RESUMEN
Selective degradation of the cyclin-dependent kinases 12 and 13 (CDK12/13) presents a novel therapeutic opportunity for triple-negative breast cancer (TNBC), but there is still a lack of dual CDK12/13 degraders. Here, we report the discovery of the first series of highly potent and selective dual CDK12/13 degraders by employing the proteolysis-targeting chimera (PROTAC) technology. The optimal compound 7f effectively degraded CDK12 and CDK13 with DC50 values of 2.2 and 2.1 nM, respectively, in MDA-MB-231 breast cancer cells. Global proteomic profiling demonstrated the target selectivity of 7f. In vitro, 7f suppressed expression of core DNA damage response (DDR) genes in a time- and dose-dependent manner. Further, 7f markedly inhibited proliferation of multiple TNBC cell lines including MFM223, with an IC50 value of 47 nM. Importantly, 7f displayed a significantly improved antiproliferative activity compared to the structurally similar inhibitor 4, suggesting the potential advantage of a CDK12/13 degrader for TNBC targeted therapy.
Asunto(s)
Proteína Quinasa CDC2 , Quinasas Ciclina-Dependientes , Neoplasias de la Mama Triple Negativas , Humanos , Proteína Quinasa CDC2/antagonistas & inhibidores , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Proteolisis , Proteómica , Neoplasias de la Mama Triple Negativas/tratamiento farmacológicoRESUMEN
The heterogeneous response of acute myeloid leukemia (AML) to current anti-leukemic therapies is only partially explained by mutational heterogeneity. We previously identified GPR56 as a surface marker associated with poor outcome across genetic groups, which characterizes two leukemia stem cell (LSC)-enriched compartments with different self-renewal capacities. How these compartments self-renew remained unclear. Here, we show that GPR56+ LSC compartments are promoted in a complex network involving epithelial-to-mesenchymal transition (EMT) regulators besides Rho, Wnt, and Hedgehog (Hh) signaling. Unexpectedly, Wnt pathway inhibition increased the more immature, slowly cycling GPR56+ CD34+ fraction and Hh/EMT gene expression, while Wnt activation caused opposite effects. Our data suggest that the crucial role of GPR56 lies in its ability to co-activate these opposing signals, thus ensuring the constant supply of both LSC subsets. We show that CDK7 inhibitors suppress both LSC-enriched subsets in vivo and synergize with the Bcl-2 inhibitor venetoclax. Our data establish reciprocal transition between LSC compartments as a novel concept underlying the poor outcome in GPR56high AML and propose combined CDK7 and Bcl-2 inhibition as LSC-directed therapy in this disease.
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Compuestos Bicíclicos Heterocíclicos con Puentes , Quinasas Ciclina-Dependientes , Leucemia Mieloide Aguda , Inhibidores de Proteínas Quinasas , Sulfonamidas , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proteína Quinasa CDC2/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Sinergismo Farmacológico , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/uso terapéutico , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/uso terapéutico , Sulfonamidas/farmacología , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Cyclin-dependent kinase 12 (CDK12) plays a crucial role in DNA-damage response gene transcription and has recently been validated as a promising target in cancer therapy. However, existing CDK12 inhibitors potently inhibit its closest isoform CDK13, which could cause potential toxicity. Therefore, the development of CDK12 inhibitors with isoform-selectivity against CDK13 continues to be a challenge. By taking advantage of the emerging PROteolysis-TArgeting Chimeras (PROTACs) approach, we have synthesized a potent PROTAC degrader PP-C8 based on the noncovalent dual inhibitors of CDK12/13 and demonstrated its specificity for CDK12 over CDK13. Notably, PP-C8 induces profound degradation of cyclin K simultaneously and downregulates the mRNA level of DNA-damage response genes. Global proteomics profiling revealed PP-C8 is highly selective toward CDK12-cyclin K complex. Importantly, PP-C8 demonstrates profound synergistic antiproliferative effects with PARP inhibitor in triple-negative breast cancer (TNBC). The potent and selective CDK12 PROTAC degrader developed in this study could potentially be used to treat CDK12-dependent cancers as combination therapy.
