RESUMEN
Pharmacologic inhibition of epigenetic enzymes can have therapeutic benefit against hematologic malignancies. In addition to affecting tumor cell growth and proliferation, these epigenetic agents may induce antitumor immunity. Here, we discovered a novel immunoregulatory mechanism through inhibition of histone deacetylases (HDAC). In models of acute myeloid leukemia (AML), leukemia cell differentiation and therapeutic benefit mediated by the HDAC inhibitor (HDACi) panobinostat required activation of the type I interferon (IFN) pathway. Plasmacytoid dendritic cells (pDC) produced type I IFN after panobinostat treatment, through transcriptional activation of IFN genes concomitant with increased H3K27 acetylation at these loci. Depletion of pDCs abrogated panobinostat-mediated induction of type I IFN signaling in leukemia cells and impaired therapeutic efficacy, whereas combined treatment with panobinostat and IFNα improved outcomes in preclinical models. These discoveries offer a new therapeutic approach for AML and demonstrate that epigenetic rewiring of pDCs enhances antitumor immunity, opening the possibility of exploiting this approach for immunotherapies. SIGNIFICANCE: We demonstrate that HDACis induce terminal differentiation of AML through epigenetic remodeling of pDCs, resulting in production of type I IFN that is important for the therapeutic effects of HDACis. The study demonstrates the important functional interplay between the immune system and leukemias in response to HDAC inhibition. This article is highlighted in the In This Issue feature, p. 1397.
Asunto(s)
Leucemia Mieloide Aguda , Diferenciación Celular , Células Dendríticas , Epigénesis Genética , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/genética , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Panobinostat/farmacologíaRESUMEN
Targeting chromatin binding proteins and modifying enzymes can concomitantly affect tumor cell proliferation and survival, as well as enhance antitumor immunity and augment cancer immunotherapies. By screening a small-molecule library of epigenetics-based therapeutics, BET (bromo- and extra-terminal domain) inhibitors (BETi) were identified as agents that sensitize tumor cells to the antitumor activity of CD8+ T cells. BETi modulated tumor cells to be sensitized to the cytotoxic effects of the proinflammatory cytokine TNF. By preventing the recruitment of BRD4 to p65-bound cis-regulatory elements, BETi suppressed the induction of inflammatory gene expression, including the key NF-κB target genes BIRC2 (cIAP1) and BIRC3 (cIAP2). Disruption of prosurvival NF-κB signaling by BETi led to unrestrained TNF-mediated activation of the extrinsic apoptotic cascade and tumor cell death. Administration of BETi in combination with T-cell bispecific antibodies (TCB) or immune-checkpoint blockade increased bystander killing of tumor cells and enhanced tumor growth inhibition in vivo in a TNF-dependent manner. This novel epigenetic mechanism of immunomodulation may guide future use of BETi as adjuvants for immune-oncology agents.
Asunto(s)
Antineoplásicos/administración & dosificación , Neoplasias Colorrectales/tratamiento farmacológico , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Nucleares/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Animales , Apoptosis/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , FN-kappa B/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
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
The RNA polymerase II (POLII)-driven transcription cycle is tightly regulated at distinct checkpoints by cyclin-dependent kinases (CDKs) and their cognate cyclins. The molecular events underpinning transcriptional elongation, processivity, and the CDK-cyclin pair(s) involved remain poorly understood. Using CRISPR-Cas9 homology-directed repair, we generated analog-sensitive kinase variants of CDK12 and CDK13 to probe their individual and shared biological and molecular roles. Single inhibition of CDK12 or CDK13 induced transcriptional responses associated with cellular growth signaling pathways and/or DNA damage, with minimal effects on cell viability. In contrast, dual kinase inhibition potently induced cell death, which was associated with extensive genome-wide transcriptional changes including widespread use of alternative 3' polyadenylation sites. At the molecular level, dual kinase inhibition resulted in the loss of POLII CTD phosphorylation and greatly reduced POLII elongation rates and processivity. These data define substantial redundancy between CDK12 and CDK13 and identify both as fundamental regulators of global POLII processivity and transcription elongation.
