RESUMO
Modern cancer therapies increased the survival rates of acute myeloid leukemia (AML) patients tremendously. However, the complexity of the disease and the identification of new targets require the adaptation of treatment protocols to reduce side effects and increase benefit for the patients. One key regulator of leukemogenesis and chemotherapy resistance in AML is the Hedgehog (HH) signaling pathway. It is deregulated in numerous cancer entities and inhibition of its downstream transcription factors GLI translates into anti-leukemic effects. One major regulator of GLI is BRD4, a BET family member with epigenetic functions. We investigated the effect of ZEN-3365, a novel BRD4 inhibitor, on AML cells in regard to the HH pathway. We show that ZEN-3365 alone or in combination with GANT-61 reduced GLI promoter activity, cell proliferation and colony formation in AML cell lines and primary cells. Our findings strongly support the evaluation of the BRD4 inhibitor ZEN-3365 as a new therapeutic option in AML.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Fatores de Transcrição/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismoRESUMO
A subgroup of castration-resistant prostate cancer (CRPC) aberrantly expresses a gastrointestinal (GI) transcriptome governed by two GI-lineage-restricted transcription factors, HNF1A and HNF4G. In this study, we found that expression of GI transcriptome in CRPC correlates with adverse clinical outcomes to androgen receptor signaling inhibitor treatment and shorter overall survival. Bromo- and extra-terminal domain inhibitors (BETi) downregulated HNF1A, HNF4G, and the GI transcriptome in multiple CRPC models, including cell lines, patient-derived organoids, and patient-derived xenografts, while AR and the androgen-dependent transcriptome were largely spared. Accordingly, BETi selectively inhibited growth of GI transcriptome-positive preclinical models of prostate cancer. Mechanistically, BETi inhibited BRD4 binding at enhancers globally, including both AR and HNF4G bound enhancers while gene expression was selectively perturbed. Restoration of HNF4G expression in the presence of BETi rescued target gene expression without rescuing BRD4 binding. This suggests that inhibition of master transcription factors expression underlies the selective transcriptional effects of BETi. SIGNIFICANCE: GI transcriptome expression in CRPC is regulated by the HNF1A-HNF4G-BRD4 axis and correlates with worse clinical outcomes. Accordingly, BET inhibitors significantly reduce tumor cell growth in multiple GI-transcriptome-positive preclinical models of CRPC. Our studies point that expression of GI transcriptome could serve as a predictive biomarker to BETi therapy response.
RESUMO
DNA damage can induce a tumor suppressive response termed cellular senescence. Damaged senescent cells permanently arrest growth, secrete inflammatory cytokines and other proteins and harbor persistent nuclear foci that contain DNA damage response (DDR) proteins. To understand how persistent damage foci differ from transient foci that mark repairable DNA lesions, we identify sequential events that differentiate transient foci from persistent foci, which we term 'DNA segments with chromatin alterations reinforcing senescence' (DNA-SCARS). Unlike transient foci, DNA-SCARS associate with PML nuclear bodies, lack the DNA repair proteins RPA and RAD51, lack single-stranded DNA and DNA synthesis and accumulate activated forms of the DDR mediators CHK2 and p53. DNA-SCARS form independently of p53, pRB and several other checkpoint and repair proteins but require p53 and pRb to trigger the senescence growth arrest. Importantly, depletion of the DNA-SCARS-stabilizing component histone H2AX did not deplete 53BP1 from DNA-SCARS but diminished the presence of MDC1 and activated CHK2. Furthermore, depletion of H2AX reduced both the p53-dependent senescence growth arrest and p53-independent cytokine secretion. DNA-SCARS were also observed following severe damage to multiple human cell types and mouse tissues, suggesting that they can be used in combination with other markers to identify senescent cells. Thus, DNA-SCARS are dynamically formed distinct structures that functionally regulate multiple aspects of the senescent phenotype.
