Figure 7.
HDAC inhibition increases Pol2 engagement and reverses silencing of a select set of microRNA genes from cluster II and III which are anticorrelated with the expression of key driver genes in CLL. (A) Volcano plots showing the differential expression of small RNA genes assayed by small RNA seq in 10 CLL samples before and after exposure to 0.4 μM, abexinostat for 24 hours. Of a total of 10 microRNA genes that were significantly upregulated (adjust P < .05 and log2 ratio > 1), 1 (miR-1248 in pink) belonged to cluster I, 5 (miR-210, miR-95, miR92b, miR-320d, miR-1296 in blue) to cluster II, and 4 (miR182-183-96 cluster, miR9-3 in red) to cluster III. One gene (miR-1303) from cluster III was downregulated (adjust P < .05 and log2 ratio < −1). (B) Genome browser tracks showing the change in HDAC1 (red), BRD4 (turquoise), H3K9Ac (green), H3K27Ac (olive), RNA Pol II (violet), and ATAC seq peaks (brown) in a representative CLL sample out of 3 at baseline and after exposure to 0.4 μM, abexinostat for 6 hours, HDAC1 (yellow), BRD4 (mint), H3K9Ac (light blue), H3K27Ac (light green), RNA Pol II (mauve), and ATAC seq peaks (orange) at miR-210 and miR-182 to 183 to 96 shown as a representative genes from cluster II that bound HDAC1 with H3K9Ac, open ATAC signatures, and RNA Pol2 and cluster III that bound HDAC1 alone. The x-axis of each track shows genomic position, and the y-axis shows the intensity of the ChIP signal (rpm/bp). (C) Effect of HDAC inhibition on RNA Pol II occupancy at promoters. Gene plots showing changes in log2 read counts per million (log2 CPM) for RNA Pol II occupancy from −5 to -1KBkb of the PP centered around the TSS to +3 to +6 KB of the GB at the miR-210 and miR-182 to 183 to 96 cluster before (blue) and after exposure to 0.4 μM abexinostat for 24 hours (red). (D) Expression levels of miR-210 from cluster II and miR-182 to 183 and 96 from cluster III in 10 CLL samples exposed to abexinostat for 24 hours. (E) The Pearson correlation between the top microRNA induced (miR-182, miR-183, and miR-96) in both primary CLL samples and CLL bearing Eμ-TCL1 mice were evaluated for negative correlation against their predicted target driver genes relevant to CLL (IKZF3, BTK, BCL2, and SYK). We show the Pearson scatter plots showing a negative correlation between miR-182, miR-183, and miR-96 and IKZF3 (P = .001 for miR-182, 0.004 for miR-183 and 0.004 for miR-96), BTK (P = .003 for miR-182, 0.01 for miR-183 and 0.003 for miR-96), BCL2 (P = .044 for miR-182, 0.048 for miR-183 and 0.06 for miR-96), and SYK (P = .013 for miR-182, 0.019 for miR-183 and 0.054 for miR-96). Rho values and slopes are shown. GB, gene body; PP, proximal promoter.

HDAC inhibition increases Pol2 engagement and reverses silencing of a select set of microRNA genes from cluster II and III which are anticorrelated with the expression of key driver genes in CLL. (A) Volcano plots showing the differential expression of small RNA genes assayed by small RNA seq in 10 CLL samples before and after exposure to 0.4 μM, abexinostat for 24 hours. Of a total of 10 microRNA genes that were significantly upregulated (adjust P < .05 and log2 ratio > 1), 1 (miR-1248 in pink) belonged to cluster I, 5 (miR-210, miR-95, miR92b, miR-320d, miR-1296 in blue) to cluster II, and 4 (miR182-183-96 cluster, miR9-3 in red) to cluster III. One gene (miR-1303) from cluster III was downregulated (adjust P < .05 and log2 ratio < −1). (B) Genome browser tracks showing the change in HDAC1 (red), BRD4 (turquoise), H3K9Ac (green), H3K27Ac (olive), RNA Pol II (violet), and ATAC seq peaks (brown) in a representative CLL sample out of 3 at baseline and after exposure to 0.4 μM, abexinostat for 6 hours, HDAC1 (yellow), BRD4 (mint), H3K9Ac (light blue), H3K27Ac (light green), RNA Pol II (mauve), and ATAC seq peaks (orange) at miR-210 and miR-182 to 183 to 96 shown as a representative genes from cluster II that bound HDAC1 with H3K9Ac, open ATAC signatures, and RNA Pol2 and cluster III that bound HDAC1 alone. The x-axis of each track shows genomic position, and the y-axis shows the intensity of the ChIP signal (rpm/bp). (C) Effect of HDAC inhibition on RNA Pol II occupancy at promoters. Gene plots showing changes in log2 read counts per million (log2 CPM) for RNA Pol II occupancy from −5 to -1KBkb of the PP centered around the TSS to +3 to +6 KB of the GB at the miR-210 and miR-182 to 183 to 96 cluster before (blue) and after exposure to 0.4 μM abexinostat for 24 hours (red). (D) Expression levels of miR-210 from cluster II and miR-182 to 183 and 96 from cluster III in 10 CLL samples exposed to abexinostat for 24 hours. (E) The Pearson correlation between the top microRNA induced (miR-182, miR-183, and miR-96) in both primary CLL samples and CLL bearing Eμ-TCL1 mice were evaluated for negative correlation against their predicted target driver genes relevant to CLL (IKZF3, BTK, BCL2, and SYK). We show the Pearson scatter plots showing a negative correlation between miR-182, miR-183, and miR-96 and IKZF3 (P = .001 for miR-182, 0.004 for miR-183 and 0.004 for miR-96), BTK (P = .003 for miR-182, 0.01 for miR-183 and 0.003 for miR-96), BCL2 (P = .044 for miR-182, 0.048 for miR-183 and 0.06 for miR-96), and SYK (P = .013 for miR-182, 0.019 for miR-183 and 0.054 for miR-96). Rho values and slopes are shown. GB, gene body; PP, proximal promoter.

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