RESUMO
Gene expression by RNA polymerase II (RNAPII) is tightly controlled by cyclin-dependent kinases (CDKs) at discrete checkpoints during the transcription cycle. The pausing checkpoint following transcription initiation is primarily controlled by CDK9. We discovered that CDK9-mediated, RNAPII-driven transcription is functionally opposed by a protein phosphatase 2A (PP2A) complex that is recruited to transcription sites by the Integrator complex subunit INTS6. PP2A dynamically antagonizes phosphorylation of key CDK9 substrates including DSIF and RNAPII-CTD. Loss of INTS6 results in resistance to tumor cell death mediated by CDK9 inhibition, decreased turnover of CDK9 phospho-substrates, and amplification of acute oncogenic transcriptional responses. Pharmacological PP2A activation synergizes with CDK9 inhibition to kill both leukemic and solid tumor cells, providing therapeutic benefit in vivo. These data demonstrate that fine control of gene expression relies on the balance between kinase and phosphatase activity throughout the transcription cycle, a process dysregulated in cancer that can be exploited therapeutically.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteína Fosfatase 2/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos NOD , Fosforilação , Ligação Proteica , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Especificidade por SubstratoRESUMO
Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.
Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , Animais , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Metilação de DNA , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
CKIα ablation induces p53 activation, and CKIα degradation underlies the therapeutic effect of lenalidomide in a pre-leukemia syndrome. Here we describe the development of CKIα inhibitors, which co-target the transcriptional kinases CDK7 and CDK9, thereby augmenting CKIα-induced p53 activation and its anti-leukemic activity. Oncogene-driving super-enhancers (SEs) are highly sensitive to CDK7/9 inhibition. We identified multiple newly gained SEs in primary mouse acute myeloid leukemia (AML) cells and demonstrate that the inhibitors abolish many SEs and preferentially suppress the transcription elongation of SE-driven oncogenes. We show that blocking CKIα together with CDK7 and/or CDK9 synergistically stabilize p53, deprive leukemia cells of survival and proliferation-maintaining SE-driven oncogenes, and induce apoptosis. Leukemia progenitors are selectively eliminated by the inhibitors, explaining their therapeutic efficacy with preserved hematopoiesis and leukemia cure potential; they eradicate leukemia in MLL-AF9 and Tet2-/-;Flt3ITD AML mouse models and in several patient-derived AML xenograft models, supporting their potential efficacy in curing human leukemia.
Assuntos
Caseína Quinase Ialfa/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Caseína Quinase Ialfa/fisiologia , Proliferação de Células/efeitos dos fármacos , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/fisiologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/fisiologia , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Elementos Facilitadores Genéticos/genética , Hematopoese , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Proteína Supressora de Tumor p53/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The positive transcription elongation factor b (P-TEFb) is composed of cyclins T1 or T2 and cyclin-dependent kinase 9 that regulate the elongation phase of transcription by RNA polymerase II. By antagonizing negative elongation factors and phosphorylating the C-terminal domain of RNA polymerase II, P-TEFb facilitates the elongation and co-transcriptional processing of nascent transcripts. This step is critical for the expression of most eukaryotic genes. In growing cells, P-TEFb is regulated negatively by its reversible associations with HEXIM1/2 in the 7SK snRNP and positively by a number of transcription factors, as well as the super elongation complex. In resting cells, P-TEFb falls apart, and cyclin T1 is degraded by the proteasome. This complex regulation of P-TEFb has evolved for the precise temporal and spatial regulation of gene expression in the organism. Its dysregulation contributes to inflammatory and neoplastic conditions.
Assuntos
Fator B de Elongação Transcricional Positiva , RNA Polimerase II , Humanos , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Células HeLa , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Ciclina T/genética , Ciclina T/metabolismo , Transcrição Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The elongation stage of transcription by RNA polymerase II (RNA Pol II) is central to the regulation of gene expression in response to developmental and environmental cues in metazoan. Dysregulated transcriptional elongation has been associated with developmental defects as well as disease and aging processes. Decades of genetic and biochemical studies have painstakingly identified and characterized an ensemble of factors that regulate RNA Pol II elongation. This review summarizes recent findings taking advantage of genetic engineering techniques that probe functions of elongation factors in vivo. We propose a revised model of elongation control in this accelerating field by reconciling contradictory results from the earlier biochemical evidence and the recent in vivo studies. We discuss how elongation factors regulate promoter-proximal RNA Pol II pause release, transcriptional elongation rate and processivity, RNA Pol II stability and RNA processing, and how perturbation of these processes is associated with developmental disorders, neurodegenerative disease, cancer, and aging.
