Combination of EZH2 inhibitor and BET inhibitor for treatment of diffuse intrinsic pontine glioma

Abstract

Background: Diffuse intrinsic pontine glioma is an infiltrative, often high-grade glioma of the brainstem that is not amenable to surgical resection. The current treatment of DIPG by radiation therapy showed dramatically improvement of patient's condition, however, the tumor recurs rapidly. More and more studies are focused on the genetic and epigenetic drivers of DIPGs, which may provide more and more novel therapy target for DIPG. EZH2 has been proved to be a potential therapeutic target for H3K27M-mutant pediatric gliomas recently. Meanwhile, BET family protein is a hot target in many different types of cancers, including DIPG. In this study, we performed the treatment of both EZH2 and BET inhibitor for DIPG cells.

Results: The combination of these two inhibitors exhibited better inhibition of the tumor growth both in vitro and in vivo compared to use the inhibitor individually. This inhibition was performed by blocking the proliferation and promoting the cell apoptosis. Meanwhile, combination treatment of these two inhibitors would also affect the epigenetic markers which were abnormal in the tumors of the certain set of genes.

Conclusion: Thus we provided a novel therapy strategy for clinical treatment of DIPG.

Keywords: BET inhibitor; DIPG; EZH2 inhibitor; Epigenetics; Tumor therapy.

Fig. 1

H3K27M make the NSCs gain the tumor activity. a Western blot showing the expression level of Flag-tagged-H3K27M or Flag-tagged-H3K27wt, HA-tagged-PDGFB, H3K27me3, H3 and GAPDH. b Soft agar colony assay of PDFGB/H3WT or PDGFB/H3K27M NSCs. Data were represented as mean ± SD, n = 3 independent experiments. ***p < 0.001. c Cck-8 kit was used to evaluate the viability of PDFGB/H3WT or PDGFB/H3K27M NSCs. Data were represented as mean ± SD, n = 3 independent experiments. ***p < 0.001

Fig. 2

H3K27M is sufficient to generate DIPG tumors. a Tumor generated by PDFGB/H3WT or PDGFB/H3K27M NSCs was measured in diameters and calculated to volume according to the time point. b Survival curve of the mice injected into the pons with PDFGB/H3WT or PDGFB/H3K27M NSCs (1 × 10⁵). Each group contains 20 mice

Fig. 3

Combination of EZH2 and BET inhibitors reduced the cell proliferation in DIPG cells. a Cell proliferation of different groups as indicated was determined by cell counting. The concentration of EPZ6438 was 3 μM and JQ-1 was 300 nM, the following in vitro assay used the same amount of the inhibitors. **p < 0.01 and ***p < 0.001. b Cck-8 kit was used to evaluate the viability of each group of the cells as indicated. Data were represented as mean ± SD; n = 3 independent experiments. **p < 0.01 and ***p < 0.001

Fig. 4

Combination of EZH2 and BET inhibitors promoted the cell apoptosis in DIPG cells. a The apoptosis of the DIPG cells with different treatment as indicated was determined by using the Annexin-V-FITC & PI Apoptosis Kit and assessed by flow cytometry. n = 3 independent experiments and this panel presented one of these repeats. b Statistic of percentage of the apoptosis cells performed in a. Data showed the Annexin-V and PI double positive cells. Data were represented as mean ± SD; n = 3 independent experiments. **p < 0.01 and ***p < 0.001

Fig. 5

Combination of EZH2 and BET inhibitors epigenetically regulated tumor-surpressor like p16^Inka4. ChIP-q-PCR analysis showing the enrichment of H3K27me3 (left panel) or H3 (right panel) as control over the p16^Ink4a gene (a), Igf2bp2 (b) and HOXA10 (c) in the NSCs with different treatment as indicated. The genes and the primer locations were presented on the top of the panel. Data were represented as mean ± SD; n = 3 independent experiments

Fig. 6

Gene expression level under the condition of combination of EZH2 and BET inhibitors. Q-PCR analysis showing the mRNA level of p16 (a), Igf2bp2 (b) and HOXA10 (c) gene with the different treatment as indicated. Data were represented as mean ± SD; n = 3 independent experiments. *p < 0.05, **p < 0.01 and ^#p > 0.05

Fig. 7

Combination of EZH2 and BET inhibitors improved survival in mice model. Survival curve of the mice injected into the pons with PDFGB/H3WT or PDGFB/H3K27M NSCs (1 × 10⁵). The inhibitor was given by intraperitoneal (ip) injection with the amount of EPZ6438 by 250 mg/kg and JQ-1 by 50 mg/kg. Each group has 20 mice