Asunto(s)
Sinergismo Farmacológico , Leucemia Mieloide Aguda , Mutación , Inhibidores de Proteínas Quinasas , Tirosina Quinasa 3 Similar a fms , Humanos , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéuticoRESUMEN
Human T-cell leukemia virus type-1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL). The trans-activator protein Tax of HTLV-1 plays crucial roles in leukemogenesis by promoting proliferation of virus-infected cells through activation of growth-promoting genes. However, critical target genes are yet to be elucidated. We show here that Tax activates the gene coding for cyclin-dependent kinase 7 (CDK7), the essential component of both CDK-activating kinase (CAK) and general transcription factor TFIIH. CAK and TFIIH play essential roles in cell cycle progression and transcription by activating CDKs and facilitating transcriptional initiation, respectively. Tax induced CDK7 gene expression not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs) along with increased protein expression. Tax stimulated phosphorylation of CDK2 and RNA polymerase II at sites reported to be mediated by CDK7. Tax activated the CDK7 promoter through the NF-κB pathway, which mainly mediates cell growth promotion by Tax. Knockdown of CDK7 expression reduced Tax-mediated induction of target gene expression and cell cycle progression. These results suggest that the CDK7 gene is a crucial target of Tax-mediated trans-activation to promote cell proliferation by activating CDKs and transcription.
Asunto(s)
Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes , Productos del Gen tax , Virus Linfotrópico T Tipo 1 Humano , Humanos , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/patogenicidad , Productos del Gen tax/genética , Productos del Gen tax/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Factores de Transcripción TFII/genética , Factores de Transcripción TFII/metabolismo , Activación Transcripcional , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , FosforilaciónRESUMEN
Cyclin-dependent kinase 7 (Cdk7) is required in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of transcription factor TFIIH. Cdk7 forms active complexes by associating with Cyclin H and Mat1, and is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates basic residues conserved in other CDKs, pS164 nucleates an arginine network unique to the ternary Cdk7 complex, involving all three subunits. We identify differential dependencies of kinase activity and substrate recognition on the individual phosphorylations. CAK function is unaffected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by T170 phosphorylation. Moreover, dual T-loop phosphorylation stimulates multisite phosphorylation of the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7 activation is a two-step process wherein S164 phosphorylation precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing processivity, while pT170 enhances activity towards key transcriptional substrates.
Asunto(s)
Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes , Fosforilación , Humanos , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/genética , Ciclina H/metabolismo , Ciclina H/química , Ciclina H/genética , Cristalografía por Rayos X , ARN Polimerasa II/metabolismo , ARN Polimerasa II/química , Factor de Transcripción TFIIH/metabolismo , Factor de Transcripción TFIIH/química , Factor de Transcripción TFIIH/genética , Modelos Moleculares , Factores de Transcripción/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Dominios Proteicos , Proteínas de Ciclo CelularRESUMEN
BACKGROUND: Targeting kinases presents a potential strategy for treating solid tumors; however, the therapeutic potential of vaccines targeting kinases remains uncertain. METHODS: Adenovirus (Ad) vaccines encoding Aurora kinase A (AURKA) or cyclin-dependent kinase 7 (CDK7) were developed, and their therapeutic potentials were investigated by various methods including western blot, flow cytometry, cytotoxic T lymphocyte assay, and enzyme-linked immunospot (ELISpot), in mouse and humanized solid tumor models. RESULTS: Co-immunization with Ad-AURKA/CDK7 effectively prevented subcutaneous tumor growth in the Renca, RM-1, MC38, and Hepa1-6 tumor models. In therapeutic tumor models, Ad-AURKA/CDK7 treatment impeded tumor growth and increased immune cell infiltration. Administration of Ad-AURKA/CDK7 promoted the induction and maturation of dendritic cell subsets and augmented multifunctional CD8+ T-cell antitumor immunity. Furthermore, the vaccine induced a long-lasting antitumor effect by promoting the generation of memory CD8+ T cells. Tumor recovery on CD8+ T-cell depletion underscored the indispensable role of these cells in the observed therapeutic effects. The potent efficacy of the Ad-AURKA/CDK7 vaccine was consistently demonstrated in lung metastasis, orthotopic, and humanized tumor models by inducing multifunctional CD8+ T-cell antitumor immune responses. CONCLUSIONS: Our findings illustrate that the Ad-AURKA/CDK7 vaccine targeting dual kinases AURKA and CDK7 emerges as a promising and effective therapeutic approach for the treatment of solid tumors.
