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S. Xie et al.
icantly improve the anti-proliferative activity of CDKI-73 in this scheme, with CI values higher than 0.8.
As EZH2i takes longer to exhibit weak proliferation inhibition in EZH2 wt cells in the in vitro assay,14 we tried another combination scheme in a panel of DLBCL cell lines. The combination groups were pretreated with a fixed dose of EPZ6438/GSK126 for 72 hours, which was adequate to inhibit H3K27me3, and had no obvious pro- liferation inhibition (the inhibitory rate was controlled below 20%). The mean CI values of treatment with CDKI-73 and EZH2i were calculated as reported29 and were also observed to be less than 0.8 in EZH2 mut DLBCL cells (Figure 4D). Even more gratifying was the observation that at least an additional effect was exhibited in SU-DHL-8, Will-1, Will-2, HT, SU-DHL-5, and U2932 cell lines which harbor wt EZH2 (Figure 4D), indicating that EZH2i can promote the anti-proliferative potency of CDKI-73 against DLBCL cell lines with the combination schedule regardless of the EZH2 phenotype. When the pretreatment time with EZH2i was extended to seven days (the inhibitory rate of EZH2i was still controlled below 20 %), the dose-response curves of all the co-treat- ment groups were apparently shifted to the left compared with the curves treated with CDKI-73 alone in EZH2 wt DLBCL cells, also indicating a synergistic effect (Figure 4E).
Thereafter, we evaluated whether EZH2i could enhance the anti-tumor ability of CDKI-73 in vivo. In Pfeiffer xenografts, CDKI-73 and EPZ6438 alone suppressed tumor growth compared to the vehicle, yielding a T/C rate of 67.69 % and 46.07 %, respectively. Combinatorial CDKI-73 and EPZ6438 therapy more potently inhibited tumor growth compared to treatment with CDKI-73, EPZ6438, and vehicle alone, yielding a T/C rate of 25.69 %, indicating 74.31 % inhibition of tumor growth (Figure 4F). Moreover, there was no obvious weight loss under the combined regimen as compared to the single treat- ments (Figure 4F). Similar results were observed in another model, SU-DHL-6 (Figure 4G). The superior in vivo anti- tumor activity of the combination therapy was also asso- ciated with a greater decline in H3K27me3, which was upregulated by CDKI-73 alone (Figure 4H).
Combination therapy synergistically induces apoptosis and DNA damage
As mentioned above, CDKI-73 alone induced apoptosis and DNA DSB in DLBCL, we next determined the com- bined effects on apoptosis and DNA damage. As expect- ed, combination treatment remarkably increased the rate of apoptosis compared to each agent alone (Figure 5A). This was associated with a synergistic increase in the PARP cleavage, and a decline of anti-apoptotic proteins including MCL1, XIAP, and BCL-XL (Figure 5B). Similar results were obtained in the in vivo assay (Figure 5C). In addition, accumulating data suggest that EZH2 also partic- ipates in modulating the DNA damage response.30 Therefore, we hypothesized that co-induction of DNA damage may be another mechanism contributing to the synergistic anti-tumor effect of the combination. As expected, both comet assay and γ-H2AX analysis indicat- ed that CDKI-73 or EPZ6438/GSK126 alone could induce DNA damage both in Pfeiffer and SU-DHL-4 cells. However, the combined therapy resulted in a dramatic increase in γH2AX accumulation, and the frequent appear- ance and expanding volume of comet tails (Figure 5D-E).
CDK9 and EZH2 inhibition also exhibited synergistic antitumor activity in multiple solid tumors
We further studied whether the combination therapy was also effective against solid tumors. At first, we found that CDKI-73/Flavopiridol elevated H3K27me3 in various kinds of cancer cells, including MCF-7 (breast cancer), MDA-MB-453 (breast cancer), SGC-7901 (gastric cancer), and SW620 (colorectal cancer) (Online Supplementary Figure S1A). This finding might partially explain the observation that the inhibition of CDK9 is insufficient to inhibit tumor growth in clinical trials.31 And as expected, EZH2i down- regulated the augmented H3K27me3 levels induced by CDK9 inhibition and synergized with CDK9i in these solid tumor models in vitro and in vivo (Figure 6 and Online Supplementary Figure S3B). We also found that depletion of both CDK9 and EZH2 in MCF-7 cells delayed cells prolif- eration, as compared with siCDK9 alone (Online Supplementary Figure S3C). And the deletion of CDK9 also diminished PRC2, JMJD3, and UTX, leading to the observed increase of H3K27me3 in these cells (Figure 6E). All these data strongly suggested that CDK9 and EZH2 inhibition also exhibit synergistic interaction in multiple solid tumors.
Discussion
The frequent observance of resistance and relapse to first line therapy by DLBCL underscores the need for novel approaches to treat this disease. CDKI-73, as an example of a highly efficacious CDK9 inhibitor, has dis- played potent anti-tumor activity in vitro and in vivo.15-17 Previous studies also have shown that this agent had little toxicity on normal T and B cells while exhibiting potent efficiency against CLL,15 suggesting it to be a far superior therapeutic agent for clinics. Here, we demonstrated that it retains efficacy in DLBCL in vitro and in vivo. CDKI-73 exhibited a broad ability to trigger apoptosis and dramat- ically repress the proliferation of 15 DLBCL cell lines, associated with lost MCL1 and XIAP proteins, owing to CDK9 inhibition. All these data provide a basis for moving forward with the clinical evaluation of CDKI-73 in DLBCL.
Deregulation of CDK has been used to develop antitu- mor target for more than 20 years. Although CDK inhibitors have always shown tremendous preclinical activity, their clinical application was limited by modest efficacy along with various side effects.31 Here, in our study, we provided a view of ‘CDK9-histone modification crosstalk’, which expands the function of CDK9. We found that CDK9 inhibition specifically elevated the trimethylation of H3K27, which may restrain the antitu- mor potency against DLBCL and other solid tumor types. H3K27me3 is a transcription-repressing epigenetic modifi- cation that has been causally associated with the initiation and development of multiple tumor types, especially DLBCL. Recently, oncogenic heterozygous mutations were identified in the SET domain of EZH2 in DLBCL patients, which could elevate H3K27me3, and drive cell proliferation.14 Moreover, selective EZH2i show preclinical and clinical efficiency against DLBCL by reducing the level of H3K27me3. Positive interim efficacy data from an ongoing phase II clinical trial of EPZ6438, as a single-agent treatment for patients with relapsed or refractory DLBCL, were released at the International Conference on Malignant Lymphoma (Lugano, Switzerland). An objec-
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