Assuntos
Doenças Neurodegenerativas , RNA Polimerase II , Animais , Humanos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fatores de Elongação da Transcrição/genética , Doenças Neurodegenerativas/genética , Transcrição Gênica , Regulação da Expressão Gênica , Envelhecimento/genética , Genes Controladores do DesenvolvimentoRESUMO
The heat shock (HS) response involves rapid induction of HS genes, whereas transcriptional repression is established more slowly at most other genes. Previous data suggested that such repression results from inhibition of RNA polymerase II (RNAPII) pause release, but here, we show that HS strongly affects other phases of the transcription cycle. Intriguingly, while elongation rates increase upon HS, processivity markedly decreases, so that RNAPII frequently fails to reach the end of genes. Indeed, HS results in widespread premature transcript termination at cryptic, intronic polyadenylation (IPA) sites near gene 5'-ends, likely via inhibition of U1 telescripting. This results in dramatic reconfiguration of the human transcriptome with production of new, previously unannotated, short mRNAs that accumulate in the nucleus. Together, these results shed new light on the basic transcription mechanisms induced by growth at elevated temperature and show that a genome-wide shift toward usage of IPA sites can occur under physiological conditions.
Assuntos
Poliadenilação , Transcriptoma , Resposta ao Choque Térmico/genética , Humanos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genéticaRESUMO
Based on in vitro studies, it has been demonstrated that the DSIF complex, composed of SPT4 and SPT5, regulates the elongation stage of transcription catalyzed by RNA polymerase II (RNA Pol II). The precise cellular function of SPT5 is not clear, because conventional gene depletion strategies for SPT5 result in loss of cellular viability. Using an acute inducible protein depletion strategy to circumvent this issue, we report that SPT5 loss triggers the ubiquitination and proteasomal degradation of the core RNA Pol II subunit RPB1, a process that we show to be evolutionarily conserved from yeast to human cells. RPB1 degradation requires the E3 ligase Cullin 3, the unfoldase VCP/p97, and a novel form of CDK9 kinase complex. Our study demonstrates that SPT5 stabilizes RNA Pol II specifically at promoter-proximal regions, permitting RNA Pol II release from promoters into gene bodies and providing mechanistic insight into the cellular function of SPT5 in safeguarding accurate gene expression.
Assuntos
Proteínas Culina/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Animais , Sobrevivência Celular , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Culina/química , Fibroblastos/metabolismo , Humanos , Ácidos Indolacéticos/química , Camundongos , Ubiquitina-Proteína Ligases Nedd4/química , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/química , Proteoma , Proteômica/métodos , Ubiquitina-Proteína Ligases/química , Proteína com Valosina/química , Proteína com Valosina/metabolismoRESUMO
In response to stress, human cells coordinately downregulate transcription and translation of housekeeping genes. To downregulate transcription, the negative elongation factor (NELF) is recruited to gene promoters impairing RNA polymerase II elongation. Here we report that NELF rapidly forms nuclear condensates upon stress in human cells. Condensate formation requires NELF dephosphorylation and SUMOylation induced by stress. The intrinsically disordered region (IDR) in NELFA is necessary for nuclear NELF condensation and can be functionally replaced by the IDR of FUS or EWSR1 protein. We find that biomolecular condensation facilitates enhanced recruitment of NELF to promoters upon stress to drive transcriptional downregulation. Importantly, NELF condensation is required for cellular viability under stressful conditions. We propose that stress-induced NELF condensates reported here are nuclear counterparts of cytosolic stress granules. These two stress-inducible condensates may drive the coordinated downregulation of transcription and translation, likely forming a critical node of the stress survival strategy.