Asunto(s)
Aurora Quinasa A , Vacunas contra el Cáncer , Animales , Ratones , Humanos , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Adenoviridae , Línea Celular Tumoral , Quinasa Activadora de Quinasas Ciclina-Dependientes , Femenino , Neoplasias/inmunología , Neoplasias/terapia , Vacunas contra el Adenovirus/inmunología , Vacunas contra el Adenovirus/uso terapéutico , Linfocitos T CD8-positivos/inmunologíaRESUMEN
Cyclin-dependent kinase 7, along with cyclin H and MAT1, forms the CDK-activating complex (CAK), which directs cell cycle progression via T-loop phosphorylation of cell cycle CDKs. Pharmacological inhibition of CDK7 leads to selective anti-cancer effects in cellular and in vivo models, motivating several ongoing clinical investigations of this target. Current CDK7 inhibitors are either reversible or covalent inhibitors of its catalytic activity. We hypothesized that small molecule targeted protein degradation (TPD) might result in differentiated pharmacology due to the loss of scaffolding functions. Here, we report the design and characterization of a potent CDK7 degrader that is comprised of an ATP-competitive CDK7 binder linked to a CRL2VHL recruiter. JWZ-5-13 effectively degrades CDK7 in multiple cancer cells and leads to a potent inhibition of cell proliferation. Additionally, compound JWZ-5-13 displayed bioavailability in a pharmacokinetic study conducted in mice. Therefore, JWZ-5-13 is a useful chemical probe to investigate the pharmacological consequences of CDK7 degradation.
Asunto(s)
Proliferación Celular , Quinasas Ciclina-Dependientes , Inhibidores de Proteínas Quinasas , Humanos , Animales , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Proliferación Celular/efectos de los fármacos , Ratones , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Relación Estructura-Actividad , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/síntesis química , Descubrimiento de Drogas , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Relación Dosis-Respuesta a Droga , Quinasa Activadora de Quinasas Ciclina-Dependientes , Proteolisis/efectos de los fármacos , Línea Celular Tumoral , Ensayos de Selección de Medicamentos AntitumoralesRESUMEN
AIMS: Colorectal cancer (CRC) remains a major global health issue, with metastatic cases presenting poor prognosis despite advances in chemotherapy and targeted therapy. Irinotecan, a key drug for advanced CRC treatment, faces challenges owing to the development of resistance. This study aimed to understand the mechanisms underlying irinotecan resistance in colorectal cancer. MAIN METHODS: We created a cell line resistant to irinotecan using HT29 cells. These resistant cells were utilized to investigate the role of the CDK7-MDK axis. We employed bulk RNA sequencing, conducted in vivo experiments with mice, and analyzed patient tissues to examine the effects of the CDK7-MDK axis on the cellular response to irinotecan. KEY FINDINGS: Our findings revealed that HT29 cells resistant to irinotecan, a crucial colorectal cancer medication, exhibited significant phenotypic and molecular alterations compared to their parental counterparts, including elevated stem cell characteristics and increased levels of cytokines and drug resistance proteins. Notably, CDK7 expression was substantially higher in these resistant cells, and targeting CDK7 effectively decreased their survival and tumor growth, enhancing irinotecan sensitivity. RNA-seq analysis indicated that suppression of CDK7 in irinotecan-resistant HT29 cells significantly reduced Midkine (MDK) expression. Decreased CDK7 and MDK levels, achieved through siRNA and the CDK7 inhibitor THZ1, enhanced the sensitivity of resistant HT29 cells to irinotecan. SIGNIFICANCE: Our study sheds light on how CDK7 and MDK influence irinotecan resistance in colorectal and highlights the potential of MDK-targeted therapies. We hypothesized that irinotecan sensitivity and overall treatment efficacy would improve by inhibiting MDK. This finding encourages a careful yet proactive investigation of MDK as a therapeutic target to enhance outcomes in colorectal cancer patients.