RESUMEN
This chapter details a compendium of protocols that collectively enable the reader to perform a pooled shRNA and/or CRISPR screen-with methods to identify and validate positive controls and subsequent hits; establish a viral titer in the cell line of choice; create and screen libraries, sequence strategies, and bioinformatics resources to analyze outcomes. Collectively, this provides an overarching resource from the start to finish of a screening project, making this technology possible in all laboratories.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Biblioteca de Genes , Ensayos Analíticos de Alto Rendimiento , Lentivirus/genética , ARN Interferente Pequeño/genética , Biología Computacional , Células HEK293 , HumanosRESUMEN
BET inhibitors (BETi) target bromodomain-containing proteins and are currently being evaluated as anti-cancer agents. We find that maximal therapeutic effects of BETi in a Myc-driven B cell lymphoma model required an intact host immune system. Genome-wide analysis of the BETi-induced transcriptional response identified the immune checkpoint ligand Cd274 (Pd-l1) as a Myc-independent, BETi target-gene. BETi directly repressed constitutively expressed and interferon-gamma (IFN-γ) induced CD274 expression across different human and mouse tumor cell lines and primary patient samples. Mechanistically, BETi decreased Brd4 occupancy at the Cd274 locus without any change in Myc occupancy, resulting in transcriptional pausing and rapid loss of Cd274 mRNA production. Finally, targeted inhibition of the PD-1/PD-L1 axis by combining anti-PD-1 antibodies and the BETi JQ1 caused synergistic responses in mice bearing Myc-driven lymphomas. Our data uncover an interaction between BETi and the PD-1/PD-L1 immune-checkpoint and provide mechanistic insight into the transcriptional regulation of CD274.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo II/inmunología , Sistema Inmunológico/inmunología , Proteínas Nucleares/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Factores de Transcripción/inmunología , Animales , Antígeno B7-H1/inmunología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Interferón gamma/inmunología , Ligandos , Linfoma de Células B/inmunología , Ratones , Proteínas Proto-Oncogénicas c-myc/inmunología , ARN Mensajero/inmunología , Transcripción Genética/inmunologíaRESUMEN
Targeting BET bromodomain proteins using small molecules is an emerging anticancer strategy with clinical evaluation of at least six inhibitors now underway. Although MYC downregulation was initially proposed as a key mechanistic property of BET inhibitors, recent evidence suggests that additional antitumor activities are important. Using the Eµ-Myc model of B-cell lymphoma, we demonstrate that BET inhibition with JQ1 is a potent inducer of p53-independent apoptosis that occurs in the absence of effects on Myc gene expression. JQ1 skews the expression of proapoptotic (Bim) and antiapoptotic (BCL-2/BCL-xL) BCL-2 family members to directly engage the mitochondrial apoptotic pathway. Consistent with this, Bim knockout or Bcl-2 overexpression inhibited apoptosis induction by JQ1. We identified lymphomas that were either intrinsically resistant to JQ1-mediated death or acquired resistance following in vivo exposure. Strikingly, in both instances BCL-2 was strongly upregulated and was concomitant with activation of RAS pathways. Eµ-Myc lymphomas engineered to express activated Nras upregulated BCL-2 and acquired a JQ1 resistance phenotype. These studies provide important information on mechanisms of apoptosis induction and resistance to BET-inhibition, while providing further rationale for the translation of BET inhibitors in aggressive B-cell lymphomas. Mol Cancer Ther; 15(9); 2030-41. ©2016 AACR.
