Figure 2.
Global epigenetic changes associated with gene expression reprogramming induced by the combination of OR21 and EPZ-6438. MT-1 and MT-2 cells were treated with OR21, EPZ, or OR21 + EPZ (Comb) for 4 days (MT-1: 0.5 μM OR21 and/or 2.5 μM EPZ; MT-2: 0.1 μM OR21 and/or 0.5 μM EPZ). (A) The LINE-1 promoter methylation level was determined using bisulfite pyrosequencing. Data are expressed as the means of 3 independent experiments ± SD. (B) Amounts of H3K27me3 were determined by immunoblotting. (C) Scatter plot showing upregulated (≥twofold) and downregulated genes (≤0.5-fold) (red and blue dots, respectively) compared with the control in global gene expression analysis using microarrays. (D) Venn diagrams showing the number of upregulated genes. (E) β-values in the TSS200 or TSS1500 region in MT-2 cells determined by global methylation analysis using GSE136189. The gray plots show β-values of all genes (n = 125 523); the red plots show β-values of OR21-up genes (n = 2536); the green plots show β-values of EPZ-up genes (n = 786); the blue plots show β-values of com-up genes (n = 180). OR21-up genes are genes upregulated by OR21 monotherapy in MT-2 cells in panel D. EPZ-up genes are genes upregulated by EPZ monotherapy in MT-2 cells in panel D. Com-up genes are genes commonly upregulated by both OR21 and EPZ in MT-2 cells in panel D. Differences between all and each group were tested using Steel test. ∗∗P < .01. (F) H3K27me3 levels (log2) around the promoter region in MT-2 cells obtained from public data (GSE71450). The gray (n = 202 178), red (n = 1759), green (n = 711), and blue (n = 186) plots indicate the same gene groups shown in panel E. Differences between all and each group were tested using Steel test. ∗∗P < .01. (G) Histogram showing the frequency of β-values under each condition (control, OR21, EPZ, Comb) in all GBs in MT-1 cells. (H) Box plots showing the β-values under each condition (control, OR21, EPZ, Comb). Left panels: β-values of hypermethylated GBs (β-value in the control condition >0.6, upper panel) and hypomethylated GBs (β-value in the control condition <0.3, lower panel). Right panels: β-values of hypermethylated (upper panel) or hypomethylated (lower panel) GBs within TSS200 and TSS1500 region in MT-1 cells. (I) Venn diagrams showing the number of upregulated genes increased by combination treatment compared with single treatment; 158 genes were common to MT-1 and MT-2 cells. Twenty-nine genes were expressed in normal T cells and downregulated in cells from patients with ATL compared with normal T cells (analyzed using GSE33615) were selected as candidate genes involved in the synergistic anti-ATL effects. (J) β-values in the TSS200 or TSS1500 region of 29 genes after OR21 and/or EPZ treatment in MT-1 cells. Differences between control and each condition were tested using Steel test. ∗∗P < .01. n.s., not significant; SD, standard deviation.

Global epigenetic changes associated with gene expression reprogramming induced by the combination of OR21 and EPZ-6438. MT-1 and MT-2 cells were treated with OR21, EPZ, or OR21 + EPZ (Comb) for 4 days (MT-1: 0.5 μM OR21 and/or 2.5 μM EPZ; MT-2: 0.1 μM OR21 and/or 0.5 μM EPZ). (A) The LINE-1 promoter methylation level was determined using bisulfite pyrosequencing. Data are expressed as the means of 3 independent experiments ± SD. (B) Amounts of H3K27me3 were determined by immunoblotting. (C) Scatter plot showing upregulated (≥twofold) and downregulated genes (≤0.5-fold) (red and blue dots, respectively) compared with the control in global gene expression analysis using microarrays. (D) Venn diagrams showing the number of upregulated genes. (E) β-values in the TSS200 or TSS1500 region in MT-2 cells determined by global methylation analysis using GSE136189. The gray plots show β-values of all genes (n = 125 523); the red plots show β-values of OR21-up genes (n = 2536); the green plots show β-values of EPZ-up genes (n = 786); the blue plots show β-values of com-up genes (n = 180). OR21-up genes are genes upregulated by OR21 monotherapy in MT-2 cells in panel D. EPZ-up genes are genes upregulated by EPZ monotherapy in MT-2 cells in panel D. Com-up genes are genes commonly upregulated by both OR21 and EPZ in MT-2 cells in panel D. Differences between all and each group were tested using Steel test. ∗∗P < .01. (F) H3K27me3 levels (log2) around the promoter region in MT-2 cells obtained from public data (GSE71450). The gray (n = 202 178), red (n = 1759), green (n = 711), and blue (n = 186) plots indicate the same gene groups shown in panel E. Differences between all and each group were tested using Steel test. ∗∗P < .01. (G) Histogram showing the frequency of β-values under each condition (control, OR21, EPZ, Comb) in all GBs in MT-1 cells. (H) Box plots showing the β-values under each condition (control, OR21, EPZ, Comb). Left panels: β-values of hypermethylated GBs (β-value in the control condition >0.6, upper panel) and hypomethylated GBs (β-value in the control condition <0.3, lower panel). Right panels: β-values of hypermethylated (upper panel) or hypomethylated (lower panel) GBs within TSS200 and TSS1500 region in MT-1 cells. (I) Venn diagrams showing the number of upregulated genes increased by combination treatment compared with single treatment; 158 genes were common to MT-1 and MT-2 cells. Twenty-nine genes were expressed in normal T cells and downregulated in cells from patients with ATL compared with normal T cells (analyzed using GSE33615) were selected as candidate genes involved in the synergistic anti-ATL effects. (J) β-values in the TSS200 or TSS1500 region of 29 genes after OR21 and/or EPZ treatment in MT-1 cells. Differences between control and each condition were tested using Steel test. ∗∗P < .01. n.s., not significant; SD, standard deviation.

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