Characterisation of a potent, selective inhibitor of CDK9

The two compounds (PC585 and PC579) used in the present study are specific inhibitors of CDK915. Tests showed that neither compound had a significant inhibitory effect on any of 235 kinases examined when used at a concentration of 1 μM (data not shown).

Administration of CDK9 inhibitors in murine arthritis models

Daily treatment with CDK9 inhibitors (PC585 and PC579; each at 10 mg/kg) had a marked impact on CIA development, progression and severity in DBA/1 mice. We compared the effects of the two orally dosed CDK9 inhibitors with those of Enbrel (a recombinant human TNF receptor p75 Fc fusion protein commonly used to treat RA). Treatment with the CDK9 inhibitors resulted in a significant delay in disease onset. The first clinical signs of arthritis presented in control animals on Day 26, whereas animals treated with PC585 showed the first symptoms on Day 31 (p = 0.02). The effect of PC585 was comparable with that of Enbrel (p = 0.008; Fig. 1A). Treatment with PC579 did not cause a significant delay in disease onset; however, we observed a 30% reduction in disease incidence (compared with the control) at the end of the experiment (Fig. 1A). The clinical score for the control group increased rapidly up until Day 39, reaching a mean arthritis index of 4.15. By contrast, mice treated with PC585 or PC579 showed a mean arthritis index of 0.7 and 1.35, respectively (Fig. 1B). Animals treated with Enbrel showed comparatively few clinical symptoms and a mean arthritis index of 0.6 (Fig. 1B). Morphological examination of joint tissues confirmed that the CDK9 inhibitors prevented the clinical signs of CIA. When compared with that in the control group, inflammatory cell infiltration and/or thickening of the synovial membrane was markedly reduced in both treatment groups (Fig. 1C,F). In addition, the level of cartilage and bone destruction was significantly reduced in all treatment groups (PC585, p < 0.01; PC579, p < 0.05; Enbrel, p < 0.01) (Fig. 1D,F). Interestingly, inhibitor treatment had no impact on the level of anti-collagen type II (CII) antibodies (Fig. 1E).

Figure 1 Treatment with CDK9 inhibitors ameliorates collagen induced arthritis. (A) Kaplan-Meier plots showing onset of arthritis during treatment with 10 mg/kg CDK9 inhibitors (PC585, n = 10 and PC579 n = 10), 100 ug anti-TNF treatment (Enbrel, n = 10) or untreated controls (n = 10). (B) Development of clinical signs of arthritis during the course of experiment. (C) Histological evaluation of synovitis and (D) erosions in joints (Controls, n = 10; PC585, n = 10; PC579, n = 9; Enbrel, n = 9). (E) Serum levels of IgG fraction of antibodies against CII at end of experiment (Controls, n = 10; PC585, n = 10; PC579, n = 9; Enbrel, n = 9). (F) Representative histological changes in the front paw joints of animals treated with PC585 and non-treated controls. Horizontal line and error bars represent the mean and SEM, respectively. Box and whiskers graphs show median and min to max values. Differences between groups were analyzed using log-rank test, two-way ANOVA or one-way ANOVA with Bonferronion post-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Full size image

CDK9 inhibition results in the loss of Mcl-1 expression by inflammatory cells

Non-specific CDK inhibitors impact cell survival by inducing apoptosis and by down-regulating expression of the anti-apoptotic protein, Mcl-110; however, there is no direct evidence to suggest that the mechanism(s) underlying these phenomena is mediated solely by CDK9. Western blot analysis of protein extracts from the spleens of arthritic animals treated only twice weekly with PC585 (30 mg/kg and 10 mg/kg, respectively) revealed that Mcl-1 expression was down-regulated in a dose-dependent manner. Notably, there was an increase in expression of the pro-apoptotic isoform, Mcl-1s (Fig. 2A). Immunohistochemical analysis of joints from CIA mice showed that Mcl-1 expression was very low in treated animals compared with that in untreated controls. The latter showed signs of active inflammation and high expression of Mcl-1 (Fig. 2B). We further analysed Mcl-1 expression in PBMC activated with anti-CD3 in vitro. Treatment with CDK9 inhibitors led to reduced Mcl-1 expression as manifested by the lack of Mcl-1 protein after 6 h and 24 h (Fig. 2C). This was confirmed by reverse transcriptase PCR analysis, which showed that inhibition of CDK9 abrogated transcription of Mcl-1 (data not shown).