Asunto(s)
Apoptosis/genética , Azepinas/farmacología , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Linfoma de Células B/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Triazoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Genes myc , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/mortalidad , Linfoma de Células B/patología , Ratones , Familia de Multigenes , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Histone deacetylase (HDAC) inhibitors (HDACis) have demonstrated activity in hematological and solid malignancies. Vorinostat, romidepsin, belinostat, and panobinostat are Food and Drug Administration-approved for hematological malignancies and inhibit class II and/or class I HDACs, including HDAC1, 2, 3, and 6. We combined genetic and pharmacological approaches to investigate whether suppression of individual or multiple Hdacs phenocopied broad-acting HDACis in 3 genetically distinct leukemias and lymphomas. Individual Hdacs were depleted in murine acute myeloid leukemias (MLL-AF9;Nras(G12D); PML-RARα acute promyelocytic leukemia [APL] cells) and Eµ-Myc lymphoma in vitro and in vivo. Strikingly, Hdac3-depleted cells were selected against in competitive assays for all 3 tumor types. Decreased proliferation following Hdac3 knockdown was not prevented by BCL-2 overexpression, caspase inhibition, or knockout of Cdkn1a in Eµ-Myc lymphoma, and depletion of Hdac3 in vivo significantly reduced tumor burden. Interestingly, APL cells depleted of Hdac3 demonstrated a more differentiated phenotype. Consistent with these genetic studies, the HDAC3 inhibitor RGFP966 reduced proliferation of Eµ-Myc lymphoma and induced differentiation in APL. Genetic codepletion of Hdac1 with Hdac2 was pro-apoptotic in Eµ-Myc lymphoma in vitro and in vivo and was phenocopied by the HDAC1/2-specific agent RGFP233. This study demonstrates the importance of HDAC3 for the proliferation of leukemia and lymphoma cells, suggesting that HDAC3-selective inhibitors could prove useful for the treatment of hematological malignancies. Moreover, our results demonstrate that codepletion of Hdac1 with Hdac2 mediates a robust pro-apoptotic response. Our integrated genetic and pharmacological approach provides important insights into the individual or combinations of HDACs that could be prioritized for targeting in a range of hematological malignancies.
Asunto(s)
Histona Desacetilasas/metabolismo , Leucemia Promielocítica Aguda/enzimología , Leucemia Promielocítica Aguda/genética , Linfoma/enzimología , Linfoma/genética , Animales , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Leucemia Promielocítica Aguda/tratamiento farmacológico , Leucemia Promielocítica Aguda/patología , Linfoma/tratamiento farmacológico , Linfoma/patología , Ratones , Células 3T3 NIH , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Cell-intrinsic effects such as induction of apoptosis and/or inhibition of cell proliferation have been proposed as the major antitumor responses to histone deacetylase inhibitors (HDACi). These compounds can also mediate immune-modulatory effects that may contribute to their anticancer effects. However, HDACi can also induce anti-inflammatory, and potentially immunosuppressive, outcomes. We therefore sought to clarify the role of the immune system in mediating the efficacy of HDACi in a physiologic setting, using preclinical, syngeneic murine models of hematologic malignancies and solid tumors. We showed an intact immune system was required for the robust anticancer effects of the HDACi vorinostat and panobinostat against a colon adenocarcinoma and two aggressive models of leukemia/lymphoma. Importantly, although HDACi-treated immunocompromised mice bearing established lymphoma succumbed to disease significantly earlier than tumor bearing, HDACi-treated wild-type (WT) mice, treatment with the conventional chemotherapeutic etoposide equivalently enhanced the survival of both strains. IFN-γ and tumor cell signaling through IFN-γR were particularly important for the anticancer effects of HDACi, and vorinostat and IFN-γ acted in concert to enhance the immunogenicity of tumor cells. Furthermore, we show that a combination of vorinostat with α-galactosylceramide (α-GalCer), an IFN-γ-inducing agent, was significantly more potent against established lymphoma than vorinostat treatment alone. Intriguingly, B cells, but not natural killer cells or CD8(+) T cells, were implicated as effectors of the vorinostat antitumor immune response. Together, our data suggest HDACi are immunostimulatory during cancer treatment and that combinatorial therapeutic regimes with immunotherapies should be considered in the clinic.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Sistema Inmunológico/fisiología , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/inmunología , Receptores de Interferón/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/inmunología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/inmunología , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/inmunología , Sistema Inmunológico/efectos de los fármacos , Inmunoterapia , Leucemia Linfoide/tratamiento farmacológico , Leucemia Linfoide/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor de Interferón gammaRESUMEN