Asunto(s)
Proteína Quinasa CDC2/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteína Quinasa CDC2/metabolismo , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Inhibidores de Poli(ADP-Ribosa) Polimerasas/síntesis química , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteolisis/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
PURPOSE: Osteosarcoma (OS) universally exhibits heterogeneity and cisplatin (CDDP) resistance. Although the Wee1/CDC2 and nuclear factor кB (NF-κB) pathways were reported to show abnormal activation in some tumor cells with CDDP resistance, whether there is any concrete connection is currently unclear. We explored it in human OS cells. MATERIALS AND METHODS: Multiple OS cell lines were exposed to a Wee1 inhibitor (AZD1775) and CDDP to assess the half-maximal inhibitory concentration values. Western blot, coimmunoprecipitation, confocal immunofluorescence, cell cycle, and Cell Counting Kit-8assays were performed to explore the connection between the Wee1/CDC2 and NF-κB pathways and their subsequent physiological contribution to CDDP resistance. Finally, CDDP-resistant PDX-OS xenograft models were established to confirm that AZD1775 restores the antitumor effects of CDDP. RESULTS: A sensitivity hierarchy of OS cells to CDDP and AZD1775 exists. In the highly CDDP-tolerant cell lines, Wee1 and RelA were physically crosslinked, which resulted in increased abundance of phosphorylated CDC2 (Y15) and RelA (S536) and consequent modulation of cell cycle progression, survival, and proliferation. Wee1 inhibition restored the effects of CDDP on these processes in CDDP-resistant OS cells. In addition, animal experiments with CDDP-resistant PDX-OS cells showed that AZD1775 combined with CDDP not only restored CDDP efficacy but also amplified AZD1775 in inhibiting tumor growth and prolonged the median survival of the mice. CONCLUSION: Simultaneous enrichment of molecules in the Wee1/CDC2 and NF-κB pathways and their consequent coactivation is a new molecular mechanism of CDDP resistance in OS cells. OS with this molecular signature may respond well to Wee1 inhibition as an alternative treatment strategy.
Asunto(s)
Resistencia a Antineoplásicos , Osteosarcoma/fisiopatología , Transducción de Señal , Animales , Proteína Quinasa CDC2/antagonistas & inhibidores , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Osteosarcoma/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidoresRESUMEN
Type I interferon (IFN-I) is a key component of the host innate immune system. To establish efficient replication, viruses have developed several strategies to escape from the host IFN response. Japanese encephalitis virus (JEV) NS1', a larger NS1-related protein, is known to inhibit the mitochondrial antiviral signaling (MAVS)-mediated IFN-ß induction by increasing the binding of transcription factors (CREB and c-Rel) to the microRNA 22 (miRNA-22) promoter. However, the mechanism by which NS1' induces the recruitment of CREB and c-Rel onto the miRNA-22 promoter is unknown. Here, we found that JEV NS1' protein interacts with the host cyclin-dependent kinase 1 (CDK1) protein. Mechanistically, NS1' interrupts the CDC25C phosphatase-mediated dephosphorylation of CDK1, which prolongs the phosphorylation status of CDK1 and leads to the inhibition of MAVS-mediated IFN-ß induction. Furthermore, the CREB phosphorylation and c-Rel activation through the IκBα phosphorylation were observed to be enhanced upon the augmentation of CDK1 phosphorylation by NS1'. The abrogation of CDK1 activity by a small-molecule inhibitor significantly suppressed the JEV replication in vitro and in vivo. Moreover, the administration of CDK1 inhibitor protected the wild-type mice from JEV-induced lethality but showed no effect on the MAVS-/- mice challenged with JEV. In conclusion, our study provides new insight into the mechanism of JEV immune evasion, which may lead to the development of novel therapeutic options to treat JEV infection. IMPORTANCE Japanese encephalitis virus (JEV) is the main cause of acute human encephalitis in Asia. The unavailability of specific treatment for Japanese encephalitis demands a better understanding of the basic cellular mechanisms that contribute to the onset of disease. The present study identifies a novel interaction between the JEV NS1' protein and the cellular CDK1 protein, which facilitates the JEV replication by dampening the cellular antiviral response. This study sheds light on a novel mechanism of JEV replication, and thus our findings could be employed for developing new therapies against JEV infection.