RESUMO
Molecular glues are proximity-inducing small molecules that have emerged as an attractive therapeutic approach. However, developing molecular glues remains challenging, requiring innovative mechanistic strategies to stabilize neoprotein interfaces and expedite discovery. Here we unveil a trans-labeling covalent molecular glue mechanism, termed 'template-assisted covalent modification'. We identified a new series of BRD4 molecular glue degraders that recruit CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). Through comprehensive biochemical, structural and mutagenesis analyses, we elucidated how pre-existing structural complementarity between DCAF16 and BRD4BD2 serves as a template to optimally orient the degrader for covalent modification of DCAF16Cys58. This process stabilizes the formation of BRD4-degrader-DCAF16 ternary complex and facilitates BRD4 degradation. Supporting generalizability, we found that a subset of degraders also induces GAK-BRD4BD2 interaction through trans-labeling of GAK. Together, our work establishes 'template-assisted covalent modification' as a mechanism for covalent molecular glues, which opens a new path to proximity-driven pharmacology.
RESUMO
BCL6, an oncogenic transcription factor (TF), forms polymers in the presence of a small-molecule molecular glue that stabilizes a complementary interface between homodimers of BCL6's broad-complex, tramtrack, and bric-à-brac (BTB) domain. The BTB domains of other proteins, including a large class of TFs, have similar architectures and symmetries, raising the possibility that additional BTB proteins self-assemble into higher-order structures. Here, we surveyed 189 human BTB proteins with a cellular fluorescent reporter assay and identified 18 ZBTB TFs that show evidence of polymerization. Through biochemical and cryoelectron microscopy (cryo-EM) studies, we demonstrate that these ZBTB TFs polymerize into filaments. We found that BTB-domain-mediated polymerization of ZBTB TFs enhances chromatin occupancy within regions containing homotypic clusters of TF binding sites, leading to repression of target genes. Our results reveal a role of higher-order structures in regulating ZBTB TFs and suggest an underappreciated role for TF polymerization in modulating gene expression.
Assuntos
Cromatina , Microscopia Crioeletrônica , Humanos , Cromatina/metabolismo , Cromatina/genética , Multimerização Proteica , Sítios de Ligação , Ligação Proteica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Polimerização , Células HEK293 , Regulação da Expressão GênicaRESUMO
Nuclear hormone receptors (NRs) are ligand-binding transcription factors that are widely targeted therapeutically. Agonist binding triggers NR activation and subsequent degradation by unknown ligand-dependent ubiquitin ligase machinery. NR degradation is critical for therapeutic efficacy in malignancies that are driven by retinoic acid and estrogen receptors. Here, we demonstrate the ubiquitin ligase UBR5 drives degradation of multiple agonist-bound NRs, including the retinoic acid receptor alpha (RARA), retinoid x receptor alpha (RXRA), glucocorticoid, estrogen, liver-X, progesterone, and vitamin D receptors. We present the high-resolution cryo-EMstructure of full-length human UBR5 and a negative stain model representing its interaction with RARA/RXRA. Agonist ligands induce sequential, mutually exclusive recruitment of nuclear coactivators (NCOAs) and UBR5 to chromatin to regulate transcriptional networks. Other pharmacological ligands such as selective estrogen receptor degraders (SERDs) degrade their receptors through differential recruitment of UBR5 or RNF111. We establish the UBR5 transcriptional regulatory hub as a common mediator and regulator of NR-induced transcription.
Assuntos
Cromatina , Fatores de Transcrição , Humanos , Ligantes , Cromatina/genética , Fatores de Transcrição/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Ubiquitinas , Ubiquitina-Proteína Ligases/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks multiple human proteins during infection and viral replication. To examine whether any viral proteins employ human E3 ubiquitin ligases, we evaluated the stability of SARS-CoV-2 proteins with inhibition of the ubiquitin proteasome pathway. Using genetic screens to dissect the molecular machinery involved in the degradation of candidate viral proteins, we identified human E3 ligase RNF185 as a regulator of protein stability for the SARS-CoV-2 envelope protein. We found that RNF185 and the SARS-CoV-2 envelope co-localize to the endoplasmic reticulum (ER). Finally, we demonstrate that the depletion of RNF185 significantly increases SARS-CoV-2 viral titer in a cellular model. Modulation of this interaction could provide opportunities for novel antiviral therapies.