Asunto(s)
Neoplasias Colorrectales , Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes , Resistencia a Antineoplásicos , Irinotecán , Irinotecán/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Humanos , Animales , Resistencia a Antineoplásicos/efectos de los fármacos , Ratones , Células HT29 , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Ratones Desnudos , Ensayos Antitumor por Modelo de Xenoinjerto , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ratones Endogámicos BALB C , Femenino , Proliferación Celular/efectos de los fármacosRESUMEN
Cyclin dependent kinase 7 (CDK7) is an important therapeutic kinase best known for its dual role in cell cycle regulation and gene transcription. Here, we describe the application of protein engineering to generate constructs leading to high resolution crystal structures of human CDK7 in both active and inactive conformations. The active state of the kinase was crystallized by incorporation of an additional surface residue mutation (W132R) onto the double phosphomimetic mutant background (S164D and T170E) that yielded the inactive kinase structure. A novel back-soaking approach was developed to determine crystal structures of several clinical and pre-clinical inhibitors of this kinase, demonstrating the potential utility of the crystal system for structure-based drug design (SBDD). The crystal structures help to rationalize the mode of inhibition and the ligand selectivity profiles versus key anti-targets. The protein engineering approach described here illustrates a generally applicable strategy for structural enablement of challenging molecular targets.
Asunto(s)
Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes , Diseño de Fármacos , Modelos Moleculares , Ingeniería de Proteínas , Inhibidores de Proteínas Quinasas , Humanos , Ingeniería de Proteínas/métodos , Cristalografía por Rayos X , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/genética , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Unión Proteica , Sitios de UniónRESUMEN
Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.
Asunto(s)
Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes , Regiones Promotoras Genéticas , ARN Polimerasa II , Iniciación de la Transcripción Genética , Humanos , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Complejo Mediador/metabolismo , Complejo Mediador/genética , Células HeLa , Factor de Transcripción TFIIH/metabolismo , Factor de Transcripción TFIIH/genética , Células HEK293RESUMEN
The targeting of cyclin-dependent kinase 7 (CDK7) has become a highly desirable therapeutic approach in the field of oncology due to its dual role in regulating essential biological processes, encompassing cell cycle progression and transcriptional control. We have previously identified a highly selective thieno[3,2-d]pyrimidine-based CDK7 inhibitor with demonstrated efficacy and safety in animal model. In this study, we sought to optimize the thieno[3,2-d]pyrimidine core to discover a novel series of CDK7 inhibitors with improved potency and pharmacokinetic (PK) properties. Through extensive structure-activity relationship (SAR) studies, compound 20 has emerged as the lead candidate due to its potent inhibitory activity against CDK7 and remarkable efficacy on MDA-MB-453 cells, a representative triple negative breast cancer (TNBC) cell line. Furthermore, 20 has demonstrated favorable oral bioavailability and exhibited highly desirable pharmacokinetic (PK) properties, making it a promising lead candidate for further structural optimization.
Asunto(s)
Antineoplásicos , Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes , Diseño de Fármacos , Inhibidores de Proteínas Quinasas , Pirimidinas , Pirimidinas/química , Pirimidinas/síntesis química , Pirimidinas/farmacología , Pirimidinas/farmacocinética , Humanos , Relación Estructura-Actividad , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Estructura Molecular , Animales , Antineoplásicos/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Línea Celular Tumoral , RatasRESUMEN
Reactivating p53 activity to restore its anticancer function is an attractive cancer treatment strategy. In this study, we designed and synthesized a series of novel PROTACs to reactivate p53 via the co-degradation of CK1α and CDK7/9 proteins. Bioactivity studies showed that the selected PROTAC 13i exhibited potency antiproliferative activity in MV4-11 (IC50 = 0.096 ± 0.012 µM) and MOLM-13 (IC50 = 0.072 ± 0.014 µM) cells, and induced apoptosis of MV4-11 cells. Western-blot analysis showed that PROTAC 13i triple CK1α and CDK7/9 protein degradation resulted in the significantly increased expression of p53. At the same time, the transcriptional repression due to the degradation significantly reduced downstream gene expression of MYC, MDM2, BCL-2 and MCL-1, and reduced the inflammatory cytokine levels of TNF-α, IL-1ß and IL-6 in PMBCs. These results indicate the beneficial impact of simultaneous CK1α and CDK7/9 degradation for acute myeloid leukemia therapy.