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Virus de la Encefalitis Japonesa (Especie)/inmunología , Evasión Inmune/inmunología , Interferón beta/inmunología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/fisiología , Animales , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína de Unión a CREB/metabolismo , Línea Celular Tumoral , Cricetinae , Encefalitis Japonesa/inmunología , Células HeLa , Humanos , Inmunidad Innata/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , Inhibidor NF-kappaB alfa/metabolismo , Fosforilación/genética , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-rel/metabolismo , Fosfatasas cdc25/metabolismoRESUMEN
BACKGROUND: Decidualization is essential to the successful pregnancy in mice. The molecular mechanisms and effects of Aurora kinase A (Aurora A) remain poorly understood during pregnancy. This study is the first to investigate the expression and role of Aurora A during mouse decidualization. METHODS: Quantitative real time polymerase chain reaction, western blotting and in situ hybridization were used to determine the expression of Aurora A in mouse uteri. Aurora A activity was inhibited by Aurora A inhibitor to explore the role of Aurora A on decidualization via regulating the Aurora A/Stat3/Plk1/Cdk1 signaling pathway. RESULTS: Aurora A was strongly expressed at implantation sites compared with inter-implantation sites. Furthermore, Aurora A was also significantly increased in oil-induced deciduoma compared with control. Both Aurora A mRNA and protein were significantly increased under in vitro decidualization. Under in vitro decidualization, Prl8a2, a marker of mouse decidualization, was significantly decreased by TC-S 7010, an Aurora A inhibitor. Additionally, Prl8a2 was reduced by Stat3 inhibitor, Plk1 inhibitor and Cdk1 inhibitor, respectively. Moreover, the protein levels of p-Stat3, p-Plk1 and p-Cdk1 were suppressed by TC-S 7010. The protein levels of p-Stat3, p-Plk1 and p-Cdk1 were also suppressed by S3I-201, a Stat3 inhibitor). SBE 13 HCl (Plk1 inhibitor) could reduce the protein levels of p-Plk1 and p-Cdk1. Collectively, Aurora A could regulate Stat3/Plk1/Cdk1 signaling pathway. CONCLUSION: Our study shows that Aurora A is expressed in decidual cells and should be important for mouse decidualization. Aurora A/Stat3/Plk1/Cdk1 signaling pathway may be involved in mouse decidualization.
Asunto(s)
Aurora Quinasa A/biosíntesis , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Decidua/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/fisiología , Animales , Aurora Quinasa A/antagonistas & inhibidores , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteínas de Ciclo Celular/antagonistas & inhibidores , Células Cultivadas , Decidua/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Ratones , Embarazo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Factor de Transcripción STAT3/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Quinasa Tipo Polo 1RESUMEN
The pathological changes and possible underlying molecular mechanisms of hepatocellular carcinoma (HCC) are currently unclear. Effective treatment of this pathological state remains a challenge. The purpose of this study is to obtain some key genes with diagnostic and prognostic meaning and to identify potential therapeutic agents for HCC treatment. Here, CDK1, CCNB1 and CCNB2 were found to be highly expressed in HCC patients and accompanied by poor prognosis, and knockdown of them by siRNA drastically induced autophagy and senescence in hepatoma cells. Simultaneously, the anti-HCC effect of lycorine was comparable to that of interfering with these three genes, and lycorine significantly promoted the decrease both in protein and mRNA expression of CDK1. Molecular validation mechanistically demonstrated that lycorine might attenuate the degradation rate of CDK1 via interaction with it, which had been confirmed by cellular thermal shift assay and drug affinity responsive targets stability assay. Taken together, these findings suggested that CDK1, CCNB1 and CCNB2 could be regarded as potential diagnostic and prognostic biomarkers for HCC, and CDK1 might serve as a promising therapeutic target for lycorine against HCC.