RESUMO
Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics1-3. Molecular glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs4-6. The discovery and development of molecular glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a trans-labeling covalent molecular glue mechanism, which we term "template-assisted covalent modification". We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). BRD4BD2, in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 Å cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4BD2 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16Cys58 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4BD2 revealed that the loop conformation around BRD4His437, rather than specific side chains, is critical for stable interaction with DCAF16 and BD2 selectivity. Together our work establishes "template-assisted covalent modification" as a mechanism for covalent molecular glues, which opens a new path to proximity driven pharmacology.
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Despite advances in the field, chronic graft-versus-host-disease (cGVHD) remains a leading cause of morbidity and mortality following allogenic hematopoietic stem cell transplant. Because treatment options remain limited, we tested efficacy of anticancer, chromatin-modifying enzyme inhibitors in a clinically relevant murine model of cGVHD with bronchiolitis obliterans (BO). We observed that the novel enhancer of zeste homolog 2 (EZH2) inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 each improved pulmonary function; impaired the germinal center (GC) reaction, a prerequisite in cGVHD/BO pathogenesis; and JQ5 reduced EZH2-mediated H3K27me3 in donor T cells. Using conditional EZH2 knockout donor cells, we demonstrated that EZH2 is obligatory for the initiation of cGVHD/BO. In a sclerodermatous cGVHD model, JQ5 reduced the severity of cutaneous lesions. To determine how the 2 drugs could lead to the same physiological improvements while targeting unique epigenetic processes, we analyzed the transcriptomes of splenic GCB cells (GCBs) from transplanted mice treated with either drug. Multiple inflammatory and signaling pathways enriched in cGVHD/BO GCBs were reduced by each drug. GCBs from JQ5- but not JQ1-treated mice were enriched for proproliferative pathways also seen in GCBs from bone marrow-only transplanted mice, likely reflecting their underlying biology in the unperturbed state. In conjunction with in vivo data, these insights led us to conclude that epigenetic targeting of the GC is a viable clinical approach for the treatment of cGVHD, and that the EZH2 inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 demonstrated clinical potential for EZH2i and BETi in patients with cGVHD/BO.
Assuntos
Bronquiolite Obliterante , Proteína Potenciadora do Homólogo 2 de Zeste , Centro Germinativo , Doença Enxerto-Hospedeiro , Proteínas , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Linfócitos B/patologia , Bronquiolite Obliterante/genética , Bronquiolite Obliterante/metabolismo , Bronquiolite Obliterante/patologia , Doença Crônica , 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 , Inibidores Enzimáticos/farmacologia , Centro Germinativo/efeitos dos fármacos , Centro Germinativo/patologia , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Humanos , Camundongos , Proteínas/metabolismo , TranscriptomaRESUMO
Gene expression is regulated by promoters and enhancers marked by histone H3 lysine 27 acetylation (H3K27ac), which is established by the paralogous histone acetyltransferases (HAT) EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma (NB) depends on EP300, whereas CBP has a limited role. EP300 controls enhancer acetylation by interacting with TFAP2ß, a transcription factor member of the lineage-defining transcriptional core regulatory circuitry (CRC) in NB. To disrupt EP300, we developed a proteolysis-targeting chimera (PROTAC) compound termed "JQAD1" that selectively targets EP300 for degradation. JQAD1 treatment causes loss of H3K27ac at CRC enhancers and rapid NB apoptosis, with limited toxicity to untransformed cells where CBP may compensate. Furthermore, JQAD1 activity is critically determined by cereblon (CRBN) expression across NB cells. SIGNIFICANCE: EP300, but not CBP, controls oncogenic CRC-driven transcription in high-risk NB by binding TFAP2ß. We developed JQAD1, a CRBN-dependent PROTAC degrader with preferential activity against EP300 and demonstrated its activity in NB. JQAD1 has limited toxicity to untransformed cells and is effective in vivo in a CRBN-dependent manner. This article is highlighted in the In This Issue feature, p. 587.
