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S. Xie et al.
(Figure 1B). Further, we assessed the impact of CDKI-73 on the expression of anti-apoptotic proteins MCL1 and XIAP, which were proved to be down-regulated by CDK9 inhibition.7 Both SU-DHL-4 and Pfeiffer cells exposed to increasing doses of CDKI-73 exhibited decreased levels of MCL1 and XIAP at both protein (Figure 1B) and mRNA levels (Figure 1D), indicating transcriptional inhibition. The pattern is similar to that of Flavopiridol. These results indicated that CDKI-73 inhibits CDK9 activity efficiently in DLBCL cell lines.
In addition to its predominant participation in tran- scription, CDK9 also acts as a DNA damage response- related protein.21 CDK9/cyclin K is directly involved in maintaining the genomic integrity.21 Hence, we assessed the ability of CDKI-73 in inducing DNA damage in DLBCL cells. Both comet assay and γ-H2AX analysis indi- cated that CDKI-73 could induce DNA double strand breaks (DSB) in a dose- and time-dependent manner, as evidenced by the elevation of γ-H2AX expression, the fre- quent appearance and expanding volume of comet tails, as well as the shrinkage of comet heads (Figure 1E-F).
The results listed above show that CDKI-73 can potent- ly inhibit the in vitro proliferation of DLBCL. Subsequently, the in vivo effect of CDKI-73 against DLBCL was assessed in Pfeiffer xenografts. The activity was weaker than expected. Orally-administered 60 mg/kg of CDKI-73 only slightly restrained the growth of subcutaneously Pfeiffer xenografts. Treatment with 80 mg/kg of CDKI-73 resulted in T/C [(mean RTVtreated)/(mean RTVvehicle)×100] of 59.36 % at day 17 (Figure 1G).
CDKI-73 increases H3K27me3 through CDK9 inhibition
Considering the pivotal role of histone modifications in the development and/or progression of DLBCL, we next explored whether CDKI-73 affected the epigenetic modi- fication of histones. We used mass spectrometry (MS) to detect the influence of CDKI-73 on 36 different epigenet- ic modifications of histone in DLBCL cell line. Notably, we found that treatment with 50 nM CDKI-73 for 24 hours could trigger upregulation of H3K18me1 (1.36-fold) and H3K27me3 (1.29-fold), while the modifications at other sites increased less than 1.20-fold (Figure 2A). In addition, the modification of some sites were suppressed, such as H3K27ac, which was downregulated 0.42-fold, ranked first in all downregulated sites (Figure 2A and Online Supplementary Table S1). In accordance with the MS profiling data, Flavopiridol and CDKI-73 all dramati- cally elevated the level of H3K27me3 accompanied by the upregulation of both H3K27me1 and H3K27me2 levels at the same site in DLBCL cells in Western blot analysis (Figure 2B and Online Supplementary Figure S1A). Meanwhile, CDKI-73 treatment did not remarkably alter other well-studied methylation sites on histone H3, including H3K36, H3K79 and H3K9 (Figure 2B and Online Supplementary Figure S1A and Table S1). To further dissect if CDKI-73 can influence H3K27me3 binding and how CDKI-73 alters the chromatin landscape of DLBCL cells, ChIP-seq was used to analyse the profile distribution of H3K27me3. As expected, we found a global up-regulation of H3K27me3 following CDKI-73 treatment for 24 hours (Figure 2C). Consistently, the transcription of the genes repressed by H3K27me3, GATA4,22 CDKN2A,23,24 HOXC88,25 and TNFRSF21,14 was dramatically decreased (Figure 2D). All of these results indicated that CDKI-73
indeed elevated H3K27me3 in DLBCL.
Next, we attempted to determine the detailed mecha-
nism underlying CDKI-73-induced H3K27me3 upregula- tion. We observed the upregulation of H3K27me3 along with the decline of MCL1 by other CDK9-specific inhibitors SNS-032, AT-7519, and Dinaciclib (Figure 2E). Furthermore the specific CDK4/6 inhibitor PD0332991 did not influence the level of H3K27me3 (Figure 2F). Then, we hypothesized that the upregulation of H3K27me3 is due to CDK9 inhibition. The lack of selec- tivity against other CDK of these small molecules led us to further confirm the mechanism by using specific RNA interference. As expected, only CDK9 depletion by RNA interference notably increased H3K27me3 in DLBCL cells, associated with the decline of MCL1 (Figure 2G). No change of H3K27me3 was displayed after CDK1, CDK2, CDK4 and CDK7 depletion (Figure 2H). Further, the transcription of the H3K27me3-targeted genes was also decreased in CDK9 silencing cells (Figure 2I). As the MS assay showed a down-regulation of H3K27ac (Figure 2A), we then investigated whether the decrease of H3K27ac is in response to CDK9 inhibition since the loss of H2K27ac may link to the increase in H3K27me3.26 To our surprise, the level of H3K27ac, which declined after treatment with CDKI-73, remained unchanged in CDK9 knock-down cells (Online Supplementary Figure S1C-D). The detail mechanism of H3K27ac downregulation needs to be further studied and may be due to other CDK inhi- bition other than CDK9. These data strongly suggested that CDK9 inhibition indeed increased the level of H3K27 trimethylation in DLBCL cells.
H3K27 methylation has been shown to be catalyzed by EZH2,10 while the demethylation is catalyzed by UTX and JMJD3.11 We therefore tried to identify the protein regulated by CDK9 to adjust H3K27 methylation. As mentioned before, CDK9 has been shown to partcipiate in transcriptional processes, therefore reducing protein expression by CDK9 inhibition is the most important proposed mechanism. As expected, the mRNA and pro- tein levels of demethylases (UTX and JMJD3) decreased after the treatment with CDK9i in a dose- and time- dependent manner (Figure 3A-B). UTX and JMJD3 almost were completely depleted when the cells were exposed for 24 hours to 0.05 μM and 0.1 μM CDKI- 73/Flavopiridol, respectively (Figure 3B and Online Supplementary Figure S2A). To our surprise, the expression of methyltransferases, EZH2, EED, and SUZ12 was also lowered. However, these two CDK9i resulted in the decline of protein and mRNA levels of UTX and JMJD3, especially JMJD3, at lower doses and at early time points of treatment. Methyltransferases were almost completely depleted after treatment with 0.4 μM CDKI- 73/Flavopiridol for 24 hours. As expected, the silencing of CDK9 and other three CDK9i (SNS-032, AT-7519, and Dinaciclib) also diminished JMJD3, UTX, and PRC2, but the effect on JMJD3 and UTX was more significant (Figure 3A-C). These data demonstrated that H3K27me3 stimulated by CDKI-73 was dependent on CDK9 inhibi- tion.
Previous studies have indicated that CDK1- and CDK2- dependent phosphorylation of EZH2 can influence the methylation of H3K27.27,28 However, we found no obvi- ous change of EZH2 phosphorylation after treatment with the indicated dose of CDKI-73 (Online Supplementary Figure S2B).
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