Assuntos
Ciclo Celular/efeitos da radiação , Núcleo Celular/efeitos da radiação , Senescência Celular/efeitos da radiação , Cromatina/metabolismo , Citocinas/metabolismo , Dano ao DNA/efeitos da radiação , Animais , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Citocinas/genética , Histonas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Raios XRESUMO
PURPOSE: Lineage plasticity in prostate cancer-most commonly exemplified by loss of androgen receptor (AR) signaling and a switch from a luminal to alternate differentiation program-is now recognized as a treatment resistance mechanism. Lineage plasticity is a spectrum, but neuroendocrine prostate cancer (NEPC) is the most virulent example. Currently, there are limited treatments for NEPC. Moreover, the incidence of treatment-emergent NEPC (t-NEPC) is increasing in the era of novel AR inhibitors. In contradistinction to de novo NEPC, t-NEPC tumors often express the AR, but AR's functional role in t-NEPC is unknown. Furthermore, targetable factors that promote t-NEPC lineage plasticity are also unclear. EXPERIMENTAL DESIGN: Using an integrative systems biology approach, we investigated enzalutamide-resistant t-NEPC cell lines and their parental, enzalutamide-sensitive adenocarcinoma cell lines. The AR is still expressed in these t-NEPC cells, enabling us to determine the role of the AR and other key factors in regulating t-NEPC lineage plasticity. RESULTS: AR inhibition accentuates lineage plasticity in t-NEPC cells-an effect not observed in parental, enzalutamide-sensitive adenocarcinoma cells. Induction of an AR-repressed, lineage plasticity program is dependent on activation of the transcription factor E2F1 in concert with the BET bromodomain chromatin reader BRD4. BET inhibition (BETi) blocks this E2F1/BRD4-regulated program and decreases growth of t-NEPC tumor models and a subset of t-NEPC patient tumors with high activity of this program in a BETi clinical trial. CONCLUSIONS: E2F1 and BRD4 are critical for activating an AR-repressed, t-NEPC lineage plasticity program. BETi is a promising approach to block this program.
Assuntos
Antagonistas de Receptores de Andrógenos/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Carcinoma Neuroendócrino/tratamento farmacológico , Fator de Transcrição E2F1/efeitos dos fármacos , Fator de Transcrição E2F1/fisiologia , Nitrilas/uso terapêutico , Feniltioidantoína/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Proteínas/antagonistas & inibidores , Linhagem Celular Tumoral , Humanos , MasculinoRESUMO
PURPOSE: ZEN-3694 is a bromodomain extraterminal inhibitor (BETi) with activity in androgen-signaling inhibitor (ASI)-resistant models. The safety and efficacy of ZEN-3694 plus enzalutamide was evaluated in a phase Ib/IIa study in metastatic castration-resistant prostate cancer (mCRPC). PATIENTS AND METHODS: Patients had progressive mCRPC with prior resistance to abiraterone and/or enzalutamide. 3+3 dose escalation was followed by dose expansion in parallel cohorts (ZEN-3694 at 48 and 96 mg orally once daily, respectively). RESULTS: Seventy-five patients were enrolled (N = 26 and 14 in dose expansion at low- and high-dose ZEN-3694, respectively). Thirty (40.0%) patients were resistant to abiraterone, 34 (45.3%) to enzalutamide, and 11 (14.7%) to both. ZEN-3694 dosing ranged from 36 to 144 mg daily without reaching an MTD. Fourteen patients (18.7%) experienced grade ≥3 toxicities, including three patients with grade 3 thrombocytopenia (4%). An exposure-dependent decrease in whole-blood RNA expression of BETi targets was observed (up to fourfold mean difference at 4 hours post-ZEN-3694 dose; P ≤ 0.0001). The median radiographic progression-free survival (rPFS) was 9.0 months [95% confidence interval (CI), 4.6-12.9] and composite median radiographic or clinical progression-free survival (PFS) was 5.5 months (95% CI, 4.0-7.8). Median duration of treatment was 3.5 months (range, 0-34.7+). Lower androgen receptor (AR) transcriptional activity in baseline tumor biopsies was associated with longer rPFS (median rPFS 10.4 vs. 4.3 months). CONCLUSIONS: ZEN-3694 plus enzalutamide demonstrated acceptable tolerability and potential efficacy in patients with ASI-resistant mCRPC. Further prospective study is warranted including in mCRPC harboring low AR transcriptional activity.
