Oncogenic mutations in gene encoding FLT3 kinase are often detected in acute myeloid leukaemia (AML) patients, and several potent kinase inhibitors have been developed. However, the FLT3 inhibitor treatment often leads to the resistance development and subsequent relapse. Targeted degradation of oncogenic protein kinases has emerged as a feasible pharmacological strategy, providing more robust effect over traditional competitive inhibitors. Based on previously developed competitive inhibitor of FLT3 and CDK9, we have designed and prepared a novel pomalidomide-based PROTAC. A series of biochemical and cellular experiments showed selectivity towards FLT3-ITD bearing AML cells and confirmed proteasome-dependent mechanism of action. Dual FLT3-ITD and CDK9 protein degradation resulted in the block of FLT3-ITD downstream signalling pathways, apoptosis activation and cell cycle arrest of FLT3-ITD AML cells. Moreover, transcriptional repression caused by CDK9 degradation significantly reduced expression of crucial genes involved in AML pathogenesis. The obtained results indicate the beneficial impact of simultaneous FLT3-ITD/CDK9 degradation for AML therapy.

• MeSH
• akutní myeloidní leukemie * patologie   MeSH
• apoptóza MeSH
• cyklin-dependentní kinasa 9 metabolismus   MeSH
• inhibitory proteinkinas farmakologie   terapeutické užití   MeSH
• lidé MeSH
• mutace MeSH
• proteolýza MeSH
• tyrosinkinasa 3 podobná fms genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The identification of the essential role of cyclin-dependent kinases (CDKs) in the control of cell division has prompted the development of small-molecule CDK inhibitors as anticancer drugs. For many of these compounds, the precise mechanism of action in individual tumor types remains unclear as they simultaneously target different classes of CDKs - enzymes controlling the cell cycle progression as well as CDKs involved in the regulation of transcription. CDK inhibitors are also capable of activating p53 tumor suppressor in tumor cells retaining wild-type p53 gene by modulating MDM2 levels and activity. In the current study, we link, for the first time, CDK activity to the overexpression of the MDM4 (MDMX) oncogene in cancer cells. Small-molecule drugs targeting the CDK9 kinase, dinaciclib, flavopiridol, roscovitine, AT-7519, SNS-032, and DRB, diminished MDM4 levels and activated p53 in A375 melanoma and MCF7 breast carcinoma cells with only a limited effect on MDM2. These results suggest that MDM4, rather than MDM2, could be the primary transcriptional target of pharmacological CDK inhibitors in the p53 pathway. CDK9 inhibitor atuveciclib downregulated MDM4 and enhanced p53 activity induced by nutlin-3a, an inhibitor of p53-MDM2 interaction, and synergized with nutlin-3a in killing A375 melanoma cells. Furthermore, we found that human pluripotent stem cell lines express significant levels of MDM4, which are also maintained by CDK9 activity. In summary, we show that CDK9 activity is essential for the maintenance of high levels of MDM4 in human cells, and drugs targeting CDK9 might restore p53 tumor suppressor function in malignancies overexpressing MDM4.

• MeSH
• cyklin-dependentní kinasa 9 antagonisté a inhibitory   metabolismus   MeSH
• genetická transkripce MeSH
• imidazoly farmakologie   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• lidé MeSH
• melanom genetika   metabolismus   patologie   MeSH
• MFC-7 buňky MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prsu genetika   metabolismus   patologie   MeSH
• piperaziny farmakologie   MeSH
• pluripotentní kmenové buňky metabolismus   MeSH
• proteiny buněčného cyklu biosyntéza   genetika   metabolismus   MeSH
• protoonkogenní proteiny c-mdm2 biosyntéza   genetika   metabolismus   MeSH
• protoonkogenní proteiny biosyntéza   genetika   metabolismus   MeSH
• roskovitin farmakologie   MeSH
• sulfonamidy farmakologie   MeSH
• synergismus léků MeSH
• transfekce MeSH
• triaziny farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Inhibitors of cyclin-dependent kinases 9 have been developed as potential anticancer drugs for the treatment of multiple myeloma. We have previously prepared a library of arylazo-3,5-diaminopyrazole inhibitors of CDKs. Here, we describe a novel member, AAP1742 (CDK9 inhibition with IC(50) = 0.28 μm), that reduces the viability of multiple myeloma cell lines in low micromolar concentrations. Consistent with inhibition of CDK9, AAP1742 decreases the phosphorylation of RNA polymerase II and inhibits mRNA synthesis of anti-apoptotic proteins Mcl-1, Bcl-2, and XIAP, followed by apoptosis in the RPMI-8226 cell line in a dose- and a time-dependent manner. These results are consistent with the biochemical profile of AAP1742 and further suggest cellular inhibition of CDK9 as a possible target for anticancer drugs.