Asunto(s)
Antineoplásicos , Caseína Quinasa Ialfa , Proliferación Celular , Quinasa 9 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes , Ensayos de Selección de Medicamentos Antitumorales , Leucemia Mieloide Aguda , Proteína p53 Supresora de Tumor , Humanos , Proteína p53 Supresora de Tumor/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Caseína Quinasa Ialfa/metabolismo , Caseína Quinasa Ialfa/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 9 Dependiente de la Ciclina/metabolismo , Relación Estructura-Actividad , Estructura Molecular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Relación Dosis-Respuesta a Droga , Apoptosis/efectos de los fármacos , Descubrimiento de Drogas , Línea Celular Tumoral , Proteolisis/efectos de los fármacos , Células Tumorales Cultivadas , Quimera Dirigida a la Proteólisis , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
AIMS: The anthracycline family of anticancer agents such as doxorubicin (DOX) can induce apoptotic death of cardiomyocytes and cause cardiotoxicity. We previously reported that DOX-induced apoptosis is accompanied by cardiomyocyte cell cycle re-entry. Cell cycle progression requires cyclin-dependent kinase 7 (CDK7)-mediated activation of downstream cell cycle CDKs. This study aims to determine whether CDK7 can be targeted for cardioprotection during anthracycline chemotherapy. METHODS AND RESULTS: DOX exposure induced CDK7 activation in mouse heart and isolated cardiomyocytes. Cardiac-specific ablation of Cdk7 attenuated DOX-induced cardiac dysfunction and fibrosis. Treatment with the covalent CDK7 inhibitor THZ1 also protected against DOX-induced cardiomyopathy and apoptosis. DOX treatment induced activation of the proapoptotic CDK2-FOXO1-Bim axis in a CDK7-dependent manner. In response to DOX, endogenous CDK7 directly bound and phosphorylated CDK2 at Thr160 in cardiomyocytes, leading to full CDK2 kinase activation. Importantly, inhibition of CDK7 further suppressed tumour growth when used in combination with DOX in an immunocompetent mouse model of breast cancer. CONCLUSION: Activation of CDK7 is necessary for DOX-induced cardiomyocyte apoptosis and cardiomyopathy. Our findings uncover a novel proapoptotic role for CDK7 in cardiomyocytes. Moreover, this study suggests that inhibition of CDK7 attenuates DOX-induced cardiotoxicity but augments the anticancer efficacy of DOX. Therefore, combined administration of CDK7 inhibitor and DOX may exhibit diminished cardiotoxicity but superior anticancer activity.
Asunto(s)
Apoptosis , Cardiotoxicidad , Quinasa 2 Dependiente de la Ciclina , Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes , Doxorrubicina , Ratones Endogámicos C57BL , Miocitos Cardíacos , Inhibidores de Proteínas Quinasas , Animales , Doxorrubicina/toxicidad , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Miocitos Cardíacos/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Femenino , Fenilendiaminas/farmacología , Transducción de Señal/efectos de los fármacos , Fosforilación , Ratones Noqueados , Cardiomiopatías/inducido químicamente , Cardiomiopatías/enzimología , Cardiomiopatías/prevención & control , Cardiomiopatías/patología , Cardiomiopatías/metabolismo , Antibióticos Antineoplásicos/toxicidad , Pirimidinas/farmacología , Humanos , Fibrosis , Línea Celular Tumoral , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/metabolismo , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
The duality of function (cell cycle regulation and gene transcription) of cyclin-dependent kinase 7 (CDK7) makes it an attractive oncology target and the discovery of CDK7 inhibitors has been a long-term pursuit by academia and pharmaceutical companies. However, achieving selective leading compounds is still difficult owing to the similarities among the ATP binding pocket. Herein, we detail the design and synthesis of a series of macrocyclic derivatives with pyrazolo[1,5-a]-1,3,5-triazine core structure as potent and selective CDK7 inhibitors. The diverse manners of macrocyclization led to distinguished selectivity profiles of the CDK family. Molecular dynamics (MD) simulation explained the binding difference between 15- and 16-membered macrocyclic compounds. Further optimization generated compound 37 exhibiting good CDK7 inhibitory activity and high selectivity over other CDKs. This work clearly demonstrated macrocyclization is a versatile method to finely tune the selectivity profile of small molecules and MD simulation can be a valuable tool in prioritizing designs of the macrocycle.