Assuntos
Resposta ao Choque Térmico/genética , Proteínas Intrinsicamente Desordenadas/genética , Processamento de Proteína Pós-Traducional , RNA Polimerase II/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Cromatina/química , Cromatina/metabolismo , Células Clonais , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Genes Reporter , Células HEK293 , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fosforilação , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Transdução de Sinais , Estresse Fisiológico , Sumoilação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Proteína Vermelha FluorescenteRESUMO
Precise control of the RNA polymerase II (RNA Pol II) cycle, including pausing and pause release, maintains transcriptional homeostasis and organismal functions. Despite previous work to understand individual transcription steps, we reveal a mechanism that integrates RNA Pol II cycle transitions. Surprisingly, KAP1/TRIM28 uses a previously uncharacterized chromatin reader cassette to bind hypo-acetylated histone 4 tails at promoters, guaranteeing continuous progression of RNA Pol II entry to and exit from the pause state. Upon chromatin docking, KAP1 first associates with RNA Pol II and then recruits a pathway-specific transcription factor (SMAD2) in response to cognate ligands, enabling gene-selective CDK9-dependent pause release. This coupling mechanism is exploited by tumor cells to aberrantly sustain transcriptional programs commonly dysregulated in cancer patients. The discovery of a factor integrating transcription steps expands the functional repertoire by which chromatin readers operate and provides mechanistic understanding of transcription regulation, offering alternative therapeutic opportunities to target transcriptional dysregulation.
Assuntos
RNA Polimerase II/metabolismo , Proteína 28 com Motivo Tripartido/metabolismo , Acetilação , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Quinase 9 Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica/genética , Histonas/metabolismo , Humanos , Oncogenes/genética , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional/genética , RNA Polimerase II/genética , Proteína Smad2/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteína 28 com Motivo Tripartido/genéticaRESUMO
CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Modelos Biológicos , Fator de Transcrição TFIIH/metabolismo , Transcrição Gênica/genética , Processamento Alternativo/genética , Sobrevivência Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Ativação Enzimática/genética , Células HL-60 , Humanos , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain ill defined. Here, we show that following genotoxic stress, the RNA-binding motif protein 7 (RBM7) stimulates RNA polymerase II (Pol II) transcription and promotes cell viability by activating the positive transcription elongation factor b (P-TEFb) via its release from the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP). This is mediated by activation of p38MAPK, which triggers enhanced binding of RBM7 with core subunits of 7SK snRNP. In turn, P-TEFb relocates to chromatin to induce transcription of short units, including key DDR genes and multiple classes of non-coding RNAs. Critically, interfering with the axis of RBM7 and P-TEFb provokes cellular hypersensitivity to DNA-damage-inducing agents due to activation of apoptosis. Our work uncovers the importance of stress-dependent stimulation of Pol II pause release, which enables a pro-survival transcriptional response that is crucial for cell fate upon genotoxic insult.
Assuntos
Fator B de Elongação Transcricional Positiva/genética , RNA Polimerase II/genética , Proteínas de Ligação a RNA/genética , Transcrição Gênica , Apoptose/genética , Sobrevivência Celular/genética , Dano ao DNA/genética , Células HEK293 , Humanos , RNA Longo não Codificante/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Cyclin-dependent kinase 9 (CDK9) plays a critical role in transcription initiation and is essential for maintaining gene silencing at heterochromatic loci. Inhibition of CDK9 increases sensitivity to immunotherapy, but the underlying mechanism remains unclear. We now report that RNF20 stabilizes LSD1 via K29-mediated ubiquitination, which is dependent on CDK9-mediated phosphorylation. This CDK9- and RNF20-dependent LSD1 stabilization is necessary for the demethylation of histone H3K4, then subsequent repression of endogenous retrovirus, and an interferon response, leading to epigenetic immunosuppression. Moreover, we found that loss of RNF20 sensitizes cancer cells to the immune checkpoint inhibitor anti-PD-1 in vivo and that this effect can be rescued by the expression of ectopic LSD1. Our findings are supported by the observation that RNF20 levels correlate with LSD1 levels in human breast cancer specimens. This study sheds light on the role of RNF20 in CDK9-dependent LSD1 stabilization, which is crucial for epigenetic silencing and immunosuppression. Our findings explore the potential importance of targeting the CDK9-RNF20-LSD1 axis in the development of new cancer therapies.
Assuntos
Quinase 9 Dependente de Ciclina , Histona Desmetilases , Tolerância Imunológica , Ubiquitina-Proteína Ligases , Humanos , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Epigênese Genética , Histona Desmetilases/metabolismo , Histonas/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Nuclear actin has been demonstrated to be essential for optimal transcription, but the molecular mechanisms and direct binding partner for actin in the RNA polymerase complex have remained unknown. By using purified proteins in a variety of biochemical assays, we demonstrate a direct and specific interaction between monomeric actin and Cdk9, the kinase subunit of the positive transcription elongation factor b required for RNA polymerase II pause-release. This interaction efficiently prevents actin polymerization, is not dependent on kinase activity of Cdk9, and is not involved with releasing positive transcription elongation factor b from its inhibitor 7SK snRNP complex. Supporting the specific role for actin in the elongation phase of transcription, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) reveals that actin interacts with genes only upon their active transcription elongation. This study therefore provides novel insights into the mechanisms by which actin facilitates the transcription process.
