can be used to monitor the uptake of the labeled glucose analog fluorodeoxyglucose (¹8F-FDG), a process that is generally believed to reflect viable tumor cell mass. The use of ¹8F-FDG PET can be helpful in documenting over time the reduction in tumor mass volume in response to anticancer drug therapy in vivo. In this protocol, we describe how to monitor the response of murine B-cell lymphomas to an inducer of apoptosis, the anticancer drug vorinostat (a histone deacetylase inhibitor). B-cell lymphoma cells are injected into recipient mice and, on tumor formation, the mice are treated with vorinostat. The tracer ¹8F-FDG is then injected into the mice at several time points, and its uptake is monitored using PET. Because the uptake of ¹8F-FDG is not a direct measure of apoptosis, an additional direct method proving that apoptotic cells are present should also be performed.
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Antineoplásicos/uso terapéutico , Fluorodesoxiglucosa F18 , Ácidos Hidroxámicos/uso terapéutico , Linfoma de Células B/diagnóstico por imagen , Linfoma de Células B/tratamiento farmacológico , Tomografía de Emisión de Positrones , Animales , Línea Celular Tumoral , Humanos , Ratones , Ratones Endogámicos C57BL , Vorinostat , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Positron emission tomography (PET) is used to monitor the uptake of the labeled glucose analogue fluorodeoxyglucose (¹8F-FDG) by solid tumor cells, a process generally believed to reflect viable tumor cell mass. The use of ¹8F-FDG exploits the high demand for glucose in tumor cells, and serves to document over time the response of a solid tumor to an inducer of apoptosis. The apoptosis inducer crizotinib is a small-molecule inhibitor of c-Met, a receptor tyrosine kinase that is often dysregulated in human tumors. In this protocol, we describe how to monitor the response of a solid tumor to crizotinib. Human gastric tumor cells (GTL-16 cells) are injected into recipient mice and, on tumor formation, the mice are treated with crizotinib. The tracer ¹8F-FDG is then injected into the mice at several time points, and its uptake is monitored using PET. Because ¹8F-FDG uptake varies widely among different tumor models, preliminary experiments should be performed with each new model to determine its basal level of ¹8F-FDG uptake. Verifying that the basal level of uptake is sufficiently above background levels will assure accurate quantitation. Because ¹8F-FDG uptake is not a direct measure of apoptosis, it is advisable to carry out an additional direct method to show the presence of apoptotic cells.
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Antineoplásicos/uso terapéutico , Fluorodesoxiglucosa F18 , Tomografía de Emisión de Positrones , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Neoplasias Gástricas/diagnóstico por imagen , Neoplasias Gástricas/tratamiento farmacológico , Animales , Línea Celular Tumoral , Crizotinib , Modelos Animales de Enfermedad , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Signal transducer and activator of transcription 1 (Stat1) mediates anti-viral responses and cytokine-driven anti-proliferative, apoptotic and immunomodulatory activities. As de-regulated Stat1 function can affect tumour progression we sought to elucidate the effects of tumour cell-intrinsic Stat1 expression on immunosurveillance. Knockout of Stat1 enhanced the development of sarcomas induced by the chemical carcinogen 3-methylcholanthrene (MCA). Growth of transplanted MCA-induced Stat1â»/â» sarcomas was suppressed in wild-type mice compared to growth in Stat1â»/â» and immunocompromised recipients. Co-depletion of NK and CD8⺠T cells from wild-type mice facilitated Stat1-deficient tumour growth whereas depletion of CD4⺠T cells and CD8⺠T cells did not. In vitro and in vivo analysis of the tumours implicated a role for NK cell-mediated, perforin-dependent killing of Stat1-deficient tumours. Interestingly, restoration of Stat1 expression in Stat1â»/â» tumours resulted in diminished involvement of NK cells and increased contribution of CD8⺠T cells in anti-tumour responses. Therefore, Stat1 expression within tumour cells modulated anti-tumour immune responses by altering the dominant immune effector cell involvement from NK cells to CD8⺠T cells in the absence or presence of Stat1 respectively.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Asesinas Naturales/inmunología , Factor de Transcripción STAT1/metabolismo , Sarcoma Experimental/inmunología , Animales , Procesos de Crecimiento Celular/genética , Células Cultivadas , Citotoxicidad Inmunológica , Regulación Neoplásica de la Expresión Génica , Humanos , Depleción Linfocítica , Metilcolantreno/toxicidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monitorización Inmunológica , Trasplante de Neoplasias , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/inmunología , Sarcoma Experimental/inducido químicamenteRESUMEN