Figure 2 Specific CDK9 inhibitor affects apoptosis by inhibition of Mcl-1 expression. (A) Mcl-1 western blot on protein extract from splenocytes of arthritic mice undergoing treatment with PC585 and control. Row one and two were run on the same western blot. (B) Immunohistochemical staining for Mcl-1 of knee joint from CIA mice treated daily with 10 mg/kg of PC585 and control. (C) Western blot of protein extract of anti-CD3 activated PBMC treated with PC585 and non-treated control cells (D) Pro-apoptotic potential of 10 μM PC585 as compared to 2 μM staurosporin. (E) PBMC were pre-incubated with PC585 for 3 h and (F) 6 h followed by removal of PC585. One-way ANOVA with Bonferroni’s post-test was used for the statistical evaluation. ****p < 0.0001. Full size image

Taken together, these results show that specific inhibition of CDK9 in several cell types prevents the transcription of Mcl-1 and reduces its expression in peripheral lymphoid organs and disease target tissue.

Inhibiting CDK9 induces apoptosis via a “hit-and-run” mechanism

As described above, treatment with CDK9 inhibitors led to a reduced expression of the anti-apoptotic protein Mcl-1. Therefore, we next examined the apoptotic effects of CDK9 inhibitors by staining PBMCs with Annexin V and 7AAD. Incubation with 10 μM PC585 induced apoptosis in PBMC to the same extent as staurosporine (2 μM) at 12 h (Fig. 2D). The mean percentage of Annexin V+ cells was 20.13% in non-treated cells and 38.28% in CDK9 inhibitor-treated cells (Fig. 2D; p < 0.0001). It is worth noting that the CDK9 inhibitors used in this study have a relatively short half-life (2 h) in plasma; therefore, to investigate whether short-term exposure is sufficient to induce the irreversible changes that lead to apoptosis, we exposed cells to 10 μM PC585 for 3 h or 6 h. The inhibitor was then removed and the number of Annexin V+ cells analysed at various time points thereafter. Compared with that in untreated controls, Annexin V expression by inhibitor-treated cells increased slightly at 3 h (Fig. 2E). The percent of apoptotic cells in the control samples only increased marginally up to 30 h when it reached 15.22%. By contrast, the rate of apoptosis in PBMCs treated for 3 h with PC585 increased; more than 30% of cells were Annexin V+ after 30 h (Fig. 2E). When cells were pre-incubated with PC585 for 6 h, 80.8% remained viable and non-apoptotic up until the point at which the inhibitor was removed. The number of apoptotic cells increased nearly 2-fold during the first 12 h following inhibitor removal and continued to increase thereafter (42.8% of cells were Annexin V+ after 30 h). By contrast, only 17.65% of control cells were Annexin V+ (Fig. 2F).

CDK9 inhibition increases the regulatory T cell population

To further examine the impact of inhibitor treatment on the immune system, we harvested splenocytes at the end of the prophylactic experiment. Flow cytometric analysis showed that the percentage of regulatory T cells (Tregs; CD4+CD25+Foxp3+) in spleens from arthritic mice receiving daily inhibitor treatment was significantly higher than that in control mice, even though the overall CD4+ population remained unchanged (Fig. 3A–C). This increase in the Treg cell population was not observed in mice receiving anti-TNF treatment. Therefore, we next treated healthy animals with 10 mg/kg of PC585 for 7 days to examine whether the CDK9 inhibitors triggered the increase in Treg cells. The results showed that the Treg cell population in the spleens of treated animals was much higher than that in non-treated healthy controls (3D).

