Page 180 - Haematologica April 2020

S. Xie et al.
cation is generated by the polycomb repressive complex 2 (PRC2), composed of the SET domain-containing histone methyltransferase EZH2, and accessory proteins EED, SUZ12.10 However, the methylation of H3K27 can be removed by the histone demethylases UTX/KMD6A and JMJD3/KDM6B.11 Given the critical role of the H3K27me in gene expression, it is not surprising that this chromatin modification plays a role in malignancies, such as lym- phoma, breast and esophageal cancer.12 Accumulating studies imply that H3K27me3 is closely engaged in the development of DLBCL, especially in the germinal center B-cell subtype,13 reducing H3K27me3 level exhibits signif- icant anti-proliferation activity against DLBCL.14 Further, highly selective EZH2 inhibitors (EZH2i), such as GSK126, EPZ6438, and CPI-1205 are currently undergoing clinical evaluation against DLBCL, and show clinical efficiency by reducing the level of H3K27me3. However, clinical bene- fits of EZH2i remain unsatisfactory against other solid tumors.
CDKI-73 (also named LS-007, QHRD107), a novel phase I clinical stage CDK inhibitor in China, mainly tar- gets CDK9 with sub-nanomolar biochemical potency and represses expression of MCL1 in multiple models of can- cer, such as chronic lymphocytic leukemia (CLL), ovarian cancer, and acute leukemia.15-17 CDKI-73 is highly cytotox- ic to primary leukemia cells from CLL patients and showed >500-fold selectivity for primary leukemia cells over normal B-lymphocytes,15 making it an attractive can- didate for clinical development. In this study, firstly, we assessed the in vitro and in vivo activity of CDKI-73 against DLBCL. However, during the research, we found that CDKI-73 significantly elevated H3K27me3 level owing to CDK9 inhibition, accompanied with more pronounced transcriptional down-regulation of H3K27me3-targeted genes. Therefore, we hypothesized that the combined treatment of CDK9 inhibitors (CDK9i) and EZH2i would show greater antitumor activity than treatment with either inhibitor alone. We found a particularly potent syn- ergy of this combination against both DLBCL and other solid tumors in vitro and in vivo, revealing that this potential therapeutic combination could be evaluated in patients.
Methods
Cell culture
All cells were purchased from ATCC (Manassas, VA, USA) or DSMZ (Brunswick, Germany) and maintained following the sup- pliers’ instructions.
Cell proliferation, apoptosis, colony formation assay, comet assay, quantitative Real-Time PCR analysis, chemicals and anti- bodies were described in the Online Supplementary Methods.
Mass Spectrometry (MS) of histone modifications
Histones of SU-DHL-4 cells from SILAC (stable isotope labeling by amino acids) were manipulated as previously described.18 Then the peptides were separated by EASY–nLC 1000 HPLC system and analyzed using Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). MS data were analyzed by Mascot software against an in-house human histone sequence database (83 sequences; 13,870 residues) generated from the UniProt database (updated on 01/27/2015). Peptides containing modifications were manually quantified using the Qual Browser (Thermo Fisher Scientific).
ChIP-Seq
H3K27me3 ChIP-seq data with spike-in was generated by Active Motif’s Epigenetic Services team (Active Motif, CA, USA). Karpas-422 cells were harvested as previously described.19 Cell pellets were snap-frozen in liquid nitrogen for 10 min, stored at - 80°C and then shipped to Active Motif on dry ice for the H3K27me3 ChIP-seq assay.
Animal experiments
All experiments were performed according to the institutional ethical guidelines on animal care and approved by the Institute of Animal Care and Use Committee at the Shanghai Institute of Materia Medica (No. 2016-04-DJ-21). Pfeiffer, SU-DHL-6 were subcutaneously injected into the right flank of SCID mice and SW620 were subcutaneously injected into the right flank of nude mice at 5 × 106 cells/mouse (six mice per group). Tumor bearing mice were randomized into groups and started dosing when average tumor volume reached 100–200 mm3. CDKI-73 (0.5% HPMC+ddH2O) and EPZ6438 (0.5% CMCNa+1% Tween80+ddH2O) were given orally daily. For combination treatment, drugs were given concurrently. Tumors and body weight were measured twice a week, and the relative tumor vol- ume (RTV) was calculated with the formula: RTV =(1⁄2×length×width2 of day n)/(1⁄2×length×width2 of day 0). The therapeutic effect of the compounds was expressed as the vol- ume ratio of treatment to control: T/C (%)=100 %×(mean RTVtreated)/(mean RTVvehicle).
Statistical analysis
Combination index (CI) values were calculated using CalcuSyn software or using the IC50 ratio obtained with CDK9i plus EZH2i compared to that obtained with CDK9i alone as previously described.20 Student’s t-test was applied for statistical comparison using GraphPad Prism. Unless otherwise indicated, the results are expressed as the mean ± standard deviation (SD) from at least three independent experiments. Differences were considered to be statistically significant at P<0.05.
Results
CDKI-73 inhibits the growth of DLBCL cells in vitro and in vivo
The activity of CDKI-73 against human DLBCL was evaluated using a panel of established DLBCL cell lines. CDKI-73 remarkably restrained the proliferation of all tested DLBCL cell lines with the mean IC50 values of 87±40 nM, which was slightly lower than that of Flavopiridol, a reported potent CDK9 inhibitor (125±40 nM) (Figure 1A).
Considering that CDKI-73 has been reported to be a potent CDK9 inhibitor,15-17 we investigated its influence on CDK9 activity in DLBCL cell lines. As expected, signif- icant suppression of phosphorylation of RNA Pol II (ser2) was also found in SU-DHL-4, Pfeiffer and WSU-DLCL-2 cells treated with CDKI-73 in a dose-dependent manner (Figure 1B). As CDK9 plays a vital role in regulating the transcription of numerous anti-apoptotic proteins, its inhibition-induced cell death has been confirmed to be through apoptosis.7 Therefore, we examined the effect of CDKI-73 on apoptosis. As expected, CDKI-73 led to apoptosis in three DLBCL cell lines, WSU-DLCL2, SU- DHL-4, and Pfeiffer, in a concentration- and time-depen- dent manner (Figure 1C). Simultaneously, PARP cleavage increased consistently with the occurrence of apoptosis
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