Assuntos
Neuroblastoma , Sequências Reguladoras de Ácido Nucleico , Acetilação , Criança , Proteína p300 Associada a E1A/genética , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , OncogenesRESUMO
Lysine demethylase 5A (KDM5A) is a negative regulator of histone H3K4 trimethylation, a histone mark associated with activate gene transcription. We identify that KDM5A interacts with the P-TEFb complex and cooperates with MYC to control MYC targeted genes in multiple myeloma (MM) cells. We develop a cell-permeable and selective KDM5 inhibitor, JQKD82, that increases histone H3K4me3 but paradoxically inhibits downstream MYC-driven transcriptional output in vitro and in vivo. Using genetic ablation together with our inhibitor, we establish that KDM5A supports MYC target gene transcription independent of MYC itself, by supporting TFIIH (CDK7)- and P-TEFb (CDK9)-mediated phosphorylation of RNAPII. These data identify KDM5A as a unique vulnerability in MM functioning through regulation of MYC-target gene transcription, and establish JQKD82 as a tool compound to block KDM5A function as a potential therapeutic strategy for MM.
Assuntos
Lisina , Mieloma Múltiplo , Quinase 9 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Genes cdc , Humanos , Metilação , Mieloma Múltiplo/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase II , Proteína 2 de Ligação ao Retinoblastoma , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Identity determining transcription factors (TFs), or core regulatory (CR) TFs, are governed by cell-type specific super enhancers (SEs). Drugs to selectively inhibit CR circuitry are of high interest for cancer treatment. In alveolar rhabdomyosarcoma, PAX3-FOXO1 activates SEs to induce the expression of other CR TFs, providing a model system for studying cancer cell addiction to CR transcription. Using chemical genetics, the systematic screening of chemical matter for a biological outcome, here we report on a screen for epigenetic chemical probes able to distinguish between SE-driven transcription and constitutive transcription. We find that chemical probes along the acetylation-axis, and not the methylation-axis, selectively disrupt CR transcription. Additionally, we find that histone deacetylases (HDACs) are essential for CR TF transcription. We further dissect the contribution of HDAC isoforms using selective inhibitors, including the newly developed selective HDAC3 inhibitor LW3. We show HDAC1/2/3 are the co-essential isoforms that when co-inhibited halt CR transcription, making CR TF sites hyper-accessible and disrupting chromatin looping.
Assuntos
Elementos Facilitadores Genéticos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Rabdomiossarcoma/genética , Acetilação/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Ensaios de Triagem em Larga Escala , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Humanos , Simulação de Dinâmica Molecular , Sondas Moleculares/química , Sondas Moleculares/farmacologia , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Cultura Primária de Células , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Rabdomiossarcoma/patologia , Análise de Sequência de RNA , Transcrição Gênica/efeitos dos fármacosRESUMO
Drug-tolerant "persister" tumor cells underlie emergence of drug-resistant clones and contribute to relapse and disease progression. Here we report that resistance to the BCL-2 targeting drug ABT-199 in models of mantle cell lymphoma and double-hit lymphoma evolves from outgrowth of persister clones displaying loss of 18q21 amplicons that harbor BCL2. Further, persister status is generated via adaptive super-enhancer remodeling that reprograms transcription and offers opportunities for overcoming ABT-199 resistance. Notably, pharmacoproteomic and pharmacogenomic screens revealed that persisters are vulnerable to inhibition of the transcriptional machinery and especially to inhibition of cyclin-dependent kinase 7 (CDK7), which is essential for the transcriptional reprogramming that drives and sustains ABT-199 resistance. Thus, transcription-targeting agents offer new approaches to disable drug resistance in B-cell lymphomas.
RESUMO
BACKGROUND: Medulloblastoma (MB) is one of the most frequent malignant brain tumors of children, and a large set of these tumors is characterized by aberrant activation of the sonic hedgehog (SHH) pathway. While some tumors initially respond to inhibition of the SHH pathway component Smoothened (SMO), tumors ultimately recur due to downstream resistance mechanisms, indicating a need for novel therapeutic options. METHODS: Here we performed a targeted small-molecule screen on a stable, SHH-dependent murine MB cell line (SMB21). Comprehensive isotype profiling of histone deacetylase (HDAC) inhibitors was performed, and effects of HDAC inhibition were evaluated in cell lines both sensitive and resistant to SMO inhibition. Lastly, distinct mouse models of SHH MB were used to demonstrate pharmacologic efficacy in vivo. RESULTS: A subset of the HDAC inhibitors tested significantly inhibit tumor growth of SMB21 cells by preventing SHH pathway activation. Isotype profiling of HDAC inhibitors, together with genetic approaches suggested that concerted inhibition of multiple class I HDACs is necessary to achieve pathway inhibition. Of note, class I HDAC inhibitors were also efficacious in suppressing growth of diverse SMO inhibitorâresistant clones of SMB21 cells. Finally, we show that the novel HDAC inhibitor quisinostat targets multiple class I HDACs, is well tolerated in mouse models, and robustly inhibits growth of SHH MB cells in vivo as well as in vitro. CONCLUSIONS: Our data provide strong evidence that quisinostat or other class I HDAC inhibitors might be therapeutically useful for patients with SHH MB, including those resistant to SMO inhibition.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Neoplasias Cerebelares/tratamento farmacológico , Proteínas Hedgehog/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Meduloblastoma/tratamento farmacológico , Anilidas , Animais , Compostos de Bifenilo , Linhagem Celular Tumoral , Neoplasias Cerebelares/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Hedgehog/metabolismo , Ensaios de Triagem em Larga Escala , Concentração Inibidora 50 , Meduloblastoma/metabolismo , Camundongos , Proteínas/genética , Piridinas , Proteínas Repressoras/genética , Transdução de Sinais , Receptor Smoothened/antagonistas & inibidores , Receptor Smoothened/metabolismoRESUMO
Proteasome inhibition is an effective treatment for multiple myeloma (MM); however, targeting different components of the ubiquitin-proteasome system (UPS) remains elusive. Our RNA-interference studies identified proteasome-associated ubiquitin-receptor Rpn13 as a mediator of MM cell growth and survival. Here, we developed the first degrader of Rpn13, WL40, using a small-molecule-induced targeted protein degradation strategy to selectively degrade this component of the UPS. WL40 was synthesized by linking the Rpn13 covalent inhibitor RA190 with the cereblon (CRBN) binding ligand thalidomide. We show that WL40 binds to both Rpn13 and CRBN and triggers degradation of cellular Rpn13, and is therefore first-in-class in exploiting a covalent inhibitor for the development of degraders. Biochemical and cellular studies show that WL40-induced Rpn13 degradation is both CRBN E3 ligase- and Rpn13-dependent. Importantly, WL40 decreases viability in MM cell lines and patient MM cells, even those resistant to bortezomib. Mechanistically, WL40 interrupts Rpn13 function and activates caspase apoptotic cascade, ER stress response and p53/p21 signaling. In animal model studies, WL40 inhibits xenografted human MM cell growth and prolongs survival. Overall, our data show the development of the first UbR Rpn13 degrader with potent anti-MM activity, and provide proof of principle for the development of degraders targeting components of the UPS for therapeutic application.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mieloma Múltiplo/metabolismo , Inibidores de Proteassoma/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose , Bortezomib/farmacologia , Sistemas CRISPR-Cas , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Células Dendríticas/citologia , Avaliação Pré-Clínica de Medicamentos , Células HCT116 , Humanos , Lenalidomida/farmacologia , Camundongos , Camundongos SCID , Mieloma Múltiplo/terapia , Transplante de Neoplasias , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , Ubiquitina/químicaRESUMO
Terminal differentiation opposes proliferation in the vast majority of tissue types. As a result, loss of lineage differentiation is a hallmark of aggressive cancers, including soft tissue sarcomas (STS). Consistent with these observations, undifferentiated pleomorphic sarcoma (UPS), an STS subtype devoid of lineage markers, is among the most lethal sarcomas in adults. Though tissue-specific features are lost in these mesenchymal tumors they are most commonly diagnosed in skeletal muscle, and are thought to develop from transformed muscle progenitor cells. We have found that a combination of HDAC (Vorinostat) and BET bromodomain (JQ1) inhibition partially restores differentiation to skeletal muscle UPS cells and tissues, enforcing a myoblast-like identity. Importantly, differentiation is partially contingent upon downregulation of the Hippo pathway transcriptional effector Yes-associated protein 1 (YAP1) and nuclear factor (NF)-κB. Previously, we observed that Vorinostat/JQ1 inactivates YAP1 and restores oscillation of NF-κB in differentiating myoblasts. These effects correlate with reduced tumorigenesis, and enhanced differentiation. However, the mechanisms by which the Hippo/NF-κB axis impact