Figure 3 CDK9 inhibition increase percentage of splenic regulatory T cells. (A) Representative dot-plots of CD25 and Foxp3 expressing CD4+ T cells. (B) Flow cytometric analysis of Foxp3 and CD25 on T cells in splenocytes from arthritic mice treated daily with PC585 (n = 8), PC579 (n = 8) or Enbrel (n = 9) (Control, n = 10). (C) Percentage of CD4+cells in spleens of mice treated daily with PC585 (n = 8), PC579 (n = 7) or Enbrel (n = 9) (Control, n = 10) (D) CD4+ T cells expressing CD25 and Foxp3 in spleen of 10 healthy treated mice and 10 controls. Box and whiskers graphs show median and min to max values. Differences between two groups were analyzed using Mann-Whitney U test and One-way ANOVA with Bonferroni’s post-test was used for comparison between multiple groups. **p < 0.01. Full size image

CDK 9 treatment prevents down-regulation of Del-1 expression in endothelial cells

Leukocyte migration/extravasation and angiogenesis are important for the pathogenesis of RA; thus, they are interesting targets for drug development. Previous studies suggest that an endogenous leukocyte-endothelial inhibitor (called developmental endothelial locus-1; Del-1) plays a role in adhesion and leukocyte migration16 and apoptosis17, thereby contributing to the development of inflammatory diseases. Thus, we next examined the expression of Del-1 in the joints of CDK9 inhibitor-treated CIA mice and corresponding controls. Del-1 expression in synovial tissue was restricted to endothelial cells, although the protein was also detected in extravascular areas (probably due to diffusion from epithelial cells) (Fig. 4). We also found that Del-1 co-localized with neutrophils present in the joint tissue. There was a significant reduction in the expression of Del-1 protein in inflamed tissues, confirming the finding of Choi et al.16 (Fig. 4B,C). Interestingly, the levels of Del-1 were higher in joint tissues (endothelium and the perivascular areas) from mice treated with the CDK9 inhibitors (PC585 and PC579) than in non-treated animals (Fig. 4E,F). This suggests that treatment with CDK9 inhibitors leads to maintained Del-1 expression in endothelial cells, which might contribute to protection against RA by limiting LFA-1-mediated neutrophil trafficking to inflamed tissues.

Figure 4 CDK 9 inhibitors protect against inflammatory Del-1 down-regulation. Representative fluorescent confocal images of slides stained for Del-1 (B,C,F,G), Ly6G (D,H) and their sequential H&E sections (A,E). Images show knee joints from arthritic DBA/1 mice treated with CDK9 inhibitor PC585 at 10 mg/kg (E–H) and arthritic controls (A–D) at day 47 of CIA. Expression is limited to endothelial cells but can be also seen also in perivascular area (V) and PMNs (arrows). Full size image

Short-term exposure to CDK9 inhibitor leads to transient transcriptional effects in vivo

ID family proteins regulate cell proliferation, differentiation and angiogenesis and their expression is up-regulated in RA patients18. To determine the role of CDK9 in angiogenesis, we harvested blood and spleens from CIA mice at endpoint 1, 3 and 5 h after the final administration of PC585.

ID3, PNUTS and TNFα were used as biomarkers to monitor the in vivo transcriptional effects of CDK9 inhibition. When analysed one hour after dosing, high exposure levels of PC585 correlated with inhibition of biomarker expression to 20–30% of normal levels. All three mRNAs returned to normal levels when analysed 3 or 5 h after dosing, correlating with reduced exposure of PC585. (Fig. 5A,B). Taken together, these data show that even a transient reduction in the levels of pro-inflammatory molecules has a profound downstream impact on the development and progression of arthritis. Interestingly, when we examined the expression of VCAM-1 and ICAM-1 in PBMCs after short-term treatment with PC585 (a 3 h pre-incubation), we observed a striking down-regulation of integrin expression, which persisted after inhibitor wash-out (Fig. 5C,D).