Histone deacetylase inhibitors (HDACi) are anticancer agents that induce hyperacetylation of histones, resulting in chromatin remodeling and transcriptional changes. In addition, nonhistone proteins, such as the chaperone protein Hsp90, are functionally regulated through hyperacetylation mediated by HDACis. Histone acetylation is thought to be primarily regulated by HDACs 1, 2, and 3, whereas the acetylation of Hsp90 has been proposed to be specifically regulated through HDAC6. We compared the molecular and biologic effects induced by an HDACi with broad HDAC specificity (vorinostat) with agents that predominantly inhibited selected class I HDACs (MRLB-223 and romidepsin). MRLB-223, a potent inhibitor of HDACs 1 and 2, killed tumor cells using the same apoptotic pathways as the HDAC 1, 2, 3, 6, and 8 inhibitor vorinostat. However, vorinostat induced histone hyperacetylation and killed tumor cells more rapidly than MRLB-223 and had greater therapeutic efficacy in vivo. FDCP-1 cells dependent on the Hsp90 client protein Bcr-Abl for survival, were killed by all HDACis tested, concomitant with caspase-dependent degradation of Bcr-Abl. These studies provide evidence that inhibition of HDAC6 and degradation of Bcr-Abl following hyperacetylation of Hsp90 is likely not a major mechanism of action of HDACis as had been previously posited.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Acetilación/efectos de los fármacos , Animales , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Proteínas de Fusión bcr-abl/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 2/antagonistas & inhibidores , Histona Desacetilasa 6 , Inhibidores de Histona Desacetilasas/administración & dosificación , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/administración & dosificación , Ácidos Hidroxámicos/farmacología , Linfoma/tratamiento farmacológico , Linfoma/metabolismo , Linfoma/mortalidad , Linfoma/patología , Ratones , Vorinostat , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Aberrant recruitment of histone deacetylases (HDACs) by the oncogenic fusion protein PML-RAR is involved in the pathogenesis of acute promyelocytic leukemia (APL). PML-RAR, however, is not sufficient to induce disease in mice but requires additional oncogenic lesions during the preleukemic phase. Here, we show that knock-down of Hdac1 and Hdac2 dramatically accelerates leukemogenesis in transgenic preleukemic mice. These events are not restricted to APL because lymphomagenesis driven by deletion of p53 or, to a lesser extent, by c-myc overexpression, was also accelerated by Hdac1 knock-down. In the preleukemic phase of APL, Hdac1 counteracts the activity of PML-RAR in (1) blocking differentiation; (2) impairing genomic stability; and (3) increasing self-renewal in hematopoietic progenitors, as all of these events are affected by the reduction in Hdac1 levels. This led to an expansion of a subpopulation of PML-RAR-expressing cells that is the major source of leukemic stem cells in the full leukemic stage. Remarkably, short-term treatment of preleukemic mice with an HDAC inhibitor accelerated leukemogenesis. In contrast, knock-down of Hdac1 in APL mice led to enhanced survival duration of the leukemic animals. Thus, Hdac1 has a dual role in tumorigenesis: oncosuppressive in the early stages, and oncogenic in established tumor cells.
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Transformación Celular Neoplásica/genética , Histona Desacetilasa 1/genética , Histona Desacetilasa 2/genética , Leucemia Promielocítica Aguda/etiología , Leucemia Promielocítica Aguda/prevención & control , Proteína p53 Supresora de Tumor/fisiología , Animales , Western Blotting , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/patología , Femenino , Citometría de Flujo , Inestabilidad Genómica , Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/antagonistas & inhibidores , Histona Desacetilasa 2/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Leucemia Promielocítica Aguda/mortalidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Células Tumorales Cultivadas , Ácido Valproico/farmacologíaRESUMEN
Increased transcription of ribosomal RNA genes (rDNA) by RNA Polymerase I is a common feature of human cancer, but whether it is required for the malignant phenotype remains unclear. We show that rDNA transcription can be therapeutically targeted with the small molecule CX-5461 to selectively kill B-lymphoma cells in vivo while maintaining a viable wild-type B cell population. The therapeutic effect is a consequence of nucleolar disruption and activation of p53-dependent apoptotic signaling. Human leukemia and lymphoma cell lines also show high sensitivity to inhibition of rDNA transcription that is dependent on p53 mutational status. These results identify selective inhibition of rDNA transcription as a therapeutic strategy for the cancer specific activation of p53 and treatment of hematologic malignancies.