Assuntos
Actinas , Quinase 9 Dependente de Ciclina , Fator B de Elongação Transcricional Positiva , Humanos , Actinas/genética , Actinas/metabolismo , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , RNA Polimerase II/metabolismo , Transcrição GênicaRESUMO
Anticancer drug development campaigns often fail due to an incomplete understanding of the therapeutic index differentiating the efficacy of the agent against the cancer and its on-target toxicities to the host. To address this issue, we established a versatile preclinical platform in which genetically defined cancers are produced using somatic tissue engineering in transgenic mice harboring a doxycycline-inducible short hairpin RNA against the target of interest. In this system, target inhibition is achieved by the addition of doxycycline, enabling simultaneous assessment of efficacy and toxicity in the same animal. As proof of concept, we focused on CDK9a cancer target whose clinical development has been hampered by compounds with poorly understood target specificity and unacceptable toxicities. We systematically compared phenotypes produced by genetic Cdk9 inhibition to those achieved using a recently developed highly specific small molecule CDK9 inhibitor and found that both perturbations led to robust antitumor responses. Remarkably, nontoxic levels of CDK9 inhibition could achieve significant treatment efficacy, and dose-dependent toxicities produced by prolonged CDK9 suppression were largely reversible upon Cdk9 restoration or drug withdrawal. Overall, these results establish a versatile in vivo target validation platform that can be employed for rapid triaging of therapeutic targets and lend support to efforts aimed at advancing CDK9 inhibitors for cancer therapy.
Assuntos
Antineoplásicos , Neoplasias , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Quinase 9 Dependente de Ciclina/metabolismo , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Interferência de RNARESUMO
Cartilage defects in the knee are often associated with the progression of degenerative osteoarthritis (OA), and cartilage repair is a useful strategy for managing this disease. However, cartilage repair is challenging because of the unique environment within the tissue. Recently, stem cell-based therapies have shed new light on this issue. In this study, we prepared exosomes (EXOs) from cartilage stem/progenitor cells (CSPCs) and found that treatment with EXOs increased the viability, migration, and proliferation of cultured primary chondrocytes. In a subacute OA rat model, the application of EXOs facilitated cartilage regeneration as evidenced by histological staining. Exosomal protein analysis together with bioinformatics suggested that cyclin-dependent kinase 9 (CDK9) is a key factor for chondrocyte growth and migration. Functional studies confirmed this prediction, that is, inhibiting CDK9 reduced the beneficial effects induced by EXOs in primary chondrocytes; while overexpression of CDK9 recapitulated the EXOs-induced phenotypes. RNA-Seq data showed that a set of genes involved in cell growth and migration were up-regulated by EXOs in chondrocytes. These changes could be partially reproduced by CDK9 overexpression. Overall, our data suggest that EXOs derived from primary CSPCs hold great therapeutic potential for treating cartilage defect-associated disorders such as degenerative OA, and that CDK9 is a key factor in this process.
Assuntos
Cartilagem Articular , Proliferação de Células , Condrócitos , Modelos Animais de Doenças , Exossomos , Animais , Exossomos/metabolismo , Ratos , Condrócitos/metabolismo , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Células-Tronco/metabolismo , Células-Tronco/citologia , Movimento Celular , Ratos Sprague-Dawley , Quinase 9 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/genética , Osteoartrite do Joelho/metabolismo , Osteoartrite do Joelho/patologia , Osteoartrite do Joelho/terapia , Masculino , Células Cultivadas , Regeneração , Osteoartrite/patologia , Osteoartrite/metabolismo , Osteoartrite/terapiaRESUMO
The persistent threat of cancer remains a significant hurdle for global health, prompting the exploration of innovative approaches in the quest for successful therapeutic interventions. Cyclin-dependent kinase 9 (CDK9), a central player in transcription regulation and cell cycle progression, has emerged as a promising target to combat cancer. Its pivotal role in oncogenic pathways and the pressing need for novel cancer treatments has propelled CDK9 into the spotlight of drug discovery efforts. This article presents a comprehensive study that connects a multidisciplinary approach, combining computational methodologies, experimental validation, and the transformative Proteolysis-Targeting Chimera (PROTAC) technology. By uniting these diverse techniques, we aim to identify, characterize, and optimize a new class of degraders targeting CDK9. We explore these compounds for targeted protein degradation, offering a novel and potentially effective approach to cancer therapy. This cohesive strategy utilizes the combination of computational predictions and experimental insights, with the goal of advancing the development of effective anticancer therapeutics, targeting CDK9.