• MeSH
• aktivace enzymů účinky léků   MeSH
• apoptóza účinky léků   MeSH
• azosloučeniny chemie   farmakologie   MeSH
• cyklin-dependentní kinasa 9 antagonisté a inhibitory   metabolismus   MeSH
• down regulace účinky léků   MeSH
• fosforylace účinky léků   MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• mnohočetný myelom metabolismus   patologie   MeSH
• nádorové buněčné linie MeSH
• protein MCL-1 genetika   metabolismus   MeSH
• protinádorové látky chemie   farmakologie   MeSH
• protoonkogenní proteiny c-bcl-2 genetika   metabolismus   MeSH
• pyrazoly chemie   farmakologie   MeSH
• RNA-polymerasa II metabolismus   MeSH
• X-vázaný inhibitor apoptózy genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cytochrome P450 1B1 (CYP1B1) is an enzyme that has a unique tumor-specific pattern of expression and is capable of bioactivating a wide range of carcinogenic compounds. We have reported previously that coordinated upregulation of CYP1B1 by inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and the aryl hydrocarbon receptor ligands, may increase bioactivation of promutagens, such as benzo[a]pyrene (BaP) in epithelial cells. Here, we extend those studies by describing a novel mechanism participating in the regulation of CYP1B1 expression, which involves activation of the p38 mitogen-activated protein kinase (p38) and mitogen- and stress-activated protein kinase 1 (MSK1). Using inhibitors of p38 and MSKs, as well as mouse embryonic cells derived from p38α-deficient and MSK1/2 double knockout mice, we show here that TNF-α potentiates CYP1B1 upregulation via the p38/MSK1 kinase cascade. Effects of this inflammatory cytokine on CYP1B1 expression further involve the positive transcription elongation factor b (P-TEFb). The inhibition of the P-TEFb subunit, cyclin-dependent kinase 9 (CDK9), which phosphorylates RNA polymerase II (RNAPII), prevented the enhanced CYP1B1 induction by a combination of BaP and inflammatory cytokine. Furthermore, using chromatin immunoprecipitation assays, we found that cotreatment of epithelial cells with TNF-α and BaP resulted in enhanced recruitment of both CDK9 and RNAPII to the Cyp1b1 gene promoter. Overall, these results have implications concerning the contribution of inflammatory factors to carcinogenesis, since enhanced CYP1B1 induction during inflammation may alter metabolism of exogenous carcinogens, as well as endogenous CYP1B1 substrates playing role in tumor development.

• MeSH
• cyklin-dependentní kinasa 9 genetika   MeSH
• cytochrom P450 CYP1B1 biosyntéza   genetika   MeSH
• cytokiny metabolismus   MeSH
• karcinogeneze účinky léků   genetika   MeSH
• karcinogeny toxicita   MeSH
• lidé MeSH
• mitogenem aktivované proteinkinasy p38 antagonisté a inhibitory   genetika   metabolismus   MeSH
• myši MeSH
• nádory chemicky indukované   genetika   patologie   MeSH
• pozitivní transkripční elongační faktor b genetika   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• RNA-polymerasa II genetika   MeSH
• signální transdukce účinky léků   MeSH
• TNF-alfa metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Deregulation of cyclin-dependent kinases (CDKs) has been associated with many cancer types and has evoked an interest in chemical inhibitors with possible therapeutic benefit. While most known inhibitors display broad selectivity towards multiple CDKs, recent work highlights CDK9 as the critical target responsible for the anticancer activity of clinically evaluated drugs. In this review, we discuss recent findings provided by structural biologists that may allow further development of highly specific inhibitors of CDK9 towards applications in cancer therapy. We also highlight the role of CDK9 in inflammatory processes and diseases.