Asunto(s)
Quinasas Ciclina-Dependientes , Diseño de Fármacos , Compuestos Macrocíclicos , Simulación de Dinámica Molecular , Inhibidores de Proteínas Quinasas , Compuestos Macrocíclicos/farmacología , Compuestos Macrocíclicos/síntesis química , Compuestos Macrocíclicos/química , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Rational design of next-generation therapeutics can be facilitated by high-resolution structures of drug targets bound to small-molecule inhibitors. However, application of structure-based methods to macromolecules refractory to crystallization has been hampered by the often-limiting resolution and throughput of cryogenic electron microscopy (cryo-EM). Here, we use high-resolution cryo-EM to determine structures of the CDK-activating kinase, a master regulator of cell growth and division, in its free and nucleotide-bound states and in complex with 15 inhibitors at up to 1.8 Å resolution. Our structures provide detailed insight into inhibitor interactions and networks of water molecules in the active site of cyclin-dependent kinase 7 and provide insights into the mechanisms contributing to inhibitor selectivity, thereby providing the basis for rational design of next-generation therapeutics. These results establish a methodological framework for the use of high-resolution cryo-EM in structure-based drug design.
Asunto(s)
Quinasa Activadora de Quinasas Ciclina-Dependientes , Diseño de Fármacos , Humanos , Microscopía por Crioelectrón/métodos , Sustancias Macromoleculares/química , Ciclo CelularRESUMEN
PURPOSE: Resistance to endocrine therapy (ET) and CDK4/6 inhibitors (CDK4/6i) is a clinical challenge in estrogen receptor (ER)-positive (ER+) breast cancer. Cyclin-dependent kinase 7 (CDK7) is a candidate target in endocrine-resistant ER+ breast cancer models and selective CDK7 inhibitors (CDK7i) are in clinical development for the treatment of ER+ breast cancer. Nonetheless, the precise mechanisms responsible for the activity of CDK7i in ER+ breast cancer remain elusive. Herein, we sought to unravel these mechanisms. EXPERIMENTAL DESIGN: We conducted multi-omic analyses in ER+ breast cancer models in vitro and in vivo, including models with different genetic backgrounds. We also performed genome-wide CRISPR/Cas9 knockout screens to identify potential therapeutic vulnerabilities in CDK4/6i-resistant models. RESULTS: We found that the on-target antitumor effects of CDK7 inhibition in ER+ breast cancer are in part p53 dependent, and involve cell cycle inhibition and suppression of c-Myc. Moreover, CDK7 inhibition exhibited cytotoxic effects, distinctive from the cytostatic nature of ET and CDK4/6i. CDK7 inhibition resulted in suppression of ER phosphorylation at S118; however, long-term CDK7 inhibition resulted in increased ER signaling, supporting the combination of ET with a CDK7i. Finally, genome-wide CRISPR/Cas9 knockout screens identified CDK7 and MYC signaling as putative vulnerabilities in CDK4/6i resistance, and CDK7 inhibition effectively inhibited CDK4/6i-resistant models. CONCLUSIONS: Taken together, these findings support the clinical investigation of selective CDK7 inhibition combined with ET to overcome treatment resistance in ER+ breast cancer. In addition, our study highlights the potential of increased c-Myc activity and intact p53 as predictors of sensitivity to CDK7i-based treatments.
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Apoptosis , Neoplasias de la Mama , Ciclo Celular , Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes , Resistencia a Antineoplásicos , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas c-myc , Receptores de Estrógenos , Transducción de Señal , Humanos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Femenino , Resistencia a Antineoplásicos/genética , Apoptosis/efectos de los fármacos , Animales , Ratones , Receptores de Estrógenos/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Quinasas Ciclina-Dependientes/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Transducción de Señal/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/genética , Sistemas CRISPR-CasRESUMEN
Dysregulation of histone methyltransferase SET and MYND domain-containing protein 2 (SMYD2) has been correlated with human developmental disorders and cancers. This research aims to investigate the roles of SMYD2 and its interacted molecules in pancreatic adenocarcinoma (PAAD). Two PAAD-related gene expression datasets were downloaded to screen key molecules involved in tumor progression. SMYD2 was expressed at high levels in PAAD tissues and cells. SMYD2 silencing suppressed while its overexpression promoted proliferation, invasiveness, migration, apoptosis resistance, and cell cycle progression of PAAD cells. Target molecules of SMYD2 were predicted by online tools and validated by chromatin immunoprecipitation and luciferase assays. SMYD2 catalyzed H3K36me2 modification at the promoter region of MNAT1 component of CDK activating kinase (MNAT1), to promote its transcription. MNAT1 was correlated with an unfavorable clinical outcome of PAAD patients. Alteration of MNAT1 alone also affected the malignant behavior of PAAD cells. Moreover, MNAT1 overexpression in cells rescued the malignant phenotype of cells suppressed by SMYD2 silencing. MNAT1 activated the phosphatidyl inositol 3-kinase/protein kinase B (PI3K/AKT) signaling. In vivo, SMYD2 silencing decreased the growth rate and weight of xenograft tumors in nude mice. Overall, this paper demonstrates that SMYD2-mediated MNAT1 upregulation is linked to PAAD tumorigenesis via PI3K/AKT pathway activation.
Asunto(s)
Adenocarcinoma , Quinasa Activadora de Quinasas Ciclina-Dependientes , Neoplasias Pancreáticas , Factores de Transcripción , Ratones , Animales , Humanos , Regulación hacia Arriba , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Adenocarcinoma/genética , Ratones Desnudos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Neoplasias Pancreáticas/genética , Proteínas de Ciclo Celular/genética , Carcinogénesis/genética , Transformación Celular Neoplásica/genética , Epigénesis Genética , Proliferación Celular/genética , Línea Celular TumoralRESUMEN
Tumor growth is driven by continued cellular growth and proliferation. Cyclin-dependent kinase 7's (CDK7) role in activating mitotic CDKs and global gene expression makes it therefore an attractive target for cancer therapies. However, what makes cancer cells particularly sensitive to CDK7 inhibition (CDK7i) remains unclear. Here, we address this question. We show that CDK7i, by samuraciclib, induces a permanent cell-cycle exit, known as senescence, without promoting DNA damage signaling or cell death. A chemogenetic genome-wide CRISPR knockout screen identified that active mTOR (mammalian target of rapamycin) signaling promotes samuraciclib-induced senescence. mTOR inhibition decreases samuraciclib sensitivity, and increased mTOR-dependent growth signaling correlates with sensitivity in cancer cell lines. Reverting a growth-promoting mutation in PIK3CA to wild type decreases sensitivity to CDK7i. Our work establishes that enhanced growth alone promotes CDK7i sensitivity, providing an explanation for why some cancers are more sensitive to CDK inhibition than normally growing cells.
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Quinasas Ciclina-Dependientes , Neoplasias , Humanos , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Quinasa Activadora de Quinasas Ciclina-Dependientes , Transducción de Señal , Ciclo Celular , Inhibidores Enzimáticos , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Línea Celular TumoralRESUMEN
Retinoblastoma (RB) is a common malignancy that primarily affects pediatric populations. Although a well-known cause of RB is RB1 mutation, MYCN amplification can also lead to the disease, which is a poor prognosis factor. Studies conducted in various tumor types have shown that MYCN inhibition is an effective approach to impede tumor growth. Various indirect approaches have been developed to overcome the difficulty of directly targeting MYCN, such as modulating the super enhancer (SE) upstream of MYCN. The drug used in this study to treat MYCN-amplified RB was THZ1, a CDK7 inhibitor that can effectively suppress transcription by interfering with the activity of SEs. The study findings confirmed the anticancer activity of THZ1 against RB in both in vitro and in vivo experiments. Therapy with THZ1 was found to affect numerous genes in RB according to the RNA-seq analysis. Moreover, the gene expression changes induced by THZ1 treatment were enriched in ribosome, endocytosis, cell cycle, apoptosis, etc. Furthermore, the combined analysis of ChIP-Seq and RNA-seq data suggested a potential role of SEs in regulating the expression of critical transcription factors, such as MYCN, OTX2, and SOX4. Moreover, ChIP-qPCR experiments were conducted to confirm the interaction between MYCN and SEs. In conclusion, THZ1 caused substantial changes in gene transcription in RB, resulting in inhibited cell proliferation, interference with the cell cycle, and increased apoptosis. The efficacy of THZ1 is positively correlated with the degree of MYCN amplification and is likely exerted by interfering with MYCN upstream SEs.
