Acute myeloid leukemia (AML) remains incurable for most patients, particularly in the elderly, and novel therapeutic approaches are needed. Genes are often epigenetically silenced in AML through the recruitment of histone deacetylases (HDACs) and the development of agents that inhibit HDACs (HDACi) are a novel treatment approach to AML. However, AML represents a spectrum of mutations and chromosomal abnormalities and it would be of benefit to target these therapies based on epigenetic profiles and responses. One HDAC inhibitor, Vorinostat (Zolinza™), is currently being used in phase i/ii clinical trials as a therapeutic strategy in multiple myeloma with evidence that combinational therapies provide an extended disease free survival; this could be an attractive option for AML. In order to find a successful combination, a fuller characterisation of Vorinostat induced histone modifications is required.
In this study, we used a combination of ChIP-seq and microarray approaches to identify epigenetically silenced genes pathways in AML cells that could be restored by treatment with the HDACi Vorinostat.
OCI-AML3 cells were treated with Vorinostat 1 μM or control conditions for 24 hours. Changes in gene expression were measured using Affymetrix Human Genome U133 Plus 2.0 and/or Leukemia AmpliChip arrays. The ChIP-seq was done on DNA extracted from the same cells and time points as for the expression study. The chromatin was cross-linked and the protein-DNA interactions at H3K9Ac were elucidated by chromatin immunoprecipitation, with anti-acetylated H3K9 histone and Pol II antibodies. Pol II was used to identify regions of actively transcribed DNA, and high throughput sequencing using the Ion Torrent PGM (Life Technologies).
After treatment with Vorinostat, expression studies showed that 180 genes were up-regulated and 441 genes were down-regulated (FC 4, p<0.05). The ChIP-seq data identified DNA sequences that were associated with de-acetylated histones following Vorinostat treatment. Bioinformatic analysis identified regions that were also associated with actively transcribed regions of DNA. Pathway analysis identified that the principle pathways induced by Vorinostat were TGFβ signalling and glycosaminoglycan degradation. Gene set enrichment analysis showed that Vorinostat down-regulated the integrin mediated signalling and p53 binding, and up-regulated stem cell differentiation, blood vessel morphogenesis and response to fibroblast growth factor stimulus.
This study would indicate that Vorinostat is an effective regulator of gene expression in AML cells and that the integrated approach can identify specific epigenetic targets of Vorinostat and could lead to the development of tailored AML therapies.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.