• MeSH
• cyklin-dependentní kinasa 9 antagonisté a inhibitory   metabolismus   MeSH
• inhibitory proteinkinas farmakologie   MeSH
• lékové transportní systémy MeSH
• lidé MeSH
• nádory farmakoterapie   enzymologie   MeSH
• protinádorové látky farmakologie   MeSH
• racionální návrh léčiv MeSH
• zánět enzymologie   patologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Embryonic stem cells (ESCs) proliferate rapidly and have a unique cell-cycle structure with a very short G1 phase. Previous reports suggested that the rapid G1 phase progression of ESCs might be underpinned by high and precocious Cdk2 activity and that Cdk2 activity might be crucial for both cell-cycle regulation and cell-fate decisions in human ESCs. However, the actual role of Cdk2 in cell-cycle progression of mouse ESCs (mESCs) has not been elucidated. In this study, we investigated the effects of down-regulation of Cdk2 activity by olomoucine II in 2 mESC lines. Olomoucine II treatment significantly increased the G1 phase cell numbers, decreased the S phase cell numbers, and inhibited DNA replication in mESCs. In nocodazole-synchronized mESCs, we show that specific down-regulation of Cdk2 activity prolongs G1 phase progression. In addition, down-regulation of Cdk2 activity in mESCs established a somatic cell-like cell cycle and induced expression of differentiation markers. Our results suggest that high Cdk2 activity is essential for rapid G1 phase progression and establishment of ESC-specific cell-cycle structure in mESCs and support the hypothesis of a link between cell-cycle regulation and pluripotency maintenance in ESCs. This study reveals olomoucine II to be an effective tool for manipulation of the cell cycle and pluripotency in ESCs and very likely also for the manipulation of other stem cell types, including cancer stem cells.

• MeSH
• buněčné linie MeSH
• buněčný cyklus účinky léků   MeSH
• buňky HT-29 MeSH
• časové faktory MeSH
• cyklin-dependentní kinasa 2 antagonisté a inhibitory   genetika   metabolismus   MeSH
• cyklin-dependentní kinasa 9 antagonisté a inhibitory   genetika   metabolismus   MeSH
• embryonální kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• G1 fáze účinky léků   MeSH
• inbrední kmeny myší MeSH
• inhibiční koncentrace 50 MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteinkinasa CDC2 antagonisté a inhibitory   genetika   metabolismus   MeSH
• průtoková cytometrie MeSH
• puriny farmakologie   MeSH
• replikace DNA účinky léků   MeSH
• viabilita buněk účinky léků   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cardiac hypertrophy allows the heart to adapt to workload, but persistent or unphysiological stimulus can result in pump failure. Cardiac hypertrophy is characterized by an increase in the size of differentiated cardiac myocytes. At the molecular level, growth of cells is linked to intensive transcription and translation. Several cyclin-dependent kinases (CDKs) have been identified as principal regulators of transcription, and among these CDK9 is directly associated with cardiac hypertrophy. CDK9 phosphorylates the C-terminal domain of RNA polymerase II and thus stimulates the elongation phase of transcription. Chronic activation of CDK9 causes not only cardiac myocyte enlargement but also confers predisposition to heart failure. Due to the long interest of molecular oncologists and medicinal chemists in CDKs as potential targets of anticancer drugs, a portfolio of small-molecule inhibitors of CDK9 is available. Recent determination of CDK9's crystal structure now allows the development of selective inhibitors and their further optimization in terms of biochemical potency and selectivity. CDK9 may therefore constitute a novel target for drugs against cardiac hypertrophy.

