Molecular mechanism of gene expression and cell apoptosis mediated by hdac inhibitor valproic acid in acute myeloid leukemia cells

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Valproic acid (VPA), an approved antiseizure drug, restores gene expression and induces cell differentiation and apoptosis. However, the molecular mechanism remains largely unknown. In t(8;21) AML, the AML1/ETO fusion protein promotes leukemogenesis by recruiting HDAC to silence target genes important for hematopoiesis, thereby representing a paradigm for HDAC-mediated leukemogenesis. In the current study, we reported that exposure to VPA resulted in a significant HDAC inhibition, global histone hyperacetylation and distinct patterns of transcription-permissive changes on histones H3 and H4 lysine residues in AML1/ETO-positive cells. Further, VPA treatment significantly decreased HDAC 1 and 2 protein levels in nuclei, suggesting this as an important mechanism for inhibition of the HDAC activity. AML1/ETO protein level was also decreased 48 hr after VPA treatment. Importantly, following exposure to VPA, chromatin immunoprecipitation identified profound changes on the promoter of IL-3, an AML1/ETO target gene, with hyperacetylation of histones H3 and H4, release of AML1/ETO and HDAC1, recruitment of HAT1 and RNA polymerase II, which led to gene transcriptional activation. Band depletion proved that AML1/ETO and HDAC1 were dissociated globally from their target genes’ promoter, and this global dissociation was further verified by the confocal images showing that nuclear proteins AML1/ETO and HDAC1 were re-located from nuclei to cytoplasm upon VPA treatment. These effects results in significant antileukemic activity, supported by cell cycle arrest via upregulation of the cyclin dependent kinase (CDK) inhibitor p21, cell partial differentiation and apoptosis. The apoptosis was mostly induced through mitochondrial-dependent pathways with terminal activation of caspase 3 and 9. Notably, other myeloid leukemia cell lines (NB-4, ML-1 and THP-1) harboring distinct fusion genes were also analyzed for comparison. Interestingly, while global and lysine-specific histone hyperacetylation, induction of p21 and cell cycle arrest occurred in all cell lines, significant apoptosis was observed only in NB4 cells expressing PML/RAR-alpha, like AML1/ETO, directly recruiting HDAC activity, but not in ML-1 and THP-1 cells harboring MLL rearrangements. These data shed a bright light on the molecular mechanism of gene regulation and cell apoptosis mediated by valproic acid and suggested that VPA is a promising drug targeting AML, particularly the AML with the aberrant recruitment of HDAC activity.