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19 inhibits not only the CDKsinvolved in cell cycle control but also CDKs involved in transcriptional regulation, itsmechanism of action in MM may possibly be a consequence of transcriptional repression. AlthoughCDK7 and CDK9 are the main transcriptional activating kinases that Celecoxib phosphorylate CTD,both CDK2 and CDK1 also phosphorylate RNA pol II CTD at serine 2 and serine 5 in vitro. Furthermore, CDK inhibition with flavopiridol and seliciclib is alsoassociated with inhibition of phosphorylation of RNA pol II CTD, resulting in a decrease intranscription. The present study demonstrates that AT7519 decreased dephosphorylation ofRNA pol II CTD at both serine 2 and serine 5 top to transcriptional repression.
Becausethe most sensitive targets of transcription inhibitors are mRNAs coding for proteins withshort half lives, we evaluated the expressionlevel of antiapoptotic proteins with rapid turnover, including Mcl1 and XIAP. As expected,AT7519 decreased the degree of Mcl1 and XIAP. Mcl1 can be a Bcl2 family members antiapoptoticprotein vital for MM cell Celecoxib survival. Inhibition of Mcl1 by antisenseoligonucleotides induces apoptosis in MM cells. XIAPoverexpression renders myeloma cells resistant to apoptosis induced by chemotherapeuticagents, and its highlevel expression has been associated having a poor prognosis. The ability of AT7519 to lower levels of both Mcl1 and XIAP demonstratedhere suggests that it may have promise in the treatment of MM.Our data demonstrated that the inhibition of RNA synthesis, measured byUridineincorporation, was only partial suggesting that other mechanisms are implicated in AT7519induced MM cytotoxicity.
The fact that CDKs are closely homologous to GSK3, led us to investigate the role of thiskinase in the biological effects of AT7519. Because of their structural similarity, many CDKinhibitors are inhibitors of GSK3in isolated biochemical assays.Offered its inhibitory role in Alogliptin the pathogenesis of cancers, GSK3had not until recently beenconsidered as a therapeutic target. A lot more recently, a number of lines of evidence have challengedthis view. Whilst GSK3promotes oncogenesis and supports cell proliferation in mixedlineage leukemia, a similar effect has not been seen in other leukemia cell lines. Inhibition of GSK3 induces apoptosis in colonprostate cancer cellsas well as in chronic lymphocytic leukemia B cells; and suppresses cell growth in MM.
AKTinhibitors HSP induce apoptosis in MM cell lines by decreasing phosphorylation of AKT andGSK3at serine 9, suggesting that it may play adual role depending on cell and cancer type. The role of GSK3 in MM cell biology has however to befully defined. Surprisingly, we observed a rapid dephosphorylation of GSK3at serine 9. Due to the fact GSK3is an essential kinase involved in a number of signalingpathways, its activity is regulated by a number of mechanisms and atmultiple levels. GSK3is constitutively active in MM cells; AKT as well as other kinases inhibitGSK3 by phosphorylating the regulatory residues at serine 21or serine 9. The substrates of GSK3include many signaling proteins and transcriptionfactors that regulate growth and survival e.gcyclin D, cyclin E, cMyc, NFKB, betacatenin, p53.
Among these substrates, cMyc, and cyclin D1 wereall downregulated whereas p53 was upregulatedby AT7519 treatment. Alogliptin Noeffect was noted on beta catenin. In contrast, the upstream pathways ofGSK3were upregulated, suggesting that the activation of GSK3wasindependent of these upstream pathways, and that GSK3was a direct target of AT7519.To further realize the role on the activation of GSK3in AT7519 induced cytotoxicity,we Celecoxib used a specific inhibitor of GSK3, ARA04414. This inhibitor elevated GSK3phosphorylation in a dosedependent manner, associated having a dephosphorylation ofglycogen synthase. Importantly, the inhibition of GSK3usingARA04414 at low doses prior to treatment with AT7519 and GSK3knock down usingshRNA resulted in partial rescue of cell death. Our findings for that reason suggest that theactivation of GSK3plays a role in the inhibition of MM cell survival.
This was interestinggiven that the in vitro kinase assay demonstrated inhibition of GSK3.Considering that AT7519 inhibits transcription, we investigated if dephosphorylation of GSK3was aconsequence of transcriptional repression by using a specific and selective inhibitor of RNApol II. Therapy with alphaamanitin Alogliptin did notcorrelate with GSK3dephosphorylation, suggesting that dephosphorylation of GSK3occurs independently from the RNA pol II inhibition induced by AT7519.In conclusion, we've demonstrated that AT7519, a novel modest molecule multiCDKinhibitor, has potent anti MM activity both in vitro and in vivo. In addition, even though theinhibition of transcription is an critical mechanism prevalent to many CDK inhibitors,molecular studies of AT7519 revealed that GSK3plays a essential role in AT7519mediatedantimyeloma effect. These outcomes hence give the rationale for future clinical trials ofAT7519 in MM patients, too as give insights into the potential role of GSK3as atherapeutic