• MeSH
• cyklin T chemie   MeSH
• cyklin-dependentní kinasa 9 antagonisté a inhibitory   chemie   metabolismus   MeSH
• inhibitory proteinkinas chemie   metabolismus   farmakologie   terapeutické užití   MeSH
• kardiomegalie farmakoterapie   enzymologie   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• racionální návrh léčiv MeSH
• sekvence aminokyselin MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Cross-linking the B cell Ag receptor (BCR) to surface Fc receptors for IgG (Fc gamma R) inhibits G1-to-S progression; the mechanism by which this occurs is not completely known. We investigated the regulation of three key cell cycle regulatory components by BCR-Fc gamma R co-cross-linking: G1-cyclins, cyclin-dependent kinases (Cdks), and the retinoblastoma gene product (Rb). Rb functions to suppress G1-to-S progression in mammalian cells. Rb undergoes cell-cycle-dependent phosphorylation, leading to its inactivation and thereby promoting S phase entry. We demonstrate in this paper for the first time that BCR-induced Rb phosphorylation is abrogated by co-cross-linking with Fc gamma R. The activation of Cdk4/6- and Cdk2-dependent Rb protein kinases is concomitantly blocked. Fc gamma R-mediated inhibition of Cdk2 activity results in part from an apparent failure to express Cdk2 protein. By contrast, inhibition of Cdk4/6 activities is not due to suppression of Cdk4/6 or cyclins D2/D3 expression or inhibition of Cdk-activating kinase activity. Cdk4- and Cdk6-immune complexes recovered from B cells following BCR-Fc gamma R co-cross-linking are devoid of coprecipitated D-type cyclins, indicating that inhibition of their Rb protein kinase activities is due in part to the absence of bound D-type cyclin. Thus, BCR-derived activation signals that up-regulate D-type cyclin and Cdk4/6 protein expression remain intact; however, Fc gamma R-mediated signals block cyclin D-Cdk4/6 assembly or stabilization. These results suggest that assembly or stabilization of D-type cyclin holoenzyme complexes 1) is an important step in the activation of Cdk4/6 by BCR signals, and 2) suffice in providing a mechanism to account for inhibition of BCR-stimulated Rb protein phosphorylation by Fc gamma R.

• MeSH
• aktivace enzymů imunologie   MeSH
• B-lymfocyty enzymologie   imunologie   metabolismus   MeSH
• buněčná diferenciace imunologie   MeSH
• cyklin D MeSH
• cyklin E antagonisté a inhibitory   biosyntéza   MeSH
• cyklin-dependentní kinasa 2 MeSH
• cyklin-dependentní kinasa 4 MeSH
• cyklin-dependentní kinasa 6 MeSH
• cyklin-dependentní kinasa 9 MeSH
• cyklin-dependentní kinasy antagonisté a inhibitory   biosyntéza   metabolismus   MeSH
• cykliny antagonisté a inhibitory   biosyntéza   MeSH
• DNA antagonisté a inhibitory   biosyntéza   MeSH
• fosforylace MeSH
• G1 fáze imunologie   MeSH
• holoenzymy biosyntéza   MeSH
• imunoglobuliny - Fab fragmenty farmakologie   MeSH
• inhibitor p27 cyklin-dependentní kinasy MeSH
• kinasy CDC2-CDC28 * MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nádorové supresorové proteiny * MeSH
• protein-serin-threoninkinasy antagonisté a inhibitory   biosyntéza   metabolismus   MeSH
• proteiny asociované s mikrotubuly biosyntéza   MeSH
• proteiny buněčného cyklu * MeSH
• protilátky anti-idiotypické farmakologie   MeSH
• protoonkogenní proteiny * MeSH
• receptory antigenů B-buněk antagonisté a inhibitory   imunologie   metabolismus   fyziologie   MeSH
• receptory IgG metabolismus   MeSH
• retinoblastomový protein antagonisté a inhibitory   metabolismus   MeSH
• RNA antagonisté a inhibitory   biosyntéza   MeSH
• upregulace imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH