Category: Clinical Pharmacology
Purpose: DNA hypomethylating agents (DHA) like 5-Azacytidine (5AC) and decitabine (DC) are among the first line therapeutic agents in the treatment of myelodysplastic syndrome (MDS). Despite their similarity in chemical structure and mechanism of action, the cellular and clinical response to 5AC and DC are quite different and not solely dependent on DNA methylation reversal. We recently demonstrated that 5AC increases the enzymatic activity of the histone deacetylase (HDAC) SIRT6 and induces a genome-wide decrease in H3K9 acetylation in primary leukemia cells. In this study, we are investigating the hypothesis that DC induces genome-wide H3K9 acetylation decreases that are comparable to its congener 5AC.
Methods: Bone marrow mononuclear cells from six de-identified naive acute myeloid leukemia (AML) patients were cultured with 500 nM DC or DMSO (control) for 72 h followed by ChIP-Seq analysis using a ChIP-validated acetylated-H3K9 (H3K9ac) antibody. Chip-Seq libraries were prepared from treated and control cells using SMARTer ThruPLEX DNA-seq kit (Takara Bio, USA) according to the manufacturer’s instructions. Libraries were purified and size-selected with AMPure XP beads at 1:1 (v/v) ratio. All libraries were pooled prior to sequencing on an Illumina HiSeq 1500. The dual-indexed single-read Rapid Run was performed with 1x120 cycles at 5 pM final concentration of the library pool. Sequence reads were aligned to the reference human genome (hg38) using Bowtie v18.104.22.168 in end-to-end alignment mode. Reads with average phred quality < 10, PCR duplicates and those aligning to blacklisted regions of the genome were filtered out using Samtools v1.4.1. H3K9ac enriched (peak) regions were identified using diffReps v1.55.4 software using input samples for background correction. Statistical significance of differential peak counts was assessed using negative binomial test using all individuals as replicates. The differential peak lists with padj< 0.2 and fold change > +/- 1.5 were used in Ingenuity Pathway Analysis software to identify significant pathways.
Results: Data pooling from the six patients showed significant acetylation changes at 182 loci after DC treatment. DC modulated H3K9 acetylation at different chromosomal regions including promoters, coding exons, introns and distal intergenic regions. Both increases and decreases in H3K9 acetylation were observed where 99/182 genes showed acetylation decrease and 83/182 showed acetylation increase. 19/182 genes demonstrated H3K9 acetylation changes in their promoter region and all of them were associated with decrease in acetylation. DC modulated H3K9 acetylation of genes associated with signaling pathways relevant to leukemia such as focal adhesion kinase (FAK) and CXCR4 signaling. Comparing the effect of DC on H3K9 acetylation to our previous findings with 5AC shows that 5AC induces more H3K9 acetylation changes compared to DC (845 vs 182 genes). In contrast to an approximately equal increase and decrease in H3K9 acetylation of genes with DC, 5AC induces mainly decreases in H3K9 acetylation. Among the genes that showed H3K9 acetylation changes, only 10 were common between both drugs (Table 1).
Conclusion: Previously, we demonstrated that both 5AC and DC increase SIRT6 enzymatic activity in vitro. In this study, we observed that DC modulates H3K9 acetylation with no significant trend in acetylation decrease. Comparing the effect of DC with 5AC on H3K9 acetylation changes shows that 5AC is more potent and shows a significant trend in acetylation decrease. This study highlights a major difference between 5AC and DC that may contribute to their therapeutic efficacy.
Hetty Carraway– Cleveland, Ohio
Sridhar Malkaram– Institute, West Virginia
Aymen Shatnawi– Charleston, West Virginia
Aladin Siddig– Associate Professor, University of Charleston School of Pharmacy, Charleston, West Virginia
Jun Fan– Huntington, West Virginia
James Denvir– Huntington, West Virginia
Donald Primerano– Huntington, West Virginia