RESUMO
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.
Assuntos
Biocatálise , Histonas/metabolismo , Oncogenes , Transcrição Gênica , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Linhagem Celular , Cromatina/metabolismo , Proteínas Correpressoras/metabolismo , Sequência Conservada , Evolução Molecular , Redes Reguladoras de Genes , Genoma , Histona Desacetilases/metabolismo , Humanos , Cinética , Metilação , Modelos Biológicos , RNA Polimerase II/metabolismoRESUMO
ABSTRACT: Chromosomal translocation (4;14), an adverse prognostic factor in multiple myeloma (MM), drives overexpression of the histone methyltransferase nuclear receptor binding SET domain protein 2 (NSD2). A genome-wide CRISPR screen in MM cells identified adenylate kinase 2 (AK2), an enzyme critical for high-energy phosphate transfer from the mitochondria, as an NSD2-driven vulnerability. AK2 suppression in t(4;14) MM cells decreased nicotinamide adenine dinucleotide phosphate (NADP[H]) critical for conversion of ribonucleotides to deoxyribonucleosides, leading to replication stress, DNA damage, and apoptosis. Driving a large genome-wide increase in chromatin methylation, NSD2 overexpression depletes S-adenosylmethionine, compromising the synthesis of creatine from its precursor, guanidinoacetate. Creatine supplementation restored NADP(H) levels, reduced DNA damage, and rescued AK2-deficient t(4;14) MM cells. As the creatine phosphate shuttle constitutes an alternative means for mitochondrial high-energy phosphate transport, these results indicate that NSD2-driven creatine depletion underlies the hypersensitivity of t(4;14) MM cells to AK2 loss. Furthermore, AK2 depletion in t(4;14) cells impaired protein folding in the endoplasmic reticulum, consistent with impaired use of mitochondrial adenosine triphosphate (ATP). Accordingly, AK2 suppression increased the sensitivity of MM cells to proteasome inhibition. These findings delineate a novel mechanism in which aberrant transfer of carbon to the epigenome creates a metabolic vulnerability, with direct therapeutic implications for t(4;14) MM.
Assuntos
Adenilato Quinase , Histona-Lisina N-Metiltransferase , Mieloma Múltiplo , Translocação Genética , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Adenilato Quinase/metabolismo , Adenilato Quinase/genética , Cromossomos Humanos Par 14/genética , Epigenoma , Cromossomos Humanos Par 4/genética , Carbono/metabolismo , Linhagem Celular Tumoral , Proteínas RepressorasRESUMO
The histone H3K27 demethylase KDM6A is a tumor suppressor in multiple cancers, including multiple myeloma (MM). We created isogenic MM cells disrupted for KDM6A and tagged the endogenous protein to facilitate genome wide studies. KDM6A binds genes associated with immune recognition and cytokine signaling. Most importantly, KDM6A binds and activates NLRC5 and CIITA encoding regulators of Major Histocompatibility Complex (MHC) genes. Patient data indicate that NLRC5 and CIITA, are downregulated in MM with low KDM6A expression. Chromatin analysis shows that KDM6A binds poised and active enhancers and KDM6A loss led to decreased H3K27ac at enhancers, increased H3K27me3 levels in body of genes bound by KDM6A and decreased gene expression. Reestablishing histone acetylation with an HDAC3 inhibitor leads to upregulation of MHC expression, offering a strategy to restore immunogenicity of KDM6A deficient tumors. Loss of Kdm6a in murine RAS-transformed fibroblasts led to increased growth in vivo associated with decreased T cell infiltration.
RESUMO
Multiple myeloma (MM) is a plasma cell neoplasm associated with a broad variety of genetic lesions. In spite of this genetic heterogeneity, MMs share a characteristic malignant phenotype whose underlying molecular basis remains poorly characterized. In the present study, we examined plasma cells from MM using a multi-epigenomics approach and demonstrated that, when compared to normal B cells, malignant plasma cells showed an extensive activation of regulatory elements, in part affecting coregulated adjacent genes. Among target genes up-regulated by this process, we found members of the NOTCH, NF-kB, MTOR signaling, and TP53 signaling pathways. Other activated genes included sets involved in osteoblast differentiation and response to oxidative stress, all of which have been shown to be associated with the MM phenotype and clinical behavior. We functionally characterized MM-specific active distant enhancers controlling the expression of thioredoxin (TXN), a major regulator of cellular redox status and, in addition, identified PRDM5 as a novel essential gene for MM. Collectively, our data indicate that aberrant chromatin activation is a unifying feature underlying the malignant plasma cell phenotype.
