An inhibitor of BRD4, GNE987, inhibits the growth of glioblastoma cells by targeting C-Myc and S100A16

Abstract

Purpose: Among children, glioblastomas (GBMs) are a relatively common type of brain tumor. BRD4 expression was elevated in GBM and negatively correlated with the prognosis of glioma. We investigated the anti-GBM effects of a novel BRD4 inhibitor GNE987.

Methods: We evaluated the anti-tumor effect of GNE987 in vitro and in vivo by Western blot, CCK8, flow cytometry detection, clone formation, the size of xenografts, and Ki67 immunohistochemical staining, and combined ChIP-seq with RNA-seq techniques to find its anti-tumor mechanism.

Results: In vitro experiments showed that GNE987 significantly degraded BRD4, inhibited the proliferation of GBM cells, blocked the cell cycle, and induced apoptosis. Similarly, in vivo experiments, GNE987 also inhibited GBM growth as seen from the size of xenografts and Ki67 immunohistochemical staining. Based on Western blotting, GNE987 can significantly reduce the protein level of C-Myc; meanwhile, we combined ChIP-seq with RNA-seq techniques to confirm that GNE987 downregulated the transcription of S100A16 by disturbing H3K27Ac. Furthermore, we validated that S100A16 is indispensable in GBM growth.

Conclusion: GNE987 may be effective against GBM that targets C-Myc expression and influences S100A16 transcription through downregulation of BRD4.

Keywords: BRD4; C-Myc; GNE987; Glioblastoma; H3K27Ac; S100A16.

Fig. 1

BRD4 is overexpressed in patients with glioma. a Expression of BRD4 mRNA in tumors and normal tissues (source: GEPIA2: http://gepia2.cancer-pku.cn). b Survival curves of high and low BRD4 expression in 222 and 58 patients with primary and recurrent glioma, respectively (http://www.cgga.org.cn/, source: CGGA database, mRNAseq-325 Dataset). c Survival curve of high(red) or low(blue) BRD4 of patients with glioma (http://r2.amc.nl). The median is the cut-off point for high or low BRD4 expression

Fig. 2

GNE987 damages the viability of GBM cells and inhibits cell proliferation. a Schematic diagram of bifunctional PROTAC molecules. b BET and VHL protein levels in GBM cells. c The IC50 value of GNE987 at 3 days in GBM cell lines. d The IC50 value of GNE987 at 5 days in GBM cell lines. e The IC50 value of GNE987 at 7 days in GBM cell lines. f Various concentrations of GNE987, JQ1, ARV825 and dBET1 affect cell viability for 3 days in U87 cells. The IC50 value of GNE987, JQ1, ARV825 and dBET1 in U87 cell line. g EdU staining of GBM cells treated with DMSO or GNE987 for 3 days; White bar, 100 μm. h The bar graph shows the percentage of positive cells of EdU. i Clone-forming ability of DMSO group and GNE987 groups. j Bar graph of the colony-forming ability of GBM cells treated with DMSO or GNE987. (Data were presented with mean ± SD of three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 3

GNE987 induces cell apoptosis, arrests cell cycle and decreases BRD4 protein levels. a The apoptosis rate of GNE987 was increased through Annexin-V/PI staining. b GNE987 induced the emergence of cleaved-PARP in GBM cells. c The cell cycle assay showed that the G2 phase increased after GNE987 treatment. d cyclinB and cdc2 mRNA levels after DMSO or GNE987 treatment. e GNE987 strongly decreased BET protein levels in GBM cells (Data were presented with mean ± SD of three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 4

GNE987's anti-tumor activity is closely related to VHL expression. a Identification of VHL overexpression or knockdown by Western blotting in GBM cells. b Identification of VHL overexpression or knockdown by RT-qPCR in GBM cells. c Effects of VHL overexpression or knockdown on cell viability after treatment of GBM cells with GNE987. d Schematic diagram of the targeted degradation of BRD4 by GNE987; GNE987 binds both BRD4 and a VHL E3-ubiquitin ligase complex. Formation of the trimeric complex results in the transfer of ubiquitin to BRD4. e Western blotting of BRD in U87 and LN229 cells treated with GNE987, MG132 and their combination. (Data were presented with mean ± SD of three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 5

GNE987 inhibits tumor growth of GBM in vivo experiments. a Photo of tumor-bearing mice. b Tumor photos of the vehicle and GNE987 groups. c Growth curve of tumor volume; the formula for tumor volume is: (length × width²)/2. d Tumor weights in the vehicle group (blue) and GNE987 group (red). e Ki67 expression in the vehicle group and GNE987 group; brown-yellow denotes positive expression and purple-blue denotes the cell nucleus (Scale bar is 50 µm). f Body weight growth curve of tumor-bearing mice in the vehicle and GNE987 groups. g H&E staining of the liver and kidney of tumor-bearing mice in the vehicle and GNE987 groups (Scale bar is 50 µm). h GNE987 was injected every 2 days from the 3rd day, and the samples were harvested 48 h after the last dose of GNE987 on the 21st day for Western blot detection. Compared to the vehicle group (blue), BRD4 expression in tumors of the GNE987 group (red) was significantly decreased. i The levels of BRD4 protein in the vehicle group (blue) and GNE987 group (red). (n.s., not significant, **P < 0.01, ***P < 0.001, n = 6)

Fig. 6

Mechanism of anti-tumor effect of GNE987. a–d GNE987 reduces C-Myc expression in U87 cells, LN229 cells, U251 cells and A172 cells. e Volcano plot shows the differential genes from the GNE987 group or DMSO group (|log2FoldChange|> 1, adjusted p < 0.05). f mRNA levels of tumor-related genes, WNT5A, ZMYND8, BCL2L1, CAV1, TBX2, STEAP3, POU2F2, EPHA2, KCNJ15, EPSTI1, PALMD, and S100A16, were significantly downregulated after GNE987 treatment. g S100A16 expression level closely correlates with survival probability in patients with primary glioma (Source: CGGA database, mRNAseq-325 dataset). h IGV view software displays gene tracks of H3K27Ac ChIP-seq occupancy at S100A16 gene loci from the GNE987 group or DMSO group; the x-axis shows the genomic location and the y-axis reflects H3K27Ac enrichment. i RT-qPCR analysis of the knockdown efficiency of shRNA of the GNE987-sensitive gene, S100A16. j Effects of S100A16 knockdown on proliferation. k, l Effects of S100A16 knockdown on clone formation (Data were presented with mean ± SD of three independent experiments, *P < 0.05, **P < 0.01, ***P < 0.001)