RESUMO
Recurrent somatic mutations in the BRG1/BRM-associated factor (BAF) chromatin remodeling complex subunit ARID1A occur frequently in advanced urothelial, endometrial, and ovarian clear cell carcinomas, creating an alternative chromatin state that may be exploited therapeutically. The histone methyltransferase EZH2 has been previously identified as targetable vulnerability in the context of ARID1A mutations. In this study, we describe the discovery of tulmimetostat, an orally available, clinical stage EZH2 inhibitor, and it elucidates the aspects of its application potential in ARID1A mutant tumors. Tulmimetostat administration achieved efficacy in multiple ARID1A mutant bladder, ovarian, and endometrial tumor models and improved cisplatin response in chemotherapy-resistant models. Consistent with its comprehensive and durable level of target coverage, tulmimetostat demonstrated greater efficacy than other PRC2-targeted inhibitors at comparable or lower exposures in a bladder cancer xenograft mouse model. Tulmimetostat mediated extensive changes in gene expression, in addition to a profound reduction in global H3K27me3 levels in tumors. Phase I clinical pharmacokinetic and pharmacodynamic data indicated that tulmimetostat exhibits durable exposure and profound target engagement. Importantly, a tulmimetostat controlled gene expression signature identified in whole blood from a cohort of 32 patients with cancer correlated with tulmimetostat exposure, representing a pharmacodynamic marker for the assessment of target coverage for PRC2-targeted agents in the clinic. Collectively, these data suggest that tulmimetostat has the potential to achieve clinical benefit in solid tumors as a monotherapy but also in combination with chemotherapeutic agents, and may be beneficial in various indications with recurrent ARID1A mutations. Significance: The EZH2 inhibitor tulmimetostat achieves comprehensive target inhibition in ARID1A mutant solid tumor models and cancer patients that can be assessed with a pharmacodynamic gene signature in peripheral blood.
Assuntos
Proteínas de Ligação a DNA , Proteína Potenciadora do Homólogo 2 de Zeste , Mutação , Fatores de Transcrição , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Camundongos , Fatores de Transcrição/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Feminino , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Linhagem Celular Tumoral , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Neoplasias/metabolismoRESUMO
The histone methyltransferase EZH2 has been the target of numerous small-molecule inhibitor discovery efforts over the last 10+ years. Emerging clinical data have provided early evidence for single agent activity with acceptable safety profiles for first-generation inhibitors. We have developed kinetic methodologies for studying EZH2-inhibitor-binding kinetics that have allowed us to identify a unique structural modification that results in significant increases in the drug-target residence times of all EZH2 inhibitor scaffolds we have studied. The unexpected residence time enhancement bestowed by this modification has enabled us to create a series of second-generation EZH2 inhibitors with sub-pM binding affinities. We provide both biophysical evidence validating this sub-pM potency and biological evidence demonstrating the utility and relevance of such high-affinity interactions with EZH2.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Descoberta de Drogas , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Células HeLa , Humanos , Camundongos SCID , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
PURPOSE: The initiation, progression, and maintenance of pancreatic ductal adenocarcinoma (PDAC) results from the interplay of genetic and epigenetic events. While the genetic alterations of PDAC have been well characterized, epigenetic pathways regulating PDAC remain, for the most part, elusive. The goal of this study was to identify novel epigenetic regulators contributing to the biology of PDAC. EXPERIMENTAL DESIGN: In vivo pooled shRNA screens targeting 118 epigenetic proteins were performed in two orthotopic PDAC xenograft models. Candidate genes were characterized in 19 human PDAC cell lines, heterotopic xenograft tumor models, and a genetically engineered mouse (GEM) model of PDAC. Gene expression, IHC, and immunoprecipitation experiments were performed to analyze the pathways by which candidate genes contribute to PDAC. RESULTS: In vivo shRNA screens identified BRD2 and BRD3, members of the BET family of chromatin adaptors, as key regulators of PDAC tumor growth. Pharmacologic inhibition of BET bromodomains enhanced survival in a PDAC GEM model and inhibited growth of human-derived xenograft tumors. BET proteins contribute to PDAC cell growth through direct interaction with members of the GLI family of transcription factors and modulating their activity. Within cancer cells, BET bromodomain inhibition results in downregulation of SHH, a key mediator of the tumor microenvironment and canonical activator of GLI. Consistent with this, inhibition of BET bromodomains decreases cancer-associated fibroblast content of tumors in both GEM and xenograft tumor models. CONCLUSIONS: Therapeutic inhibition of BET proteins offers a novel mechanism to target both the neoplastic and stromal components of PDAC. Clin Cancer Res; 22(16); 4259-70. ©2016 AACR.
Assuntos
Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas de Ligação a RNA/metabolismo , Microambiente Tumoral , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Genes myc , Proteínas Hedgehog/metabolismo , Xenoenxertos , Humanos , Camundongos , Neoplasias Pancreáticas/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Transdução de Sinais , Carga Tumoral , Microambiente Tumoral/genéticaRESUMO
Covalent modification of histones is a fundamental mechanism of regulated gene expression in eukaryotes, and interpretation of histone modifications is an essential feature of epigenetic control. Bromodomains are specialized binding modules that interact with acetylated histones, linking chromatin recognition to gene transcription. Because of their ability to function in a domain-specific fashion, selective disruption of bromodomain:acetylated histone interactions with chemical probes serves as a powerful means for understanding biological processes regulated by these chromatin adaptors. Here we describe the discovery and characterization of potent and selective small molecule inhibitors for the bromodomains of CREBBP/EP300 that engage their target in cellular assays. We use these tools to demonstrate a critical role for CREBBP/EP300 bromodomains in regulatory T cell biology. Because regulatory T cell recruitment to tumors is a major mechanism of immune evasion by cancer cells, our data highlight the importance of CREBBP/EP300 bromodomain inhibition as a novel, small molecule-based approach for cancer immunotherapy.
Assuntos
Proteína de Ligação a CREB/antagonistas & inibidores , Proteína p300 Associada a E1A/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Proteína de Ligação a CREB/química , Proteína de Ligação a CREB/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Proteína p300 Associada a E1A/química , Proteína p300 Associada a E1A/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Histonas/metabolismo , Humanos , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína/efeitos dos fármacos , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
Inhibition of the bromodomains of the BET family, of which BRD4 is a member, has been shown to decrease myc and interleukin (IL) 6 in vivo, markers that are of therapeutic relevance to cancer and inflammatory disease, respectively. Herein we report substituted benzo[b]isoxazolo[4,5-d]azepines and benzotriazolo[4,3-d][1,4]diazepines as fragment-derived novel inhibitors of the bromodomain of BRD4. Compounds from these series were potent and selective in cells, and subsequent optimization of microsomal stability yielded representatives that demonstrated dose- and time-dependent reduction of plasma IL-6 in mice.
RESUMO
Pharmacological inhibition of chromatin co-regulatory factors represents a clinically validated strategy to modulate oncogenic signaling through selective attenuation of gene expression. Here, we demonstrate that CBP/EP300 bromodomain inhibition preferentially abrogates the viability of multiple myeloma cell lines. Selective targeting of multiple myeloma cell lines through CBP/EP300 bromodomain inhibition is the result of direct transcriptional suppression of the lymphocyte-specific transcription factor IRF4, which is essential for the viability of myeloma cells, and the concomitant repression of the IRF4 target gene c-MYC. Ectopic expression of either IRF4 or MYC antagonizes the phenotypic and transcriptional effects of CBP/EP300 bromodomain inhibition, highlighting the IRF4/MYC axis as a key component of its mechanism of action. These findings suggest that CBP/EP300 bromodomain inhibition represents a viable therapeutic strategy for targeting multiple myeloma and other lymphoid malignancies dependent on the IRF4 network.