Assuntos
Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo/genética , Plasmócitos/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Humanos , NF-kappa B/metabolismo , Osteogênese/genética , Receptores Notch/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Tiorredoxinas/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regulação para CimaRESUMO
PURPOSE OF REVIEW: Next generation sequencing and large-scale analysis of patient specimens has created a more complete picture of multiple myeloma (MM) revealing that epigenetic deregulation is a prominent factor in MM pathogenesis. RECENT FINDINGS: Over half of MM patients have mutations in genes encoding epigenetic modifier enzymes. The DNA methylation profile of MM is related to the stage of the disease and certain classes of mutations in epigenetic modifiers are more prevalent upon disease relapse, suggesting a role in disease progression. Many small molecules targeting regulators of epigenetic machinery have been developed and clinical trials are underway for some of these in MM. SUMMARY: Recent findings suggest that epigenetic targeting drugs could be an important strategy to cure MM. Combining these agents along with other strategies to affect the MM cell such as immunomodulatory drugs and proteasome inhibitors may enhance efficacy of combination regimens in MM.
Assuntos
Antineoplásicos/uso terapêutico , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mutação , Acetilação/efeitos dos fármacos , Antineoplásicos/farmacologia , Montagem e Desmontagem da Cromatina , Ensaios Clínicos como Assunto , Metilação de DNA/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Metilação/efeitos dos fármacos , Terapia de Alvo Molecular , Mieloma Múltiplo/metabolismoRESUMO
The histone H3K27 demethylase KDM6A is a tumor suppressor in multiple cancers, including multiple myeloma (MM). We created isogenic MM cells disrupted for KDM6A and tagged the endogenous protein to facilitate genome wide studies. KDM6A binds genes associated with immune recognition and cytokine signaling. Most importantly, KDM6A binds and activates NLRC5 and CIITA encoding regulators of Major Histocompatibility Complex (MHC) genes. Patient data indicate that NLRC5 and CIITA, are downregulated in MM with low KDM6A expression. Chromatin analysis shows that KDM6A binds poised and active enhancers and KDM6A loss led to decreased H3K27ac at enhancers, increased H3K27me3 levels in body of genes bound by KDM6A and decreased gene expression. Reestablishing histone acetylation with an HDAC3 inhibitor leads to upregulation of MHC expression, offering a strategy to restore immunogenicity of KDM6A deficient tumors. Loss of Kdm6a in murine RAS-transformed fibroblasts led to increased growth in vivo associated with decreased T cell infiltration. Statement of significance: We show that KDM6A participates in immune recognition of myeloma tumor cells by directly regulating the expression of the master regulators of MHC-I and II, NLRC5 and CIITA. The expression of these regulators can by rescued by the HDAC3 inhibitors in KDM6A-null cell lines.
RESUMO
Despite the success of BCMA-targeting CAR-Ts in multiple myeloma, patients with high-risk cytogenetic features still relapse most quickly and are in urgent need of additional therapeutic options. Here, we identify CD70, widely recognized as a favorable immunotherapy target in other cancers, as a specifically upregulated cell surface antigen in high risk myeloma tumors. We use a structure-guided design to define a CD27-based anti-CD70 CAR-T design that outperforms all tested scFv-based CARs, leading to >80-fold improved CAR-T expansion in vivo. Epigenetic analysis via machine learning predicts key transcription factors and transcriptional networks driving CD70 upregulation in high risk myeloma. Dual-targeting CAR-Ts against either CD70 or BCMA demonstrate a potential strategy to avoid antigen escape-mediated resistance. Together, these findings support the promise of targeting CD70 with optimized CAR-Ts in myeloma as well as future clinical translation of this approach.
RESUMO
Melanomas can adopt multiple transcriptional states. Little is known about the epigenetic drivers of these cell states, limiting our ability to regulate melanoma heterogeneity. Here, we identify stress-induced HDAC8 activity as driving melanoma brain metastasis development. Exposure of melanocytes and melanoma cells to multiple stresses increases HDAC8 activation leading to a neural crest-stem cell transcriptional state and an amoeboid, invasive phenotype that increases seeding to the brain. Using ATAC-Seq and ChIP-Seq we show that increased HDAC8 activity alters chromatin structure by increasing H3K27ac and enhancing accessibility at c-Jun binding sites. Functionally, HDAC8 deacetylates the histone acetyltransferase EP300, causing its enzymatic inactivation. This, in turn, increases binding of EP300 to Jun-transcriptional sites and decreases binding to MITF-transcriptional sites. Inhibition of EP300 increases melanoma cell invasion, resistance to stress and increases melanoma brain metastasis development. HDAC8 is identified as a mediator of transcriptional co-factor inactivation and chromatin accessibility that drives brain metastasis.
Assuntos
Neoplasias Encefálicas , Proteína p300 Associada a E1A , Histona Desacetilases , Melanoma , Humanos , Neoplasias Encefálicas/secundário , Cromatina/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Melanócitos/metabolismo , Melanoma/patologia , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Clinical progress in multiple myeloma (MM), an incurable plasma cell (PC) neoplasia, has been driven by therapies that have limited applications beyond MM/PC neoplasias and do not target specific oncogenic mutations in MM. Instead, these agents target pathways critical for PC biology yet largely dispensable for malignant or normal cells of most other lineages. Here we systematically characterized the lineage-preferential molecular dependencies of MM through genome-scale clustered regularly interspaced short palindromic repeats (CRISPR) studies in 19 MM versus hundreds of non-MM lines and identified 116 genes whose disruption more significantly affects MM cell fitness compared with other malignancies. These genes, some known, others not previously linked to MM, encode transcription factors, chromatin modifiers, endoplasmic reticulum components, metabolic regulators or signaling molecules. Most of these genes are not among the top amplified, overexpressed or mutated in MM. Functional genomics approaches thus define new therapeutic targets in MM not readily identifiable by standard genomic, transcriptional or epigenetic profiling analyses.
Assuntos
Mieloma Múltiplo , Humanos , Mieloma Múltiplo/genética , Genômica , Genoma , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genéticaRESUMO
Diffuse large B-cell lymphoma (DLBCL) accounts for 40% of non-Hodgkin lymphoma, and 30% to 40% of patients will succumb to relapsed/refractory disease (rrDLBCL). Patients with rrDLBCL generally have low long-term survival rates due to a lack of efficient salvage therapies. Small-molecule inhibitors targeting the histone methyltransferase EZH2 represent an emerging group of novel therapeutics that show promising clinical efficacy in patients with rrDLBCL. The mechanisms that control acquired resistance to this class of targeted therapies, however, remain poorly understood. Here, we develop a model of resistance to the EZH2 inhibitor (EZH2i) GSK343 and use RNA-seq data and in vitro investigation to show that GCB (germinal center B-cell)-DLBCL cell lines with acquired drug resistance differentiate toward an ABC (activated B-cell)-DLBCL phenotype. We further observe that the development of resistance to GSK343 is sufficient to induce cross-resistance to other EZH2i. Notably, we identify the immune receptor SLAMF7 as upregulated in EZH2i-resistant cells, using chromatin immunoprecipitation profiling to uncover the changes in chromatin landscape remodeling that permit this altered gene expression. Collectively, our data reveal a previously unreported response to the development of EZH2i resistance in DLBCL, while providing strong rationale for pursuing investigation of dual-targeting of EZH2 and SLAMF7 in rrDLBCL.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteína Potenciadora do Homólogo 2 de Zeste , Indazóis , Linfoma Difuso de Grandes Células B , Piridonas , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Humanos , Indazóis/farmacologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Fenótipo , Piridonas/farmacologiaRESUMO
Mutations in epigenetic regulators are common in relapsed pediatric acute lymphoblastic leukemia (ALL). Here, we uncovered the mechanism underlying the relapse of ALL driven by an activating mutation of the NSD2 histone methyltransferase (p.E1099K). Using high-throughput drug screening, we found that NSD2-mutant cells were specifically resistant to glucocorticoids. Correction of this mutation restored glucocorticoid sensitivity. The transcriptional response to glucocorticoids was blocked in NSD2-mutant cells due to depressed glucocorticoid receptor (GR) levels and the failure of glucocorticoids to autoactivate GR expression. Although H3K27me3 was globally decreased by NSD2 p.E1099K, H3K27me3 accumulated at the NR3C1 (GR) promoter. Pretreatment of NSD2 p.E1099K cell lines and patient-derived xenograft samples with PRC2 inhibitors reversed glucocorticoid resistance in vitro and in vivo. PRC2 inhibitors restored NR3C1 autoactivation by glucocorticoids, increasing GR levels and allowing GR binding and activation of proapoptotic