Characterization of HDACI OSU42 as a Novel Histone Deacetylase Inhibitor in AML Cell Lines.

Histone acetylation plays a key role in the regulation of gene expression. Histone hyperacetylation is associated with chromatin opening and gene transcription, while histone hypoacetylation is associated with chromatin condensation and gene silencing. Abnormal histone hypoacetylation mediated by aberrant activity of histone deacetylases (HDACs) has been found to be associated with silencing of tumor suppressor and growth inhibitory genes in malignant cells. HDAC inhibitors (HDACIs) can relieve HDAC-mediated gene silencing and thereby induce normal patterns of cell cycle, differentiation and apoptosis in malignant cells. HDACI OSU 42 is a novel hydroxamate tethered phenylbutyrate derivative that was designed and synthesized at our institution, and exhibited IC50s at submicromolar level, compared with millimolar level for other members of this classes of HDACIs such as valproic acid (VPA). We characterized the activity of this compound in acute myeloid leukemia (AML) cells. It is known that the fusion proteins AML1/ETO and PML / RAR alpha that characterized t(8;21) and t(15;17) AML silence target genes through recruitment of HDACs to their promoter regions. Therefore we utilized AML1/ETO-positive Kasumi-1 and PML/RARA-positive NB4 cells to test the activity of HDACI OSU 42 and used THP-1 cells, characterized by AF9/MLL fusion gene, as a control. We hypothesized that by virtue of the fusion genes, Kasumi-1 and NB4 are more susceptible to HDACI treatment. IC50s for proliferation inhibition in Kasumi-1 cells treated with HDACI OSU42 were 71.8±14.3nM for 24hr and 31.3± 0.4nM for 48hr, significantly lower than VPA (2.0mM for 24hr, 0.9mM for 48hr). The IC50s for NB4 were 237.7±6.5nM for 24hr and 119±6.4nM for 48hr. As a contrast, IC50 for THP-1 was 507.3±68.3nM for 48hr. HDACI OSU42 inhibited 80% of total HDAC activity at 125nM in both Kasumi-1 and NB4; 30nM HDACI OSU42 induced hyperacetylation of histone H3 and H4. Apoptosis analysis showed that nearly 60% more of Kasumi-1 and NB4 underwent apoptosis after treated with 1μM of HDACI OSU42 for 24hr, compared with their untreated control. On the other hand, the same treatment only induced 15% more of THP-1 undergoing apoptosis. Apoptotic effect of HDACI OSU42 was mediated by activation of caspase 9 and caspase 3. Cell cycle analysis demonstrated that treatment of Kasumi-1 and NB4 with 150nM of HDACI OSU 42 inhibited cell cycle progression and arrested 20% to 30% more cells at S phase or G2/M phase, whereas this treatment had not effect on cell cycle progression of THP-1. This was consistent with the up-regulated expression of p21 at both transcription level and protein level. Q-PCR data suggested that Kasumi-1 and NB4 treated with HDACI OSU42 expressed ~10 folds of p21 higher than untreated cells. Chromatin immunoprecipitation assay revealed 10 to 50 folds increase in acetylation level of histone H3 and H4 associated with p21 promoter. Kasumi-1 and NB4 cells also show differentiation ability (increase in CD14 and CD 13 expression by flow cytometry) when treated with 30nM of HDACI OSU42, whereas THP-1 remained undifferentiated. These results support the activity of HDACI OSU42 as a new potent HDACI in AML.