Asunto(s)
Neoplasias/metabolismo , ARN Polimerasa I/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis , Benzotiazoles/farmacología , ADN Ribosómico/genética , Femenino , Ratones , Ratones Transgénicos , Naftiridinas/farmacología , Neoplasias/genética , Neoplasias/patología , ARN Ribosómico/genética , Transcripción GenéticaRESUMEN
The concept of personalized anticancer therapy is based on the use of targeted therapeutics through in-depth knowledge of the molecular mechanisms of action of these agents when used alone and in combination. We have identified the apoptotic proteins and pathways necessary for synergistic tumor cell apoptosis and in vivo antitumor responses seen when the HDAC inhibitor vorinostat is combined with the BH3-mimetic ABT-737 in lymphomas overexpressing Bcl-2. Vorinostat "primes" tumors overexpressing Bcl-2 for rapid ABT-737-mediated apoptosis by inducing expression of the BH3-only gene bmf. Moreover, these synergistic effects of vorinostat/ABT-737 were blunted in cells with an inactive p53 pathway or in cells lacking expression of the p53 target gene, noxa. These studies show the important and complex functional interaction between specific proapoptotic BH3-only proteins and the BH3-mimetic compound ABT-737 and provide the most comprehensive functional link between tumor genotype and the apoptotic and therapeutic effects of HDACi combined with ABT-737.
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Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Animales , Apoptosis , Compuestos de Bifenilo/farmacología , Línea Celular Tumoral , Femenino , Genes p53 , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Nitrofenoles/farmacología , Fragmentos de Péptidos/metabolismo , Piperazinas/farmacología , Proteínas Proto-Oncogénicas/metabolismo , Sulfonamidas/farmacología , Proteína p53 Supresora de Tumor/genética , VorinostatRESUMEN
Asymmetric cell division is a potential means by which cell fate choices during an immune response are orchestrated. Defining the molecular mechanisms that underlie asymmetric division of T cells is paramount for determining the role of this process in the generation of effector and memory T cell subsets. In other cell types, asymmetric cell division is regulated by conserved polarity protein complexes that control the localization of cell fate determinants and spindle orientation during division. We have developed a tractable, in vitro model of naive CD8(+) T cells undergoing initial division while attached to dendritic cells during Ag presentation to investigate whether similar mechanisms might regulate asymmetric division of T cells. Using this system, we show that direct interactions with APCs provide the cue for polarization of T cells. Interestingly, the immunological synapse disseminates before division even though the T cells retain contact with the APC. The cue from the APC is translated into polarization of cell fate determinants via the polarity network of the Par3 and Scribble complexes, and orientation of the mitotic spindle during division is orchestrated by the partner of inscuteable/G protein complex. These findings suggest that T cells have selectively adapted a number of evolutionarily conserved mechanisms to generate diversity through asymmetric cell division.
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Presentación de Antígeno/inmunología , División Celular/inmunología , Secuencia Conservada/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Animales , Células Presentadoras de Antígenos/citología , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Adhesión Celular/inmunología , Polaridad Celular/inmunología , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Subgrupos de Linfocitos T/metabolismoRESUMEN
LAQ824 and LBH589 (panobinostat) are histone deacetylase inhibitors (HDACi) developed as cancer therapeutics and we have used the Emu-myc lymphoma model to identify the molecular events required for their antitumor effects. Induction of tumor cell death was necessary for these agents to mediate therapeutic responses in vivo and both HDACi engaged the intrinsic apoptotic cascade that did not require p53. Death receptor pathway blockade had no effect on the therapeutic activities of LAQ824 and LBH589; however, overexpression of Bcl-2 or Bcl-X(L) protected lymphoma cells from HDACi-induced killing and suppressed their therapeutic activities. Deletion of Apaf-1 or Caspase-9 delayed HDACi-induced lymphoma killing in vitro and in vivo, associated with suppression of many biochemical indicators of apoptosis, but did not provide long-term resistance to these agents and failed to inhibit their therapeutic activities. Emu-myc lymphomas lacking a functional apoptosome displayed morphologic and biochemical features of autophagy after treatment with LAQ824 and LBH589, indicating that, in the absence of a complete intrinsic apoptosis pathway involving apoptosome formation, these HDACi can still mediate a therapeutic response. Our data indicate that damage to the mitochondria is the key event necessary for LAQ824 and LBH589 to mediate tumor cell death and a robust therapeutic response.