Asunto(s)
Alcaloides de Amaryllidaceae/farmacología , Antineoplásicos/farmacología , Proteína Quinasa CDC2/antagonistas & inhibidores , Carcinoma Hepatocelular/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Neoplasias Hepáticas/tratamiento farmacológico , Fenantridinas/farmacología , Alcaloides de Amaryllidaceae/química , Alcaloides de Amaryllidaceae/uso terapéutico , Animales , Antineoplásicos/uso terapéutico , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Línea Celular Tumoral , Senescencia Celular , Ciclina B1/genética , Ciclina B1/metabolismo , Ciclina B2/genética , Ciclina B2/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Estructura Molecular , Fenantridinas/química , Fenantridinas/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A DNA replication program, which ensures that the genome is accurately and wholly replicated, is established during G1, before the onset of S phase. In G1, replication origins are licensed, and upon S phase entry, a subset of these will form active replisomes. Tight regulation of the number of active replisomes is crucial to prevent replication stress-induced DNA damage. TICRR/TRESLIN is essential for DNA replication initiation, and the level of TICRR and its phosphorylation determine the number of origins that initiate during S phase. However, the mechanisms regulating TICRR protein levels are unknown. Therefore, we set out to define the TICRR/TRESLIN protein dynamics throughout the cell cycle. Here, we show that TICRR levels are high during G1 and dramatically decrease as cells enter S phase and begin DNA replication. We show that degradation of TICRR occurs specifically during S phase and depends on ubiquitin ligases and proteasomal degradation. Using two targeted siRNA screens, we identify CRL4DTL as a cullin complex necessary for TICRR degradation. We propose that this mechanism moderates the level of TICRR protein available for replication initiation, ensuring the proper number of active origins as cells progress through S phase.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Fase S , Ubiquitina-Proteína Ligasas/metabolismo , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteínas Portadoras/fisiología , Ciclo Celular , Proteínas de Ciclo Celular/fisiología , Línea Celular Tumoral , Proteínas Cullin/metabolismo , Proteínas Cullin/fisiología , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Proteínas de Unión al ADN/fisiología , Humanos , Antígeno Nuclear de Célula en Proliferación/fisiología , Ubiquitina-Proteína Ligasas/fisiologíaRESUMEN
In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex1. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD212-4). Separase is activated by degradation of its inhibitors, securin5 and cyclin B6, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans7 and yeast8, human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalytic sites of both separase and CDK19,10. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine6 that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation.
Asunto(s)
Proteína Quinasa CDC2/química , Proteína Quinasa CDC2/metabolismo , Ciclina B1/química , Ciclina B1/metabolismo , Securina/química , Securina/metabolismo , Separasa/química , Separasa/metabolismo , Secuencias de Aminoácidos , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína Quinasa CDC2/ultraestructura , Quinasas CDC2-CDC28/química , Quinasas CDC2-CDC28/metabolismo , Quinasas CDC2-CDC28/ultraestructura , Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , Microscopía por Crioelectrón , Ciclina B1/ultraestructura , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Fosfoserina/metabolismo , Unión Proteica , Dominios Proteicos , Securina/ultraestructura , Separasa/antagonistas & inhibidores , Separasa/ultraestructura , Especificidad por SustratoRESUMEN
Malignant tumor is a disease with high mortality. Traditional treatment methods have many disadvantages, such as side-effects, drug resistance. Because cyclin-dependent kinase 1 (CDK1) plays an indispensable role in cell cycle regulation, it became an attractive target in rational anti-cancer drug discovery. Herein, we reported a series of baicalein derivatives, which remarkably repressed the proliferation of MCF-7 tumor cells and the activity of CDK1/cyclin B kinase. Among them, compound 4a displayed better inhibition rate than flavopiridol against MCF-7 proliferation at the concentration of 50 µg/ml, comparable to compound CGP74514A, while compound 3o possessed the best activity against CDK1/cyclin B kinase (IC50 = 1.26 µM). The inhibitory activities toward the kinase well correlated with anti-proliferative activities. Molecular docking results suggested that compound 3o can interact with the key amino acid residues, E81, L83, and D146, of CDK1 through hydrogen bond just like flavopiridol does. And it can also form an extra hydrogen bond with D146 by its introduced 7-acrylate group, which flavopiridol does not have. These findings proved that baicalein derivatives can be used as CDK1 inhibitors fighting against cancer.
Asunto(s)
Antineoplásicos/síntesis química , Proteína Quinasa CDC2/antagonistas & inhibidores , Flavanonas/síntesis química , Inhibidores de Proteínas Quinasas/síntesis química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclina B/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Flavanonas/farmacología , Flavonoides/farmacología , Flavonoides/normas , Humanos , Células MCF-7 , Simulación del Acoplamiento Molecular , Piperidinas/farmacología , Piperidinas/normas , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Relación Estructura-ActividadRESUMEN
To identify novel prognostic and therapeutic targets for osteosarcoma patients, we compared the gene expression profiles of osteosarcoma and control tissues from the GSE42352 dataset in the Gene Expression Omnibus. Differentially expressed genes were subjected to Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Gene Set Enrichment and protein-protein interaction network analyses. Survival curve analyses indicated that osteosarcoma patients with lower mRNA levels of cyclin-dependent kinase 1 (CDK1) and topoisomerase II alpha had better prognoses. Various computer-aided techniques were used to identify potential CDK1 inhibitors for osteosarcoma patients, and PHA-793887 was predicted to be a safe drug with a high binding affinity for CDK1. In vitro, MTT and colony formation assays demonstrated that PHA-793887 reduced the viability and clonogenicity of osteosarcoma cells, while a scratch assay suggested that PHA-793887 impaired the migration of these cells. Flow cytometry experiments revealed that PHA-793887 dose-dependently induced apoptosis in osteosarcoma cells. Western blotting and enzyme-linked immunosorbent assays indicated that CDK1 expression in osteosarcoma cells declined with increasing PHA-793887 concentrations. These results suggest that PHA-793887 could be a promising new treatment for osteosarcoma.
Asunto(s)
Biología Computacional , Simulación del Acoplamiento Molecular , Osteosarcoma/tratamiento farmacológico , Pirazoles/uso terapéutico , Pirroles/uso terapéutico , Sitios de Unión , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína Quinasa CDC2/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes/efectos de los fármacos , Humanos , Ligandos , Osteosarcoma/genética , Osteosarcoma/patología , Mapas de Interacción de Proteínas/genética , Inhibidores de Proteínas Quinasas/efectos adversos , Pirazoles/química , Pirazoles/farmacología , Pirroles/química , Pirroles/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Análisis de SupervivenciaRESUMEN
Ovarian cancer is often detected at the advanced stages at the time of initial diagnosis. Early-stage diagnosis is difficult due to its asymptomatic nature, where less than 30% of 5-year survival has been noticed. The underlying molecular events associated with the disease's pathogenesis have yet to be fully elucidated. Thus, the identification of prognostic biomarkers as well as developing novel therapeutic agents for targeting these markers become relevant. Herein, we identified 264 differentially expressed genes (DEGs) common in four ovarian cancer datasets (GSE14407, GSE18520, GSE26712, GSE54388), respectively. We constructed a protein-protein interaction (PPI) interaction network with the overexpressed genes (72 genes) and performed gene enrichment analysis. In the PPI networks, three proteins; TTK Protein Kinase (TTK), NIMA Related Kinase 2 (NEK2), and cyclin-dependent kinase (CDK1) with higher node degrees were further evaluated as therapeutic targets for our novel multi-target small molecule NSC777201. We found that the upregulated DEGs were enriched in KEGG and gene ontologies associated with ovarian cancer progression, female gamete association, otic vesicle development, regulation of chromosome segregation, and therapeutic failure. In addition to the PPI network, ingenuity pathway analysis also implicate TTK, NEK2, and CDK1 in the elevated salvage pyrimidine and pyridoxal pathways in ovarian cancer. The TTK, NEK2, and CDK1 are over-expressed, demonstrating a high frequency of genetic alterations, and are associated with poor prognosis of ovarian cancer cohorts. Interestingly, NSC777201 demonstrated anti-proliferative and cytotoxic activities (GI50 = 1.6 µM~1.82 µM and TGI50 = 3.5 µM~3.63 µM) against the NCI panels of ovarian cancer cell lines and exhibited a robust interaction with stronger affinities for TTK, NEK2, and CDK1, than do the standard drug, paclitaxel. NSC777201 displayed desirable properties of a drug-like candidate and thus could be considered as a novel small molecule for treating ovarian carcinoma.
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Biología Computacional , Descubrimiento de Drogas , Perfilación de la Expresión Génica , Variación Genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Inhibidores de Proteínas Quinasas/química , Biomarcadores de Tumor , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína Quinasa CDC2/química , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/química , Biología Computacional/métodos , Descubrimiento de Drogas/métodos , Femenino , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Quinasas Relacionadas con NIMA/antagonistas & inhibidores , Quinasas Relacionadas con NIMA/química , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/mortalidad , Pronóstico , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/química , Relación Estructura-Actividad , TranscriptomaRESUMEN
Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers as these compounds inhibit transcription of DNA damage response (DDR) genes. We previously described THZ531, a covalent inhibitor with selectivity for CDK12/13. In order to elucidate structure-activity relationship (SAR), we have undertaken a medicinal chemistry campaign and established a focused library of THZ531 analogs. Among these analogs, BSJ-01-175 demonstrates exquisite selectivity, potent inhibition of RNA polymerase II phosphorylation, and downregulation of CDK12-targeted genes in cancer cells. A 3.0 Å co-crystal structure with CDK12/CycK provides a structural rational for selective targeting of Cys1039 located in a C-terminal extension from the kinase domain. With moderate pharmacokinetic properties, BSJ-01-175 exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft (PDX) mouse model following 10 mg/kg once a day, intraperitoneal administration. Taken together, BSJ-01-175 represents the first selective CDK12/13 covalent inhibitor with in vivo efficacy reported to date.
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Anilidas/farmacología , Proteína Quinasa CDC2/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Anilidas/síntesis química , Anilidas/química , Animales , Proteína Quinasa CDC2/metabolismo , Células Cultivadas , Quinasas Ciclina-Dependientes/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Ratones , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-ActividadRESUMEN
Oncogenic KIT or PDGFRA receptor tyrosine kinase mutations are compelling therapeutic targets in gastrointestinal stromal tumor (GIST), and treatment with the KIT/PDGFRA inhibitor imatinib is the standard of care for patients with advanced GIST. Polyclonal emergence of KIT/PDGFRA secondary mutations is the main mechanism of imatinib progression, making it challenging to overcome KIT/PDGFRA-inhibitor resistance. It is unclear whether there are other therapeutic targets in advanced GIST. Using genome-wide transcriptomic profiling of advanced versus early-stage GIST and CRISPR knockout functional screens, we demonstrate that CDK1 is frequently highly expressed in advanced GIST but not in early-stage GIST across three patient cohorts. High expression of CDK1 was associated with malignancy in GIST. CDK1 was critically required for advanced GIST, including imatinib-resistant GIST. CDK1 ablation led to robust proliferation inhibition. A mass spectrometry-based proteomics screen further revealed that AKT is a novel substrate of CDK1 kinase in GIST. CDK1 bound AKT and regulated its phosphorylation, thereby promoting GIST proliferation and progression. Importantly, a pharmacologic inhibitor of CDK1, RO-3306, disrupted GIST cell proliferation in CDK1 highly expressed GIST but not in CDK1-negative GIST cells and nontransformed fibroblast cells. Treatment with RO-3306 reduced tumor growth in both imatinib-resistant and imatinib-sensitive GIST xenograft mouse models. Our findings suggest that CDK1 represents a druggable therapeutic target in GIST and warrants further testing in clinical trials. SIGNIFICANCE: These findings propose CDK1 as a novel cell-cycle-independent vulnerability in gastrointestinal stromal tumors, representing a new therapeutic opportunity for patients with advanced disease.
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Antineoplásicos/administración & dosificación , Proteína Quinasa CDC2/metabolismo , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/metabolismo , Mesilato de Imatinib/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Quinolinas/administración & dosificación , Tiazoles/administración & dosificación , Adulto , Anciano , Animales , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína Quinasa CDC2/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/genética , Estudios de Cohortes , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Tumores del Estroma Gastrointestinal/patología , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Transfección , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVES: The promoting roles of cyclin dependent kinase 1 (CDK1) have been revealed in various tumors, however, its effects in the progression of cancer stem cells are still confusing. This work aims to explore the roles of CDK1 in regulating the stemness of lung cancer cells. METHODS: Online dataset analysis was performed to evaluate the correlation between CDK1 exression and the survival of lung cancer patients. RT-qPCR, western blot, cell viability, sphere-formation analysis and ALDH activity detection were used to investigate the roles of CDK1 on lung cancer cell stemness, viability and chemotherapeutic sensitivity. Immunocoprecipitation (Co-IP) analysis and rescuing experiments were performed to reveal the underlying mechanisms contributing to CDK1-mediated effects on lung cancer cell stemness. RESULTS: CDK1 mRNA expression was negatively correlated with the overall survival of lung cancer patients and remarkably increased in tumor spheres formed by lung cancer cells compared to the parental cells. Additionally, CDK1 positively regulated the stemness of lung cancer cells. Mechanistically, CDK1 could interact with Sox2 protein, but not other stemness markers (Oct4, Nanog and CD133). Furthermore, CDK1 increased the phosphorylation, cytoplasm-nuclear translocation and transcriptional activity of Sox2 protein in lung cancer cells. Moreover, CDK1 positively regulated the stemness of lung cancer cells in a Sox2-dependent manner. Finally, we revealed that inhibition of CDK1 enhanced the chemotherapeutic sensitivity, which was also rescued by Sox2 overexpression. CONCLUSIONS: This work reveals a novel CDK1/Sox2 axis responsible for maintaining the stemness of lung cancer cells.
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Proteína Quinasa CDC2/metabolismo , Neoplasias Pulmonares/patología , Células Madre Neoplásicas/patología , Factores de Transcripción SOXB1/metabolismo , Células A549 , Antígeno AC133/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Aldehído Deshidrogenasa/metabolismo , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína Quinasa CDC2/genética , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Supervivencia Celular , Progresión de la Enfermedad , Humanos , Inmunoprecipitación/métodos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Proteína Homeótica Nanog/metabolismo , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Esferoides Celulares/patologíaRESUMEN
CDK1 plays key roles in cell cycle progression through the G2/M phase transition and activation of homologous recombination (HR) DNA repair pathway. Accordingly, various CDK1 inhibitors have been developed for cancer therapy that induce prolonged G2 arrest and/or sensitize cells to DNA damaging agents in tumor cells, resulting in cell death. However, CDK1 inhibition can induce resistance to DNA damage in certain conditions. The mechanism of different DNA damage sensitivity is not completely understood. We performed immunofluorescence and flow cytometry analysis to investigate DNA damage responses in human tumor cells during low and high dose treatments with RO-3306, a selective CDK1 inhibitor. This comparative investigation demonstrated that RO-3306-induced G2 arrest prevented cells with DNA double-strand breaks from transitioning into the M-phase and that the cells maintained their DNA repair capacity in G2-phase, even under RO-3306 dose-dependent DNA repair inhibition. These findings reveal that CDK1 inhibitor-induced DNA repair inhibition and cell cycle control, which regulate each other during the G2/M phase transition determine the cellular sensitivity to DNA damage, providing insight useful for developing clinical strategies targeting CDK1 inhibition in tumor cells.
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Proteína Quinasa CDC2/antagonistas & inhibidores , División Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Línea Celular Tumoral , Humanos , Quinolinas/farmacología , Reparación del ADN por Recombinación/efectos de los fármacos , Tiazoles/farmacologíaRESUMEN
Lung cancer (LC) and pancreatic cancer (PC) are the first and fourth leading causes of cancerrelated deaths in the US. Deregulated cell cycle progression is the cornerstone for rapid cell proliferation, tumor development, and progression. Here, we provide evidence that a novel combinatorial miR treatment inhibits cell cycle progression at two phase transitions, through their activity on the CDK4 and CDK1 genes. Following transfection with miR143 and miR506, we analyzed the differential gene expression of CDK4 and CDK1, using qPCR or western blot analysis, and evaluated cell cycle inhibition, apoptosis and cytotoxicity. The combinatorial miR143/506 treatment downregulated CDK4 and CDK1 levels, and induced apoptosis in LC cells, while sparing normal lung fibroblasts. Moreover, the combinatorial miR treatment demonstrated a comparable activity to clinically tested cell cycle inhibitors in inhibiting cell cycle progression, by presenting substantial inhibition at the G1/S and G2/M cell cycle transitions. More importantly, the miR143/506 treatment presented a broader application, effectively downregulating CDK1 and CDK4 levels, and reducing cell growth in PC cells. These findings suggest that the miR143/506 combination acts as a promising approach to inhibit cell cycle progression for cancer treatment with minimal toxicity to normal cells.
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Quinasas Ciclina-Dependientes/metabolismo , Neoplasias Pulmonares/metabolismo , MicroARNs/metabolismo , Neoplasias Pancreáticas/metabolismo , Aminopiridinas/farmacología , Antineoplásicos/farmacología , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína Quinasa CDC2/genética , Proteína Quinasa CDC2/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Procesos de Crecimiento Celular , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Regulación hacia Abajo , Fibroblastos/citología , Fibroblastos/metabolismo , Flavonoides/farmacología , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , MicroARNs/administración & dosificación , MicroARNs/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/terapia , Piperidinas/farmacología , Purinas/farmacología , Transfección , Regulación hacia ArribaRESUMEN
Prostate cancer with high Gleason grade is prone to metastasis, which is one of the factors that seriously threaten the survival of patients, and it is also a treatment difficulty. In this study, we first revealed the potential connection between TPX2 and prostate cancer metastasis. We found that TPX2 is highly expressed in high-grade prostate cancer and is significantly related to poor prognosis. Depletion of TPX2 can significantly inhibit cell activity and migration, and in vivo experiments show that knockdown of TPX2 can significantly inhibit tumor growth. In terms of mechanism, we found that knocking down TPX2 can inhibit the expression of CDK1, repress the phosphorylation of ERK/GSK3ß/SNAIL signaling pathway, and thereby inhibit tumor epithelial-mesenchymal transition. Subsequently, we found that after rescuing TPX2, all related proteins and phenotype changes were restored, and this effect can be inhibited by CDK1 inhibitor, RO-3306. Our findings suggest the potential of TPX2 as an important target in anti-tumor metastasis therapy, which is conducive to precision medicine for prostate cancer.