genes. These findings suggest a new therapeutic approach to relapsed ALL associated with NSD2 mutation. SIGNIFICANCE: NSD2 histone methyltransferase mutations observed in relapsed pediatric ALL drove glucocorticoid resistance by repression of the GR and abrogation of GR gene autoactivation due to accumulation of K3K27me3 at its promoter. Pretreatment with PRC2 inhibitors reversed resistance, suggesting a new therapeutic approach to these patients with ALL.This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Glucocorticoides/uso terapêutico , Histona Metiltransferases/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Repressoras/genética , Linhagem Celular Tumoral/efeitos dos fármacos , Sobrevivência Celular , Criança , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Feminino , Glucocorticoides/farmacologia , Humanos , Masculino , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologiaRESUMO
PURPOSE: In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. EXPERIMENTAL DESIGN: Leukemia cell lines, genetic mouse models, and isogenic cells with and without DNMT3A(mut) were used to evaluate sensitivity to nucleoside analogues such as cytarabine in vitro and in vivo, followed by analysis of DNA damage and signaling, replication restart, and cell-cycle progression on treatment and after drug removal. Transcriptome profiling identified pathways deregulated by DNMT3A(mut) expression. RESULTS: We found increased sensitivity to pharmacologically induced replication stress in cells expressing DNMT3A(R882)-mutant, with persistent intra-S-phase checkpoint activation, impaired PARP1 recruitment, and elevated DNA damage, which was incompletely resolved after drug removal and carried through mitosis. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine washout demonstrated a higher rate of fork collapse in DNMT3A(mut)-expressing cells. RNA-seq studies supported deregulated cell-cycle progression and p53 activation, along with splicing, ribosome biogenesis, and metabolism. CONCLUSIONS: Together, our studies show that DNMT3A mutations underlie a defect in recovery from replication fork arrest with subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during replication stress. See related commentary by Viny, p. 573.
Assuntos
Dano ao DNA , DNA Metiltransferase 3A , Replicação do DNA , Leucemia Mieloide Aguda , Animais , DNA Metiltransferase 3A/genética , Replicação do DNA/genética , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Camundongos , Mutação , PrognósticoRESUMO
Transcriptional activation by nuclear hormone receptors is well characterized, but their cooperation with other signaling pathways to activate transcription remains poorly understood. Tumor necrosis factor alpha (TNFalpha) and all-trans retinoic acid (RA) induce monocytic differentiation of acute promyelocytic leukemia (APL) cells in a synergistic manner. We used the promoter of DIF2, a gene involved in monocytic differentiation, to model the mechanism underlying the cooperative induction of target genes by RA and TNFalpha. We show a functional RA response element in the DIF2 promoter, which is constitutively bound by PML/RARalpha in APL cells. RA stimulates release of corepressors and recruitment of chromatin modifying proteins and additional transcription factors to the promoter, but these changes cause only a modest induction of DIF2 mRNA. Co-stimulation with RA plus TNFalpha facilitates binding of NF-kappaB to the promoter, which is crucial for full induction of transcription. Furthermore, RA plus TNFalpha greatly enhanced the level of RNA Pol II phosphorylation on the DIF2 promoter, via synergistic recruitment of TFIIH. We propose that RA mediates remodeling of chromatin to facilitate binding of transcription factors, which cooperate to enhance Pol II phosphorylation, providing a mechanism whereby nuclear receptors interact with other signaling pathways on the level of transcription.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Montagem e Desmontagem da Cromatina , Proteínas de Membrana/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Tretinoína/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , NF-kappa B/metabolismo , RNA Polimerase II/metabolismo , Elementos de Resposta , Transdução de Sinais , Fator de Transcrição RelA/metabolismo , Fator de Transcrição TFIIH/metabolismo , Fatores de Transcrição/metabolismo , Células U937RESUMO
Peroxisomes are a critical rheostat of reactive oxygen species (ROS), yet their role in drug sensitivity and resistance remains unexplored. Gene expression analysis of clinical lymphoma samples suggests that peroxisomes are involved in mediating drug resistance to the histone deacetylase inhibitor (HDACi) Vorinostat (Vor), which promotes ROS-mediated apoptosis. Vor augments peroxisome numbers in cultured lymphoma cells, concomitant with increased levels of peroxisomal proteins PEX3, PEX11B, and PMP70. Genetic inhibition of peroxisomes, using PEX3 knockdown, reveals that peroxisomes protect lymphoma cells against Vor-mediated cell death. Conversely, Vor-resistant cells were tolerant to elevated ROS levels and possess upregulated levels of (1) catalase, a peroxisomal antioxidant, and (2) plasmalogens, ether glycerophospholipids that represent peroxisome function and serve as antioxidants. Catalase knockdown induces apoptosis in Vor-resistant cells and potentiates ROS-mediated apoptosis in Vor-sensitive cells. These findings highlight the role of peroxisomes in resistance to therapeutic intervention in cancer, and provide a novel modality to circumvent drug resistance.
