Page 184 - Haematologica April 2020

S. Xie et al.
reverse the upregulation of H3K27me3, and then syner- gize the antitumor effect of CDKI-73. As expected, specif- ic EZH2i, EPZ6438 or GSK126 exactly reversed the aug- mented H3K27me3 evoked by CDKI-73 (Figure 4A). Furthermore the transcription of all these four examined target genes of H3K27me3 was reactivated in co-treated cells compared with cells treated with CDKI-73 alone (Figure 4B). Next, we investigated whether EZH2i could potentiate the anticancer activity induced by CDKI-73. For this, DLBCL cell lines (Will-1, U2932, Karpas-422, SU- DHL-4, Pfeiffer, and SU-DHL-6) were treated with CDKI- 73 alone, or in combination with EPZ6438/GSK126 for 72 hours. The combination effect was evaluated using the CI
A
value according to the CalcuSyn software.20 The effect is usually considered as synergistic when the CI value is less than 0.8, additive when the CI value is between 0.8-1.2, and antagonistic when the CI value is above 1.2.20 In EZH2 mut cell lines, Pfeiffer (A677G), SU-DHL-4 (Y641S), Karpas-422 (Y641F), and SU-DHL-6 (Y641N), the average CI values were all below 0.8 (Figure 4C), indicating a syn- ergistic interaction between CDKI-73 and EZH2i in EZH2 mut DLBCL cells. We also found that the IC50 for EZH2i was lower in CDK9 knock-down cells compared with par- ent Pfeiffer cells (Online Supplementary Figure S3A). However, in Will-1 and U2932 cells, which harbor wild- type (wt) EZH2, the combination with EZH2i didn’t signif-
B
C
Figure 3. Transcriptional repression contributed to H3K27me3 elevation via CDK9 inhibition. (A) The relative mRNA levels of H3K27-related methyltransferases and demethylases after exposure to 0.1 μM CDKI-73 for 2 and 4 hours (h) or siCDK9. (B-C) The protein levels of H3K27-related methyltransferases and demethylases after exposure to CDKI-73/Flavopiridol, other cyclin-dependent kinases (CDK) inhibitors or siCDK9. All data are representative of at least three independent experi- ments. ***P<0.001, **P<0.01, *P<0.05 compared with the control group.
1026
haematologica | 2020; 105(4)