Assuntos
Androstenos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Benzamidas/administração & dosagem , Nitrilas/administração & dosagem , Feniltioidantoína/administração & dosagem , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Androstenos/efeitos adversos , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Benzamidas/efeitos adversos , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Nitrilas/efeitos adversos , Feniltioidantoína/efeitos adversos , Intervalo Livre de Progressão , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/genética , Resultado do TratamentoRESUMO
Metastatic malignant melanoma continues to be a challenging disease despite clinical translation of the comprehensive understanding of driver mutations and how melanoma cells evade immune attack. In Myc-driven lymphoma, efficacy of epigenetic inhibitors of the bromodomain and extra-terminal domain (BET) family of bromodomain proteins can be enhanced by combination therapy with inhibitors of the DNA damage response kinase ATR. Whether this combination is active in solid malignancies like melanoma, and how it relates to immune therapy, has not previously investigated. To test efficacy and molecular consequences of combination therapies cultured melanoma cells were used. To assess tumor responses to therapies in vivo we use patient-derived xenografts and B6 mice transplanted with B16F10 melanoma cells. Concomitant inhibition of BET proteins and ATR of cultured melanoma cells resulted in similar effects as recently shown in lymphoma, such as induction of apoptosis and p62, implicated in autophagy, senescence-associated secretory pathway and ER stress. In vivo, apoptosis and suppression of subcutaneous growth of patient-derived melanoma and B16F10 cells were observed. Our data suggest that ATRI/BETI combination therapies are effective in melanoma.
Assuntos
Antineoplásicos/uso terapêutico , Melanoma/tratamento farmacológico , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Quinolinas/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Linfoma/tratamento farmacológico , Linfoma/genética , Melanoma/genética , Camundongos , Proteínas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
This corrects the article DOI: 10.1038/cddis.2017.383.
RESUMO
Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element-containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishes nucleolar associations, whereas PCNA or HP1 interaction sites within p150 are not required for these interactions. In addition, acute depletion of SUMO-2 or the SUMO E2 ligase Ubc9 reduces α-satellite DNA association with nucleoli. The nucleolar functions of p150 are separable from its interactions with the other subunits of the CAF-1 complex because an N-terminal fragment of p150 (p150N) that cannot interact with other CAF-1 subunits is sufficient for maintaining nucleolar chromosome and protein associations. Therefore these data define novel functions for a separable domain of the p150 protein, regulating protein and DNA interactions at the nucleolus.
Assuntos
Nucléolo Celular/metabolismo , Fator 1 de Modelagem da Cromatina/fisiologia , Cromossomos Humanos/metabolismo , Células HeLa , Humanos , Antígeno Ki-67/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Fatores de TranscriçãoRESUMO
Increased synthesis of Apolipoprotein A-I (ApoA-I) and HDL is believed to provide a new approach to treating atherosclerosis through the stimulation of reverse cholesterol transport. RVX-208 increases the production of ApoA-I in hepatocytes in vitro, and in vivo in monkeys and humans, which results in increased HDL-C, but the molecular target was not previously reported. Using binding assays and X-ray crystallography, we now show that RVX-208 selectively binds to bromodomains of the BET (Bromodomain and Extra Terminal) family, competing for a site bound by the endogenous ligand, acetylated lysine, and that this accounts for its pharmacological activity. siRNA experiments further suggest that induction of ApoA-I mRNA is mediated by BET family member BRD4. These data indicate that RVX-208 increases ApoA-I production through an epigenetic mechanism and suggests that BET inhibition may be a promising new approach to the treatment of atherosclerosis.
Assuntos
Apolipoproteína A-I/biossíntese , Proteínas Nucleares/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Quinazolinas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Animais , Apolipoproteína A-I/genética , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular , Linhagem Celular , Cristalografia por Raios X , Epigênese Genética/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/genética , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Quinazolinas/química , Quinazolinonas , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The discovery of RNA interference (RNAi) and the development of technologies exploiting its biology have enabled scientists to rapidly examine the consequences of depleting a particular gene product in a cell or an animal. The availability of genome-wide RNAi libraries targeting the mouse and human genomes has made it possible to carry out large scale, phenotype-based screens, which have yielded seminal information on diverse cellular processes ranging from virology to cancer biology. Today, several strategies are available to perform RNAi screens, each with their own technical and monetary considerations. Special care and budgeting must be taken into account during the design of these screens in order to obtain reliable results. In this review, we discuss a number of critical aspects to consider when planning an effective RNAi screening strategy, including selecting the right biological system, designing an appropriate selection scheme, optimizing technical aspects of the screen, and validating and verifying the hits. Similar to an artistic production, what happens behind the screen has a direct impact on its success.