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Neoplasias de la Retina , Retinoblastoma , Niño , Humanos , Proteína Proto-Oncogénica N-Myc/genética , Retinoblastoma/tratamiento farmacológico , Retinoblastoma/genética , Quinasa Activadora de Quinasas Ciclina-Dependientes , Quinasas Ciclina-Dependientes/genética , Factores de Transcripción SOXCRESUMEN
Cyclin dependent kinase 7 (CDK7) is an attractive target in tumor indications via regulating both cell cycle and transcription. Here, SHR5428 was discovered as a selective and noncovalent CDK7 inhibitor with highly potent CDK7 enzymatic activity and triple negative breast cancer cellular activity on MDA-MB-468 cell. SHR5428 also displayed favorable pharmacokinetic properties in different preclinical species such as mouse, rat and dog, and showed high selectivity over CDK1, CDK2, CDK4, CDK6, CDK9, CDK12 in CDK family. Furthermore, the computational modeling has shed some light on this mechanism. Additionally the in vivo efficacy study in a breast cancer cell line (HCC70 cell) derived xenograft mouse model proved SHR5428 to be orally efficacious with dose-dependent tumor growth inhibition.
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Quinasa Activadora de Quinasas Ciclina-Dependientes , Inhibidores de Proteínas Quinasas , Animales , Perros , Humanos , Ratones , Ratas , Ciclo Celular , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Quinasa Activadora de Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Fosforilación , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Cancer is a complex disease that develops when abnormal cells divide uncontrollably as a consequence of unregulated cell cycle protein activity. Therefore, the cell cycle is crucial for maintaining homeostasis inside the cells during DNA replication and cell division. The presence of mutations within specific genes can disrupt the equilibrium within cells, ultimately leading to the growth of cancer. CDK20 (Cyclin-Dependent Kinase 20) is recently identified as a major controller of cell cycle checkpoints, which regulate cell growth and proliferation and perform a role in the development of many malignancies. CCRK (Cell-Cycle Related Kinase) has recently been renamed CDK20. Emerging studies proclaimed that the upregulation of CDK20 was identified in cancers of the ovary, brain, colon, stomach, liver, and lung. CDK20 was thought to have Cyclin-dependent activating kinase (CAK) activity for CDK2 when it is complexed with Cyclin H. Furthermore, recent studies revealed that CDK20 is involved in the Wnt, EZH2/NF-B, and KEAP1-NRF2 signaling pathways, all of which are interconnected to cancer formation and proliferation. In addition, the structure of CDK20 was predicted using ColabFold, a powerful software integrating AlphaFold's advanced AI system. The present review focuses on a systematic overview of the current knowledge on CDK20 derived from in vitro and in vivo studies and emphasizes its role in carcinogenesis. The validation comparison of the existing CDK20 AlphaFold structure with the ColabFold was found to be exceptionally fast and accurate in generating reliable models.
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Quinasas Ciclina-Dependientes , Neoplasias , Femenino , Humanos , Carcinogénesis/genética , Quinasa Activadora de Quinasas Ciclina-Dependientes , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias/genética , Factor 2 Relacionado con NF-E2/metabolismoRESUMEN
Emerging evidence suggests that genetically highly specific triple-negative breast cancer (TNBC) possesses a relatively uniform transcriptional program that is abnormally dependent on cyclin-dependent kinase 7 (CDK7). In this study, we obtained an inhibitor of CDK7, N76-1, by attaching the side chain of the covalent CDK7 inhibitor THZ1 to the core of the anaplastic lymphoma kinase inhibitor ceritinib. This study aimed to elucidate the role and underlying mechanism of N76-1 in TNBC and evaluate its potential value as an anti-TNBC drug. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays showed that N76-1 inhibited the viability of TNBC cells. Kinase activity and cellular thermal shift assays showed that N76-1 directly targeted CDK7. Flow cytometry results revealed that N76-1 induced apoptosis and cell cycle arrest in the G2/M phase. N76-1 also effectively inhibited the migration of TNBC cells by high-content detection. The RNA-seq analysis showed that the transcription of genes, especially those related to transcriptional regulation and cell cycle, was suppressed after N76-1 treatment. Moreover, N76-1 markedly inhibited the growth of TNBC xenografts and phosphorylation of RNAPII in tumor tissues. In summary, N76-1 exerts potent anticancer effects in TNBC by inhibiting CDK7 and provides a new strategy and research basis for the development of new drugs for TNBC.