RESUMO
Current antiretroviral therapy for HIV-1 infection does not represent a cure for infection as viral rebound inevitably occurs following discontinuation of treatment. The "block and lock" therapeutic strategy is intended to enforce proviral latency and durably suppress viremic reemergence in the absence of other intervention. The transcription-associated cyclin-dependent protein kinases (tCDKs) are required for expression from the 5´ HIV-1 long-terminal repeat, but the therapeutic potential of inhibiting these kinases for enforcing HIV-1 latency has not been characterized. Here, we expanded previous observations to directly compare the effect of highly selective small molecule inhibitors of CDK7 (YKL-5-124), CDK9 (LDC000067), and CDK8/19 (Senexin A), and found each of these prevented HIV-1 provirus expression at concentrations that did not cause cell toxicity. Inhibition of CDK7 caused cell cycle arrest, whereas CDK9 and CDK8/19 inhibitors did not, and could be continuously administered to establish proviral latency. Upon discontinuation of drug administration, HIV immediately rebounded in cells that had been treated with the CDK9 inhibitor, while proviral latency persisted for several days in cells that had been treated with CDK8/19 inhibitors. These results identify the mediator kinases CDK8/CDK19 as potential "block and lock" targets for therapeutic suppression of HIV-1 provirus expression.
Assuntos
HIV-1 , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Quinases Ciclina-Dependentes/farmacologia , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Ciclinas/metabolismo , Ciclinas/farmacologiaRESUMO
CDK9 is a kinase critical for the productive transcription of protein-coding genes by RNA polymerase II (pol II). As part of P-TEFb, CDK9 phosphorylates the carboxyl-terminal domain (CTD) of pol II and elongation factors, which allows pol II to elongate past the early elongation checkpoint (EEC) encountered soon after initiation. We show that, in addition to halting pol II at the EEC, loss of CDK9 activity causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, indicating that this kinase/phosphatase pair regulates transcription elongation and RNA processing at the end of protein-coding genes. We also confirm the splicing factor SF3B1 as a target of CDK9 and show that SF3B1 in complex with polyadenylation factors is lost from chromatin after CDK9 inhibition. These results emphasize the important roles that CDK9 plays in coupling transcription elongation and termination to RNA maturation downstream of the EEC.
Assuntos
Fator B de Elongação Transcricional Positiva , RNA Polimerase II , Cromatina/genética , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , RNA , RNA Polimerase II/metabolismo , Fatores de Processamento de RNA/genética , Transcrição Gênica , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Emerging clinical evidence indicates that selective CDK9 inhibition may provide clinical benefits in the management of certain cancers. Many CDK9 selective inhibitors have entered clinical developments, and are being investigated. No clear winner has emerged because of unforeseen toxicity often observed in clinic with these agents. Therefore, a novel agent with differentiated profiles is still desirable. Herein, we report our design, syntheses of a novel azaindole series of selective CDK9 inhibitors. SAR studies led to a preclinical candidate YK-2168. YK2168 exhibited improved CDK9 selectivity over AZD4573 and BAY1251152; also showed differentiated intravenous PK profile and remarkable solid tumor efficacy in a mouse gastric cancer SNU16 CDX model in preclinical studies. YK-2168 is currently in clinical development in China (CTR20212900).
RESUMO
Cyclin-dependent kinase 9 (CDK9) plays a role in transcriptional regulation, which had become an attractive target for discovery of antitumor agent. In this work, beyond traditional CDK9 inhibitor with bidentate ligands in ATP binding domain, a series of novel CDK9 inhibitor with tridentate ligand were designed and synthesized. Surprisingly, this unique tridentate ligand structure endows better CDK9 inhibition selectivity compared to other CDK subtypes, and the lead candidate compound Z4-7a showed effective proliferation inhibition in HCT116 cells with acceptable pharmacokinetic properties. Research on the mechanism indicated that Z4-7a could induce apoptosis in the HCT116 cell line by inhibiting phosphorylation of RNA polymerase II at Ser2, which resulted in the inhibition of apoptosis-related genes and proteins expression. In brief, introduction of tridentate ligand might work as a promising strategy for the development of novel selective CDK9 inhibitor.