differentiation remained unknown. Here, we report that YAP1 and NF-κB activity suppress circadian clock function, inhibiting differentiation and promoting proliferation. In most tissues, clock activation is antagonized by the unfolded protein response (UPR). However, skeletal muscle differentiation requires both Clock and UPR activity, suggesting the molecular link between them is unique in muscle. In skeletal muscle-derived UPS, we observed that YAP1 suppresses PERK and ATF6-mediated UPR target expression as well as clock genes. These pathways govern metabolic processes, including autophagy, and their disruption shifts metabolism toward cancer cell-associated glycolysis and hyper-proliferation. Treatment with Vorinostat/JQ1 inhibited glycolysis/MTOR signaling, activated the clock, and upregulated the UPR and autophagy via inhibition of YAP1/NF-κB. These findings support the use of epigenetic modulators to treat human UPS. In addition, we identify specific autophagy, UPR, and muscle differentiation-associated genes as potential biomarkers of treatment efficacy and differentiation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagia/genética , Proteínas de Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Musculares/genética , NF-kappa B/genética , Sarcoma/genética , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Autofagia/efeitos dos fármacos , Azepinas/farmacologia , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Linhagem Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Relógios Circadianos/efeitos dos fármacos , Relógios Circadianos/genética , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Musculares/tratamento farmacológico , Neoplasias Musculares/metabolismo , Neoplasias Musculares/patologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Mioblastos/patologia , NF-kappa B/metabolismo , Sarcoma/tratamento farmacológico , Sarcoma/metabolismo , Sarcoma/patologia , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células-Tronco/patologia , Triazóis/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Vorinostat/farmacologia , Proteínas de Sinalização YAP , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
To date, no consistent oncogenic driver mutations have been identified in most adult soft tissue sarcomas; these tumors are thus generally insensitive to existing targeted therapies. Here we investigated alternate mechanisms underlying sarcomagenesis to identify potential therapeutic interventions. Undifferentiated pleomorphic sarcoma (UPS) is an aggressive tumor frequently found in skeletal muscle where deregulation of the Hippo pathway and aberrant stabilization of its transcriptional effector yes-associated protein 1 (YAP1) increases proliferation and tumorigenesis. However, the downstream mechanisms driving this deregulation are incompletely understood. Using autochthonous mouse models and whole genome analyses, we found that YAP1 was constitutively active in some sarcomas due to epigenetic silencing of its inhibitor angiomotin (AMOT). Epigenetic modulators vorinostat and JQ1 restored AMOT expression and wild-type Hippo pathway signaling, which induced a muscle differentiation program and inhibited sarcomagenesis. YAP1 promoted sarcomagenesis by inhibiting expression of ubiquitin-specific peptidase 31 (USP31), a newly identified upstream negative regulator of NFκB signaling. Combined treatment with epigenetic modulators effectively restored USP31 expression, resulting in decreased NFκB activity. Our findings highlight a key underlying molecular mechanism in UPS and demonstrate the potential impact of an epigenetic approach to sarcoma treatment.Significance: A new link between Hippo pathway signaling, NFκB, and epigenetic reprogramming is highlighted and has the potential for therapeutic intervention in soft tissue sarcomas. Cancer Res; 78(10); 2705-20. ©2018 AACR.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transformação Celular Neoplásica/patologia , NF-kappa B/metabolismo , Fosfoproteínas/metabolismo , Sarcoma/patologia , Neoplasias de Tecidos Moles/patologia , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/biossíntese , Angiomotinas , Animais , Antineoplásicos/farmacologia , Azepinas/farmacologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Via de Sinalização Hippo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Músculo Esquelético/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Sarcoma/genética , Transdução de Sinais/genética , Neoplasias de Tecidos Moles/genética , Fatores de Transcrição , Triazóis/farmacologia , Vorinostat/farmacologia , Proteínas de Sinalização YAPRESUMO
The release of paused RNA polymerase II into productive elongation is highly regulated, especially at genes that affect human development and disease. To exert control over this rate-limiting step, we designed sequence-specific synthetic transcription elongation factors (Syn-TEFs). These molecules are composed of programmable DNA-binding ligands flexibly tethered to a small molecule that engages the transcription elongation machinery. By limiting activity to targeted loci, Syn-TEFs convert constituent modules from broad-spectrum inhibitors of transcription into gene-specific stimulators. Here we present Syn-TEF1, a molecule that actively enables transcription across repressive GAA repeats that silence frataxin expression in Friedreich's ataxia, a terminal neurodegenerative disease with no effective therapy. The modular design of Syn-TEF1 defines a general framework for developing a class of molecules that license transcription elongation at targeted genomic loci.