The recent development of novel targeted anticancer therapeutics such as histone deacetylase inhibitors (HDACi) and activators of the TRAIL pathway provide opportunities for the introduction of new treatment regimens in oncology. HDACi and recombinant TRAIL or agonistic anti-TRAIL receptor antibodies have been shown to induce synergistic tumor cell apoptosis and some therapeutic activity in vivo. Herein, we have used syngeneic preclinical models of human solid cancers to demonstrate that the HDACi panobinostat can sensitize tumor cells to apoptosis mediated by the anti-mouse TRAIL receptor antibody MD5-1. We demonstrate that the combination of panobinostat and MD5-1 can eradicate tumors grown subcutaneously and orthotopically in immunocompetent mice, while single agent treatment has minimal effect. However, escalation of the dose of panobinostat to enhance antitumor activity resulted in on-target MD5-1-mediated gastrointestinal toxicities that were fatal to the treated mice. Studies performed in mice with knockout of the TRAIL receptor showed that these mice could tolerate doses of the panobinostat/MD5-1 combination that were lethal in wild type mice resulting in superior tumor clearance. Given that clinical studies using HDACi and activators of the TRAIL pathway have been initiated, our preclinical data highlight the potential toxicities that could limit the use of such a treatment regimen. Our studies also demonstrate the power of using syngeneic in vivo tumor models as physiologically relevant preclinical systems to test the antitumor effects and identify potential side effects of novel anticancer regimens.
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Anticuerpos Monoclonales/uso terapéutico , Ácidos Hidroxámicos/uso terapéutico , Neoplasias Mamarias Experimentales/terapia , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Animales , Apoptosis , Western Blotting , Terapia Combinada , Sinergismo Farmacológico , Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/química , Histona Desacetilasas/metabolismo , Humanos , Etiquetado Corte-Fin in Situ , Indoles , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Panobinostat , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Histone deacetylase inhibitors (HDACi) and agents such as recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TRAIL receptor (TRAIL-R) antibodies are anticancer agents that have shown promise in preclinical settings and in early phase clinical trials as monotherapies. Although HDACi and activators of the TRAIL pathway have different molecular targets and mechanisms of action, they share the ability to induce tumor cell-selective apoptosis. The ability of HDACi to induce expression of TRAIL-R death receptors 4 and 5 (DR4/DR5), and induce tumor cell death via the intrinsic apoptotic pathway provides a molecular rationale to combine these agents with activators of the TRAIL pathway that activate the alternative (death receptor) apoptotic pathway. Herein, we demonstrate that the HDACi vorinostat synergizes with the mouse DR5-specific monoclonal antibody MD5-1 to induce rapid and robust tumor cell apoptosis in vitro and in vivo. Importantly, using a preclinical mouse breast cancer model, we show that the combination of vorinostat and MD5-1 is safe and induces regression of established tumors, whereas single agent treatment had little or no effect. Functional analyses revealed that rather than mediating enhanced tumor cell apoptosis via the simultaneous activation of the intrinsic and extrinsic apoptotic pathways, vorinostat augmented MD5-1-induced apoptosis concomitant with down-regulation of the intracellular apoptosis inhibitor cellular-FLIP (c-FLIP). These data demonstrate that combination therapies involving HDACi and activators of the TRAIL pathway can be efficacious for the treatment of cancer in experimental mouse models.