Fig. 7: HDAC3 inhibition alleviates the myeloid differentiation block induced by ERG forced expression in hematopoietic progenitor cells.

A Analysis of myeloid differentiation in human ERG-expressing ER-Hoxb8 cells. GR-1 percentage was measured using flow cytometry after withdrawal of beta-estradiol (E2). n = 10,000 cells were analyzed using flow cytometry for each biological independent sample for n = 4 independent experiments, data is represented as mean ± SEM, Two-way ANOVA, and Sidak’s multiple comparison test. The P-value comparing BB vs. WT-ERG at 48 and 72 h after E2 withdraws are P < 0.0001. B Analysis of myeloid differentiation of ER-Hoxb8 cells expressing human ERG following inhibition of HDAC3 with BRD3308 (left panel) and RGFP966 (right panel). n = 10,000 cells were analyzed for using flow cytometry for each biological independent sample for 7 independent experiments for BRD3008, n = 3 independent experiments for RGFP966. Data is represented as mean ± SEM for both BRD3008 and RGFP966, Two-way ANOVA, and Tukey’s multiple comparison test. The P-value comparing Backbone DMSO vs. Backbone BRD3308 (2.5 µM) is 0.0165. The P-value comparing ERG DMSO vs. ERG BRD3308 (1 µM) is 0.008. The P-value comparing ERG DMSO vs. ERG BRD3308 (2.5 µM) is <0.0001. The P-value comparing Backbone DMSO vs. Backbone RGFP966 (800 nM) is 0.0169. The P-value comparing ERG DMSO vs. ERG RGFP966 (800 nM) is 0.0023. C RNA sequencing in human ERG-expressing ER-Hoxb8 cells treated with either DMSO or 2.5 µM BRD3308. An MA plot illustrates the effect of BRD3308 treatment on gene expression. Differentially expressed genes (P_adj < 0.05) are indicated. D GSEA of genetic and chemical perturbation (C2.CGP) and gene ontology biological process gene sets (C5.BP) following HDAC3 inhibition in human ERG-expressing ER-Hoxb8 cells. Most significant upregulated gene sets are indicated. E Most significant differentially expressed genes following HDAC3 inhibition in human ERG-expressing ER-Hoxb8 cells. Genes in red indicate log2 fold-change >1 and < − 1 and FDR < 0.01. F Gene Ontology on differentially expressed genes presented in E. G Gene expression matrix of myeloid differentiation-related genes in ER-Hoxb8 cells expressing ERG variants. DEG are presented (P_adj < 0.05). CPM—counts per million. NES—Normalized enrichment score. FDR—false-discovery rate. DEG—differentially expressed genes. ns—non-significant, * denotes P ≤ 0.05, ** denotes P ≤ 0.01, *** denotes P ≤ 0.001, **** denotes P ≤ 0.0001.