Assuntos
Apoptose/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Linfoma/patologia , Peroxissomos/metabolismo , Catalase/metabolismo , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Ácidos Hidroxâmicos/farmacologia , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , VorinostatRESUMO
Loss or inactivation of the histone H3K27 demethylase UTX occurs in several malignancies, including multiple myeloma (MM). Using an isogenic cell system, we found that loss of UTX leads to deactivation of gene expression ultimately promoting the proliferation, clonogenicity, adhesion, and tumorigenicity of MM cells. Moreover, UTX mutant cells showed increased in vitro and in vivo sensitivity to inhibition of EZH2, a histone methyltransferase that generates H3K27me3. Such sensitivity was related to a decrease in the levels of IRF4 and c-MYC and an activation of repressors of IRF4 characteristic of germinal center B cells such as BCL6 and IRF1. Rebalance of H3K27me3 levels at specific genes through EZH2 inhibitors may be a therapeutic strategy in MM cases harboring UTX mutations.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Histona Desmetilases/deficiência , Mieloma Múltiplo/patologia , Proteínas Nucleares/deficiência , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/patologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Desdiferenciação Celular/efeitos dos fármacos , Desdiferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Clonais , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Desmetilases/metabolismo , Histonas/metabolismo , Indazóis/farmacologia , Fatores Reguladores de Interferon/metabolismo , Lisina/metabolismo , Metilação , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mieloma Múltiplo/genética , Mutação/genética , Proteínas Nucleares/metabolismo , Fenótipo , Piridonas/farmacologia , Transcrição Gênica/efeitos dos fármacosAssuntos
Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica/métodos , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Proteassoma/farmacologia , Animais , Antineoplásicos/farmacologia , Compostos de Boro/farmacologia , Bortezomib/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Masculino , Camundongos SCID , Prognóstico , Células U937 , Vorinostat , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML) cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC) reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents.
Assuntos
Antineoplásicos/farmacologia , Dano ao DNA , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Leucemia Mieloide Aguda/patologia , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fase G2/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , VorinostatRESUMO
The steroid and xenobiotic receptor SXR (human pregnane X receptor) is a nuclear receptor that plays a key role in the body's detoxification response by regulating genes involved in drug metabolism and transport. SXR ligands include a wide range of compounds, which induce transcription of SXR target genes via activation of a heterodimeric transcription factor consisting of SXR and the related nuclear receptor retinoid X receptor (RXR). We investigated the effect of RXR-selective ligands, rexinoids, on SXR/RXR activity. In agreement with previous reports, we found that rexinoids are weak activators of SXR, but we also found that they can antagonize SXR activation by the potent SXR agonist rifampicin. This antagonism included suppression of rifampicin-induced expression of SXR target genes, as well as reduced binding of SXR/RXR to SXR response elements both in vivo and in vitro. Interestingly, two rexinoids, bexarotene (LGD1069/Targretin) and LG100268, caused a rapid and sustained decrease in the protein levels of both SXR and RXR. The decrease in SXR level was due to an enhanced rate of protein degradation and was dependent on calpain activity, as opposed to rexinoid-induced RXR degradation, which is mediated via the proteasome. Thus, we have demonstrated a novel, rexinoid-modulated mechanism regulating SXR protein stability, which may explain why rexinoids are only weak activators of SXR/RXR-mediated transcription, despite reports that they bind to SXR with high affinity. We suggest that the ability of rexinoids to induce degradation of both SXR and RXR, in combination with competition for binding to SXR, can also explain why rexinoids antagonize the activation of SXR by drugs like rifampicin.