Selective Inhibitors of Histone Deacetylases 1 and 2 Synergize with Azacitidine in Acute Myeloid Leukemia
Fig 6
Gene expression profiling identifies differentially regulated genes.
(A–B) MV-4-11 cells were treated with vehicle DMSO, azacitidine at 1 μM, ACY-1035 at 1 μM and the combination for 48 hours. Total RNA was prepared and analyzed by Affymetrix GeneChip PrimeView™ Human Gene Expression Array. (A) Genes that are up-regulated (red) or down-regulated (blue) by single or combo treatment relative to vehicle. (B) Genes exhibiting additive or synergistic response in the combination treatment relative to each single agent treatment were clustered into 8 categories and represented in a heatmap: A, genes unaffected by single agent treatments and up-regulated in the combination; B, genes unaffected by single agent treatments and down-regulated in the combination; C, genes up-regulated by azacitidine, unaffected by ACY-1035 and up-regulated in the combination more than azacitidine alone; D, genes down-regulated by azacitidine, unaffected by ACY-1035 and down-regulated by the combination more than azacitidine alone; E, genes unaffected by azacitidine, up-regulated by ACY-1035 and up-regulated by the combination more than ACY-1035 alone; F, genes unaffected by azacitidine, down-regulated by ACY-1035 and down-regulated by the combination more than ACY-1035 alone; G, genes up-regulated by azacitidine, up-regulated by ACY-1035 and up-regulated by the combination more than either single agent alone; H, genes down-regulated by azacitidine, down-regulated by ACY-1035 and down-regulated by the combination more than either single agent alone. (C) MV-4-11 and HL-60 cells were treated for 48 hours with the indicated doses of ACY-957, ACY-1035, azacitidine or the combinations. qPCR analysis was performed for the following genes from category G: CDKN1A, CDKN1C, GATA2 and HES1. ACTB was used as the normalizing control. Combinations of ACY-957 plus azacitidine or ACY-1035 plus azacitidine further increased the expression of these genes compared to single agent treatments. Mean ± SD, n = 3. Data shows one representative of 3 independent experiments. (D–E) MV-4-11 cells were transduced with GATA2 or GFP overexpression vectors and stable cell lines derived. (D) Overexpression of GATA2 was confirmed by qPCR. Mean ± SD, n = 3. (E) Stable cells were subjected to proliferation assay in 96-well plates for 6 days. Cell viability was determined at the indicated time points and viable cell numbers plotted. GATA2 overexpressing cells grew much slower than GFP expressing cells. Mean ± SD, n = 3. Data shows one representative of 3 independent experiments. (F) GSEA was performed on the array data using the ‘C3 motif TFT: transcription factor genes’ gene set database. Genes containing GATA binding sites within their promoter regions were enriched in the combination of ACY-1035 and azacitidine compared to azacitidine alone. Significant enrichment is illustrated by the positive running enrichment score (ES) marked by the green line, normalized enrichment score (NES) = 1.6, and false discovery rate (FDR) = 0.03.