Assuntos
Testes Genéticos , Mamíferos/genética , Interferência de RNA , Animais , Humanos , Projetos Piloto , Reprodutibilidade dos TestesRESUMO
The cyclin-dependent kinase inhibitor p16(INK4a) (CDKN2A) is an important tumor suppressor gene frequently inactivated in human tumors. p16 suppresses the development of cancer by triggering an irreversible arrest of cell proliferation termed cellular senescence. Here, we describe another anti-oncogenic function of p16 in addition to its ability to halt cell cycle progression. We show that transient expression of p16 stably represses the hTERT gene, encoding the catalytic subunit of telomerase, in both normal and malignant breast epithelial cells. Short-term p16 expression increases the amount of histone H3 trimethylated on lysine 27 (H3K27) bound to the hTERT promoter, resulting in transcriptional silencing, likely mediated by polycomb complexes. Our results indicate that transient p16 exposure may prevent malignant progression in dividing cells by irreversible repression of genes, such as hTERT, whose activity is necessary for extensive self-renewal.
Assuntos
Neoplasias da Mama/enzimologia , Mama/enzimologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Telomerase/antagonistas & inibidores , Mama/citologia , Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Inativação Gênica , Histonas/metabolismo , Humanos , Metilação , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/genética , Telomerase/metabolismoRESUMO
Cellular senescence suppresses cancer by stably arresting the proliferation of damaged cells. Paradoxically, senescent cells also secrete factors that alter tissue microenvironments. The pathways regulating this secretion are unknown. We show that damaged human cells develop persistent chromatin lesions bearing hallmarks of DNA double-strand breaks (DSBs), which initiate increased secretion of inflammatory cytokines such as interleukin-6 (IL-6). Cytokine secretion occurred only after establishment of persistent DNA damage signalling, usually associated with senescence, not after transient DNA damage responses (DDRs). Initiation and maintenance of this cytokine response required the DDR proteins ATM, NBS1 and CHK2, but not the cell-cycle arrest enforcers p53 and pRb. ATM was also essential for IL-6 secretion during oncogene-induced senescence and by damaged cells that bypass senescence. Furthermore, DDR activity and IL-6 were elevated in human cancers, and ATM-depletion suppressed the ability of senescent cells to stimulate IL-6-dependent cancer cell invasiveness. Thus, in addition to orchestrating cell-cycle checkpoints and DNA repair, a new and important role of the DDR is to allow damaged cells to communicate their compromised state to the surrounding tissue.
Assuntos
Senescência Celular/fisiologia , Citocinas/metabolismo , Dano ao DNA , Transdução de Sinais/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Células Cultivadas , Quinase do Ponto de Checagem 2 , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ensaio de Imunoadsorção Enzimática , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Interleucina-6/metabolismo , Masculino , Microscopia de Fluorescência , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA) or microRNA (miRNA) and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen) whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage. Finally, we determined that both isoforms of the AT-Rich Interacting Domain 4B (ARID4B) protein could induce G1 arrest when overexpressed. As new technologies emerge, the vectors in this collection can be easily modified and adapted without the need for extensive recloning.
Assuntos
Vetores Genéticos , Proteínas/genética , Retroviridae/genética , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Imunofluorescência , Humanos , RNA/genética , Interferência de RNA , Recombinação GenéticaRESUMO
Loss of a nonenzymatic function of XPG results in defective transcription-coupled repair (TCR), Cockayne syndrome (CS), and early death, but the molecular basis for these phenotypes is unknown. Mutation of CSB, CSA, or the TFIIH helicases XPB and XPD can also cause defective TCR and CS. We show that XPG interacts with elongating RNA polymerase II (RNAPII) in the cell and binds stalled RNAPII ternary complexes in vitro both independently and cooperatively with CSB. XPG binds transcription-sized DNA bubbles through two domains not required for incision and functionally interacts with CSB on these bubbles to stimulate its ATPase activity. Bound RNAPII blocks bubble incision by XPG, but an ATP hydrolysis-dependent process involving TFIIH creates access to the junction, allowing incision. Together, these results implicate coordinated recognition of stalled transcription by XPG and CSB in TCR initiation and suggest that TFIIH-dependent remodeling of stalled RNAPII without release may be sufficient to allow repair.