The critical role played by cyclin D and CDK4/6 deregulation in MM pathogenesis led us to study the pharmacology of CDK inhibitors in models of the disease. One such inhibitor is AT7519, which inhibits CDKs 1, 2, 4, 5, 6 and 9 with lower potency against CDK3 and 7 in in vitro kinase assays. Our results demonstrate that AT7519 induces apoptosis not only by a mechanism similar to other CDK inhibitors tested in MM (e.g., Seliciclib), i.e., via the dephosphorylation of the CTD of the large subunit of RNA pol II, but also, unlike other CDK inhibitors, through the rapid dephosphorylation and subsequent activation of GSK-3β at serine 9 which was in contrast to in vitro kinase assay data.
This study investigated the hypothesis that, because AT7519 inhibits not only the CDKs involved in cell cycle control but also CDKs involved in transcriptional regulation, its mechanism of action in MM may be a consequence of transcriptional repression. Although CDK7 and CDK9 are the primary transcriptional activating kinases that phosphorylate CTD, both CDK2 and CDK1 also phosphorylate RNA pol II CTD at serine 2 and serine 5 in vitro
(Cai et al., 2006
). Moreover, CDK inhibition with flavopiridol and seliciclib is also associated with inhibition of phosphorylation of RNA pol II CTD, resulting in a decrease in transcription. The present study demonstrates that AT7519 decreased dephosphorylation of RNA pol II CTD at both serine 2 and serine 5 leading to transcriptional repression. Because the most sensitive targets of transcription inhibitors are mRNAs coding for proteins with short half lives (Chen et al., 2005
; MacCallum et al., 2005
), we evaluated the expression level of antiapoptotic proteins with rapid turnover, such as Mcl-1 and XIAP. As expected, AT7519 decreased the level of Mcl-1 and XIAP. Mcl-1 is a Bcl-2 family antiapoptotic protein essential for MM cell survival (Zhang et al., 2002
). Inhibition of Mcl-1 by antisense oligonucleotides induces apoptosis in MM cells (Derenne et al., 2002
). XIAP overexpression renders myeloma cells resistant to apoptosis induced by chemotherapeutic agents, and its high-level expression has been associated with a poor prognosis (Nakagawa et al., 2006
). The ability of AT7519 to reduce levels of both Mcl-1 and XIAP demonstrated here suggests that it may have promise in the treatment of MM.
Our data demonstrated that the inhibition of RNA synthesis, measured by [3H] Uridine incorporation, was only partial suggesting that other mechanisms are implicated in AT7519 induced MM cytotoxicity. The fact that CDKs are closely homologous to GSK-3β (Cohen & Frame, 2001
; Leclerc et al., 2001
; Wyatt et al., 2008
), led us to investigate the role of this kinase in the biological effects of AT7519. Because of their structural similarity, many CDK inhibitors are inhibitors of GSK-3β in isolated biochemical assays (Knockaert et al., 2002
). Given its inhibitory role in the pathogenesis of cancers, GSK-3β had not until recently been considered as a therapeutic target. More recently, several lines of evidence have challenged this view. Whilst GSK-3β promotes oncogenesis and supports cell proliferation in mixed lineage leukemia (MLL), a similar effect has not been seen in other leukemia cell lines (Wang et al., 2008
). Inhibition of GSK-3 induces apoptosis in colon (Shakoori et al., 2005
) prostate cancer cells (Vene et al., 2008
) as well as in chronic lymphocytic leukemia B cells (Ougolkov et al., 2007
); and suppresses cell growth in MM (G-Amlak et al., 2002
). AKT inhibitors induce apoptosis in MM cell lines by decreasing phosphorylation of AKT and GSK-3β at serine 9 (Hideshima et al., 2007
; Neri et al., 2008
), suggesting that it may play a dual role based on cell and cancer type. The role of GSK-3 in MM cell biology has yet to be fully defined. Surprisingly, we observed a rapid dephosphorylation of GSK-3β at serine 9 (its active form). Because GSK-3β is an important kinase involved in several signaling pathways (Grimes & Jope, 2001
), its activity is regulated by several mechanisms and at multiple levels. GSK-3β is constitutively active in MM cells; AKT and other kinases inhibit GSK-3 by phosphorylating the regulatory residues at serine 21 (alpha isoform) or serine 9 (beta isoform). The substrates of GSK-3β include many signaling proteins and transcription factors that regulate growth and survival e.g., cyclin D, cyclin E, c-Myc, NF-KB, beta catenin, p53 (Cohen & Frame, 2001
). Among these substrates, c-Myc, and cyclin D1 were all downregulated whereas p53 was upregulated (data not shown) by AT7519 treatment. No effect was noted on beta catenin (data not shown). In contrast, the upstream pathways of GSK-3 (AKT and p70S6K) were upregulated, suggesting that the activation of GSK-3β was independent of these upstream pathways, and that GSK-3β was a direct target of AT7519. To further understand the role of the activation of GSK-3β in AT7519 induced cytotoxicity, we used a specific inhibitor of GSK-3β, AR-A04414. This inhibitor increased GSK-3β phosphorylation in a dose-dependent manner, associated with a dephosphorylation of glycogen synthase (a primary GSK-3 substrate). Importantly, the inhibition of GSK-3β using AR-A04414 at low doses prior to treatment with AT7519 and GSK-3β knock down using shRNA resulted in partial rescue of cell death. Our findings therefore suggest that the activation of GSK-3β plays a role in the inhibition of MM cell survival. This was interesting given that the in vitro kinase assay demonstrated inhibition of GSK-3β.
Since AT7519 inhibits transcription, we investigated if dephosphorylation of GSK-3β was a consequence of transcriptional repression by using a specific and selective inhibitor of RNA pol II (alpha-amanitin) (Wieland & Faulstich, 1991
). Treatment with alpha-amanitin did not correlate with GSK-3β dephosphorylation, suggesting that dephosphorylation of GSK-3β occurs independently from the RNA pol II inhibition induced by AT7519.
In conclusion, we have demonstrated that AT7519, a novel small molecule multi-CDK inhibitor, has potent anti MM activity both in vitro and in vivo. In addition, although the inhibition of transcription is an important mechanism common to many CDK inhibitors, molecular studies of AT7519 revealed that GSK-3β plays a crucial role in AT7519-mediated antimyeloma effect. These results thus provide the rationale for future clinical trials of AT7519 in MM patients, as well as provide insights into the potential role of GSK-3β as a therapeutic target in cancer treatment.