Assuntos
Antineoplásicos/farmacologia , Proteína p300 Associada a E1A/antagonistas & inibidores , Fatores Reguladores de Interferon/metabolismo , Mieloma Múltiplo/fisiopatologia , Fragmentos de Peptídeos/antagonistas & inibidores , Sialoglicoproteínas/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , HumanosRESUMO
Small-molecule inhibitors of the bromodomain and extraterminal (BET) family of proteins are being tested in clinical trials for a variety of cancers, but patient selection strategies remain limited. This challenge is partly attributed to the heterogeneous responses elicited by BET inhibition (BETi), including cellular differentiation, senescence, and death. In this study, we performed phenotypic and gene-expression analyses of treatment-naive and engineered tolerant cell lines representing human melanoma and leukemia to elucidate the dominant features defining response to BETi. We found that de novo and acquired tolerance to BETi is driven by the robustness of the apoptotic response, and that genetic or pharmacologic manipulation of the apoptotic signaling network can modify the phenotypic response to BETi. We further reveal that the expression signatures of the apoptotic genes BCL2, BCL2L1, and BAD significantly predict response to BETi. Taken together, our findings highlight the apoptotic program as a determinant of response to BETi, and provide a molecular basis for patient stratification and combination therapy development.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HL-60 , Células HT29 , Humanos , Células K562 , Leucemia/tratamento farmacológico , Leucemia/metabolismo , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
In recent years, inhibition of the interaction between the bromodomain and extra-terminal domain (BET) family of chromatin adaptors and acetyl-lysine residues on chromatin has emerged as a promising approach to regulate the expression of important disease-relevant genes, including MYC, BCL-2, and NF-κB. Here we describe the identification and characterization of a potent and selective benzoisoxazoloazepine BET bromodomain inhibitor that attenuates BET-dependent gene expression in vivo, demonstrates antitumor efficacy in an MV-4-11 mouse xenograft model, and is currently undergoing human clinical trials for hematological malignancies (CPI-0610).
Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Azepinas/química , Azepinas/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Azepinas/farmacocinética , Azepinas/farmacologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , Cães , Genes myc/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Ratos , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this report we detail the evolution of our previously reported thiophene isoxazole BET inhibitor chemotype exemplified by CPI-3 to a novel bromodomain selective chemotype (the methyl isoxazoleazepine chemotype) exemplified by carboxamide 23. The methyl isoxazoleazepine chemotype provides potent inhibition of the bromodomains of the BET family, excellent in vivo PK across species, low unbound clearance, and target engagement in a MYC PK-PD model.
Assuntos
Azepinas/farmacologia , Desenho de Fármacos , Proteínas Nucleares/antagonistas & inibidores , Oxazóis/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Azepinas/síntese química , Azepinas/química , Proteínas de Ciclo Celular , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Oxazóis/síntese química , Oxazóis/química , Relação Estrutura-AtividadeRESUMO
The identification of a novel series of small molecule BET inhibitors is described. Using crystallographic binding modes of an amino-isoxazole fragment and known BET inhibitors, a structure-based drug design effort lead to a novel isoxazole azepine scaffold. This scaffold showed good potency in biochemical and cellular assays and oral activity in an in vivo model of BET inhibition.
RESUMO
The MYC transcription factor is a master regulator of diverse cellular functions and has been long considered a compelling therapeutic target because of its role in a range of human malignancies. However, pharmacologic inhibition of MYC function has proven challenging because of both the diverse mechanisms driving its aberrant expression and the challenge of disrupting protein-DNA interactions. Here, we demonstrate the rapid and potent abrogation of MYC gene transcription by representative small molecule inhibitors of the BET family of chromatin adaptors. MYC transcriptional suppression was observed in the context of the natural, chromosomally translocated, and amplified gene locus. Inhibition of BET bromodomain-promoter interactions and subsequent reduction of MYC transcript and protein levels resulted in G(1) arrest and extensive apoptosis in a variety of leukemia and lymphoma cell lines. Exogenous expression of MYC from an artificial promoter that is resistant to BET regulation significantly protected cells from cell cycle arrest and growth suppression by BET inhibitors. MYC suppression was accompanied by deregulation of the MYC transcriptome, including potent reactivation of the p21 tumor suppressor. Treatment with a BET inhibitor resulted in significant antitumor activity in xenograft models of Burkitt's lymphoma and acute myeloid leukemia. These findings demonstrate that pharmacologic inhibition of MYC is achievable through targeting BET bromodomains. Such inhibitors may have clinical utility given the widespread pathogenetic role of MYC in cancer.