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Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/farmacología , Linfoma/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Histona Desacetilasas/metabolismo , Humanos , Indoles , Linfoma/patología , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Trasplante de Neoplasias , Panobinostat , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores de Muerte Celular/metabolismo , Transducción de Señal/efectos de los fármacos , Tasa de Supervivencia , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The apoptotic and therapeutic activities of the histone deacetylase inhibitor (HDACi) vorinostat are blocked by overexpression of Bcl-2 or Bcl-X(L). Herein, we used the small molecule inhibitor ABT-737 to restore sensitivity of Emu-myc lymphomas overexpressing Bcl-2 or Bcl-X(L) to vorinostat and valproic acid (VPA). Combining low-dose ABT-737 with vorinostat or VPA resulted in synergistic apoptosis of these cells. ABT-737 was ineffective against Emu-myc/Mcl-1 and Emu-myc/A1 cells either as a single agent or in combination with HDACi. However, in contrast to the reported binding specificity data, Emu-myc/Bcl-w lymphomas were insensitive to ABT-737 used alone or in combination with HDACi, indicating that the regulatory activity of ABT-737 is restricted to Bcl-2 and Bcl-X(L). Emu-myc lymphomas that expressed Bcl-2 throughout the tumorigenesis process were especially sensitive to ABT-737, while those forced to overexpress Mcl-1 were not. This supports the notion that tumor cells "addicted" to ABT-737 target proteins (ie, Bcl-2 or Bcl-X(L)) are likely to be the most sensitive target cell population. Our studies provide important preclinical data on the binding specificity of ABT-737 and its usefulness against primary hematologic malignancies when used as a single agent and in combination with HDACi.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Compuestos de Bifenilo/farmacología , Inhibidores Enzimáticos/administración & dosificación , Inhibidores de Histona Desacetilasas , Linfoma/tratamiento farmacológico , Nitrofenoles/farmacología , Sulfonamidas/farmacología , Animales , Compuestos de Bifenilo/administración & dosificación , Supervivencia Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Genes bcl-2 , Genes myc , Ácidos Hidroxámicos/administración & dosificación , Linfoma/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nitrofenoles/administración & dosificación , Piperazinas/administración & dosificación , Piperazinas/farmacología , Especificidad por Sustrato , Sulfonamidas/administración & dosificación , VorinostatRESUMEN
Histone deacetylase inhibitors (HDACi) are compounds that target the epigenome and cause tumor cell-selective apoptosis. A large number of these agents that have different chemical structures and can target multiple HDACs are being testing in clinical trials and vorinostat is now an approved drug for the treatment of cutaneous T-cell lymphoma. Although these agents are showing promise for the treatment of hematologic malignancies, it is possible that different drugs may have different mechanistic, biological, and therapeutic activities. When comparing an HDACi belonging to the hydroxamic acid class of compounds (vorinostat) with a cyclic tetrapeptide (romidepsin), we showed that these agents regulate the expression of a common set of cellular genes, but certain genes specifically responded to each agent. Using the Emu-myc mouse model of B-cell lymphoma, we showed previously that overexpression of the prosurvival proteins Bcl-2 and Bcl-XL inhibited the apoptotic and therapeutic activities of the vorinostat. Herein, we compared and contrasted the apoptotic-inducing activities of the hydroxamic acid oxamflatin with romidepsin. Like vorinostat, oxamflatin was unable to kill lymphomas overexpressing Bcl-2 and Bcl-XL, indicating that these proteins can generally protect cells against this class of HDACi. In contrast, romidepsin was able to induce apoptosis in lymphomas overexpressing Bcl-2 with delayed kinetics of cell death and could mediate therapeutic responses against these lymphomas. However, romidepsin was inactive when Bcl-XL was overexpressed. These data provide strong support that HDACi of different chemical classes may have subtle yet potentially important differences in their molecular and biological activities.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Apoptosis , Depsipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Animales , Antibióticos Antineoplásicos/uso terapéutico , Depsipéptidos/uso terapéutico , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/uso terapéutico , Ácidos Hidroxámicos/farmacología , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/enzimología , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMEN
Elevated expression of members of the BCL-2 pro-survival family of proteins can confer resistance to apoptosis in cancer cells. Small molecule obatoclax (GX15-070), which is predicted to occupy a hydrophobic pocket within the BH3 binding groove of BCL-2, antagonizes these members and induces apoptosis, dependent on BAX and BAK. Reconstitution in yeast confirmed that obatoclax acts on the pathway and overcomes BCL-2-, BCL-XL-, BCL-w-, and MCL-1-mediated resistance to BAX or BAK. The compound potently interfered with the direct interaction between MCL-1 and BAK in intact mitochondrial outer membrane and inhibited the association between MCL-1 and BAK in intact cells. MCL-1 has been shown to confer resistance to the BCL-2/BCL-XL/BCL-w-selective antagonist ABT-737 and to the proteasome inhibitor bortezomib. In both cases, this resistance was overcome by obatoclax. These findings support a rational clinical development opportunity for the compound in cancer indications or treatments where MCL-1 contributes to resistance to cell killing.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Pirroles/farmacología , Animales , Ácidos Borónicos/farmacología , Bortezomib , Línea Celular Tumoral , Inhibidores de Cisteína Proteinasa/farmacología , Humanos , Indoles , Melanoma/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Inhibidores de Proteasoma , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Pirazinas/farmacología , Proteína Destructora del Antagonista Homólogo bcl-2/antagonistas & inhibidores , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismoRESUMEN
To determine the importance of suppressor of cytokine signaling-3 (SOCS3) in the regulation of hematopoietic growth factor signaling generally, and of G-CSF-induced cellular responses specifically, we created mice in which the Socs3 gene was deleted in all hematopoietic cells. Although normal until young adulthood, these mice then developed neutrophilia and a spectrum of inflammatory pathologies. When stimulated with G-CSF in vitro, SOCS3-deficient cells of the neutrophilic granulocyte lineage exhibited prolonged STAT3 activation and enhanced cellular responses to G-CSF, including an increase in cloning frequency, survival, and proliferative capacity. Consistent with the in vitro findings, mutant mice injected with G-CSF displayed enhanced neutrophilia, progenitor cell mobilization, and splenomegaly, but unexpectedly also developed inflammatory neutrophil infiltration into multiple tissues and consequent hind-leg paresis. We conclude that SOCS3 is a key negative regulator of G-CSF signaling in myeloid cells and that this is of particular significance during G-CSF-driven emergency granulopoiesis.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/inmunología , Granulocitos/inmunología , Inflamación/patología , Proteínas Represoras/inmunología , Factores de Transcripción/inmunología , Animales , Diferenciación Celular , Células Cultivadas , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Factor Estimulante de Colonias de Granulocitos/farmacología , Granulocitos/efectos de los fármacos , Granulocitos/metabolismo , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Inflamación/inmunología , Ratones , Ratones Transgénicos , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/metabolismo , Factor de Transcripción STAT3 , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Transactivadores/efectos de los fármacos , Transactivadores/inmunología , Transactivadores/metabolismo , Factores de Transcripción/deficienciaRESUMEN
The haematopoietic-specific RhoGTPase, Rac2, has been indirectly implicated in T-lymphocyte development and function, and as a pivotal regulator of T Helper 1 (T(H)1) responses. In other haematopoietic cells it regulates cytoskeletal rearrangement downstream of extracellular signals. Here we demonstrate that Rac2 deficiency results in an abnormal distribution of T lymphocytes in vivo and defects in T-lymphocyte migration and filamentous actin generation in response to chemoattractants in vitro. To investigate the requirement for Rac2 in IFN-gamma production and TH1 responses in vivo, Rac2-deficient mice were challenged with Leishmania major and immunized with ovalbumin-expressing cytomegalovirus. Despite a minor skewing towards a T(H)2 phenotype, Rac2-deficient mice displayed no increased susceptibility to L. major infection. Cytotoxic T-lymphocyte responses to cytomegalovirus and ovalbumin were also normal. Although Rac2 is required for normal T-lymphocyte migration, its role in the generation of T(H)1 responses to infection in vivo is largely redundant.