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Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Transición Epitelial-Mesenquimal , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Asociadas a Microtúbulos/metabolismo , Neoplasias de la Próstata/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo , Animales , Proteína Quinasa CDC2/antagonistas & inhibidores , Ciclo Celular , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Ratones Desnudos , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Metástasis de la Neoplasia/tratamiento farmacológico , Fosforilación/efectos de los fármacos , Pronóstico , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Quinolinas/farmacología , Tiazoles/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Neuroepithelial transforming gene 1 (Net1) is a RhoA subfamily guanine nucleotide exchange factor that is overexpressed in a number of cancers and contributes to cancer cell motility and proliferation. Net1 also plays a Rho GTPase independent role in mitotic progression, where it promotes centrosomal activation of Aurora A and Pak2, and aids in chromosome alignment during prometaphase. To understand regulatory mechanisms controlling the mitotic function of Net1, we examined whether it was phosphorylated by the mitotic kinase Cdk1. We observed that Cdk1 phosphorylated Net1 on multiple sites in its N-terminal regulatory domain and C-terminus in vitro. By raising phospho-specific antibodies to two of these sites, we also demonstrated that both endogenous and transfected Net1 were phosphorylated by Cdk1 in cells. Substitution of the major Cdk1 phosphorylation sites with aliphatic or acidic residues inhibited the interaction of Net1 with RhoA, and treatment of metaphase cells with a Cdk1 inhibitor increased Net1 activity. Cdk1 inhibition also increased Net1 localization to the plasma membrane and stimulated cortical F-actin accumulation. Moreover, Net1 overexpression caused spindle polarity defects that were reduced in frequency by acidic substitution of the major Cdk1 phosphorylation sites. These data indicate that Cdk1 phosphorylates Net1 during mitosis and suggest that this negatively regulates its ability to signal to RhoA and alter actin cytoskeletal organization.
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Proteína Quinasa CDC2/metabolismo , Mitosis , Proteínas Oncogénicas/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Citoesqueleto de Actina , Actinas/metabolismo , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína Quinasa CDC2/genética , Membrana Celular/metabolismo , Células HeLa , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Oncogénicas/antagonistas & inhibidores , Proteínas Oncogénicas/genética , Fosforilación , Estabilidad Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Huso Acromático/fisiología , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal form of cancer characterized by drug resistance, urging new therapeutic strategies. In recent years, protein kinases have emerged as promising pharmacological targets for the treatment of several solid and hematological tumors. Interestingly, cyclin-dependent kinase 1 (CDK1) is overexpressed in PDAC tissues and has been correlated to the aggressive nature of these tumors because of its key role in cell cycle progression and resistance to the induction of apoptosis. For these reasons, CDK1 is one of the main causes of chemoresistance, representing a promising pharmacological target. In this study, we report the synthesis of new 1,2,4-oxadiazole compounds and evaluate their ability to inhibit the cell growth of PATU-T, Hs766T, and HPAF-II cell lines and a primary PDAC cell culture (PDAC3). Compound 6b was the most active compound, with IC50 values ranging from 5.7 to 10.7 µM. Molecular docking of 6b into the active site of CDK1 showed the ability of the compound to interact effectively with the adenosine triphosphate binding pocket. Therefore, we assessed its ability to induce apoptosis (which increased 1.5- and 2-fold in PATU-T and PDAC3 cells, respectively) and to inhibit CDK1 expression, which was reduced to 45% in Hs766T. Lastly, compound 6b passed the ADME prediction, showing good pharmacokinetic parameters. These data demonstrate that 6b displays cytotoxic activity, induces apoptosis, and targets CDK1, supporting further studies for the development of similar compounds against PDAC.