Assuntos
Apoptose/fisiologia , Linfoma de Burkitt/tratamento farmacológico , Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Leucemia Mieloide Aguda/tratamento farmacológico , Fatores de Transcrição/metabolismo , Animais , Apoptose/genética , Azepinas/farmacologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Relação Dose-Resposta a Droga , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína/genética , RNA Interferente Pequeno/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Triazóis/farmacologiaRESUMO
Mouse mammary tumor virus (MMTV) is a complex murine retrovirus that encodes an HIV Rev-like export protein, Rem, from a doubly spliced version of envelope (Env) mRNA. Previously, the N-terminal 98-amino acid sequence of Rem, which is identical to Env signal peptide (SP), and full-length Rem were shown to be functional in a reporter assay that measures a postexport function. Here we show that MMTV-infected cells or cells transfected with rem or env cDNAs express SP, which is the active component in the reporter assay. Uncleaved Rem was partially glycosylated, but mutations in both glycosylation sites within the C terminus prevented Rem function. Mutations that reduced Rem or Env cleavage by signal peptidase greatly reduced SP levels and functional activity in the reporter assay and allowed accumulation of the uncleaved protein. Fluorescence microscopy revealed that GFP-tagged cleavage-site mutants are unstable and lack fluorescence compared with wild-type Rem, suggesting improper folding. Proteasome inhibitors allowed accumulation of uncleaved Rem relative to SP and increased reporter activity, consistent with SP retrotranslocation and proteasome escape before nuclear entry. Expression of a dominant-negative p97 ATPase did not alter levels of unprocessed Rem and SP but decreased reporter activity, suggesting p97-facilitated retrotranslocation of SP. Our results provide an example of a SP that is processed by signal peptidase and retrotranslocated to allow nuclear localization and function.
Assuntos
Núcleo Celular/metabolismo , Vírus do Tumor Mamário do Camundongo/metabolismo , Proteínas de Membrana/metabolismo , Serina Endopeptidases/metabolismo , Proteínas do Envelope Viral/metabolismo , Transporte Ativo do Núcleo Celular , Processamento Alternativo , Animais , Sítios de Ligação/genética , Western Blotting , Linhagem Celular , Glicosilação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células Jurkat , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Microscopia de Fluorescência , Mutação , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/genética , Proteínas do Envelope Viral/genéticaRESUMO
Mouse mammary tumor virus (MMTV) is a complex retrovirus that encodes at least three regulatory and accessory proteins, including Rem. Rem is required for nuclear export of unspliced viral RNA and efficient expression of viral proteins. Our previous data indicated that sequences at the envelope-3' long terminal repeat junction are required for proper export of viral RNA. To further map the Rem-responsive element (RmRE), reporter vectors containing various portions of the viral envelope gene and the 3' long terminal repeat were tested in the presence and absence of Rem in transient transfection assays. A 476-bp fragment that spans the envelope-long terminal repeat junction had activity equivalent to the entire 3'-end of the mouse mammary tumor virus genome, but further deletions at the 5'- or 3'-ends reduced Rem responsiveness. RNase structure mapping of the full-length RmRE and a 3'-truncation suggested multiple domains with local base pairing and intervening single-stranded segments. A secondary structure model constrained by these data is reminiscent of the RNA response elements of other complex retroviruses, with numerous local stem-loops and long-range base pairs near the 5'- and 3'-boundaries, and differs substantially from an earlier model generated without experimental constraints. Covariation analysis provides limited support for basic features of our model. Reporter assays in human and mouse cell lines revealed similar boundaries, suggesting that the RmRE does not require cell type-specific proteins to form a functional structure.
Assuntos
Genoma Viral/fisiologia , Vírus do Tumor Mamário do Camundongo/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , RNA Viral/metabolismo , Elementos de Resposta/fisiologia , Sequências Repetidas Terminais/fisiologia , Animais , Linhagem Celular , Mapeamento Cromossômico/métodos , Humanos , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , RNA Viral/genética , Ratos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
BACKGROUND: Mouse mammary tumor virus (MMTV) encodes the Rem protein, an HIV Rev-like protein that enhances nuclear export of unspliced viral RNA in rodent cells. We have shown that Rem is expressed from a doubly spliced RNA, typical of complex retroviruses. Several recent reports indicate that MMTV can infect human cells, suggesting that MMTV might interact with human retroviruses, such as human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), and human endogenous retrovirus type K (HERV-K). In this report, we test whether the export/regulatory proteins of human complex retroviruses will increase expression from vectors containing the Rem-responsive element (RmRE). RESULTS: MMTV Rem, HIV Rev, and HTLV Rex proteins, but not HERV-K Rec, enhanced expression from an MMTV-based reporter plasmid in human T cells, and this activity was dependent on the RmRE. No RmRE-dependent reporter gene expression was detectable using Rev, Rex, or Rec in HC11 mouse mammary cells. Cell fractionation and RNA quantitation experiments suggested that the regulatory proteins did not affect RNA stability or nuclear export in the MMTV reporter system. Rem had no demonstrable activity on export elements from HIV, HTLV, or HERV-K. Similar to the Rem-specific activity in rodent cells, the RmRE-dependent functions of Rem, Rev, or Rex in human cells were inhibited by a dominant-negative truncated nucleoporin that acts in the Crm1 pathway of RNA and protein export. CONCLUSION: These data argue that many retroviral regulatory proteins recognize similar complex RNA structures, which may depend on the presence of cell-type specific proteins. Retroviral protein activity on the RmRE appears to affect a post-export function of the reporter RNA. Our results provide additional evidence that MMTV is a complex retrovirus with the potential for viral interactions in human cells.
Assuntos
Expressão Gênica , Produtos do Gene rex/metabolismo , Vírus do Tumor Mamário do Camundongo/fisiologia , RNA Viral/metabolismo , Proteínas do Envelope Viral/metabolismo , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismo , Animais , Linhagem Celular , Genes Reporter , Humanos , Camundongos , Ligação ProteicaRESUMO
Type B leukemogenic virus (TBLV), a mouse mammary tumor virus (MMTV) variant, often induces T-cell leukemias and lymphomas by c-myc activation following viral DNA integration. Transfection assays using a c-myc reporter plasmid indicated that the TBLV long terminal repeat (LTR) enhancer is necessary for T-cell-specific increases in basal reporter activity. The sequence requirements for this effect were studied using mutations of the 62-bp enhancer region in an MMTV LTR reporter vector. Deletion of a nuclear factor A-binding site dramatically reduced reporter activity in Jurkat T cells. However, a 41-bp enhancer missing the RUNX1 site still retained minimal enhancer function. DNA affinity purification using a TBLV enhancer oligomer containing the RUNX1 binding site followed by mass spectrometry resulted in the identification of ALY. Subsequent experiments focused on the reconstitution of enhancer activity in epithelial cells. ALY overexpression synergized with RUNX1B on TBLV enhancer activity, and synergism required the RUNX1B-binding site. A predicted c-Myb binding site in the enhancer was confirmed after c-myb overexpression elevated TBLV LTR reporter activity, and overexpression of c-Myb and RUNX1B together showed additive effects on reporter gene levels. ALY also synergized with c-Myb, and coimmunoprecipitation experiments demonstrated an interaction between ALY and c-Myb. These experiments suggest a central role for ALY in T-cell enhancer function and oncogene activation.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Proteínas de Ligação a RNA/metabolismo , Retroviridae/genética , Retroviridae/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Elementos Facilitadores Genéticos , Humanos , Camundongos , Dados de Sequência Molecular , Mutação/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas de Ligação a RNA/genética , Retroviridae/classificação , Fatores de Transcrição/genética , Regulação para CimaRESUMO
Mouse mammary tumor virus (MMTV) has been classified as a simple retrovirus with two accessory genes, dut and sag. Cloned MMTV proviruses carrying a trimethoprim (trim) cassette in the envelope gene were defective for Gag protein production and the nuclear export of unspliced gag-pol RNA. Complementation experiments indicated that a trans-acting product was responsible for the Gag defect of such mutants. Analysis of MMTV-infected cells revealed the presence of a novel, doubly spliced RNA that encodes a putative product of 301 amino acids. Overexpression of cDNA from this RNA increased Gag levels from env mutant proviruses or reporter gene expression from unspliced mRNAs and allowed detection of a 33-kDa protein product, which has been named regulator of export of MMTV mRNA, or Rem. The Rem N terminus has motifs similar to the Rev-like export proteins of complex retroviruses, and mutation of the nuclear localization signal (NLS) abolished RNA export and detection within the nucleus. The Rem C terminus has few identifiable features, but removal of this domain increased Rem-mediated export, suggesting an autoregulatory function. A reporter vector developed from the 3' end of the MMTV provirus was Rem responsive and required both the presence of the MMTV env-U3 junction and a functional Crm1 pathway. The identification of a third accessory protein from a doubly spliced transcript suggests that MMTV is the first murine complex retrovirus to be documented. Manipulation of the MMTV genome may provide mouse models for human retroviral diseases, such as AIDS.
Assuntos
Vírus do Tumor Mamário do Camundongo/metabolismo , Proteínas Virais/metabolismo , Animais , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Virais/genéticaRESUMO
Type B leukemogenic virus (TBLV) is highly related to mouse mammary tumor virus but induces rapidly appearing T-cell lymphomas in mice. Unlike other T-cell tumors induced by retroviruses, only 5 to 10% of TBLV-induced lymphomas have detectable viral integrations near c-myc by Southern blotting, whereas Northern blotting has shown that most tumors have two- to sixfold overexpression of c-myc RNA. In this report, PCR was used to demonstrate that at least 30% of these lymphomas have TBLV insertions near c-myc. Some tumors contained multiple TBLV proviruses in different locations and orientations, suggesting that the tumors are polyclonal. The integrated proviruses near c-myc had different numbers (two to four) of long terminal repeat (LTR) enhancer repeats, although LTRs with three-repeat enhancers dominated the proviral population. Passage of polyclonal tumors in immunocompetent mice and semiquantitative PCR revealed that only cells with particular integrations were selected for growth. In three of six tumors tested, proviruses containing four-repeat enhancers near c-myc were selected during tumor passage. Since tumor cell selection may be accomplished by overexpression of c-myc RNA due to proximity to the unique TBLV LTR enhancer, we inserted LTRs at various locations within a plasmid containing the entire c-myc locus and cellular flanking sequences. To quantitatively measure effects on transcription, the Renilla luciferase gene was substituted for most of c-myc exon 2, and transient transfections were performed with c-myc reporter constructs in two different T-cell lines. As expected, insertion of a TBLV LTR with three-repeat enhancers in either orientation, 5" and 3", of the myc gene elevated reporter activity from 2- to 160-fold, consistent with enhancer function, but four-repeat LTRs had lower levels of expression compared to three-repeat LTRs. Surprisingly, LTR insertions that gave maximal c-myc expression in transient-transfection assays declined in tumor cells selected for growth in vivo. Selection for clonal growth may occur in tumor cells that have modest c-myc overexpression after proviral insertion to prevent apoptosis.