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A.G. Gilmartin et al.
methylated cytosines in the promoters of HBG1 and HBG2 that become hypomethylated in response to DNMT1 gene silencing in EPC.31,38 We have confirmed the hypomethyla- tion of a number of the same methylcytosines in response to DNMT1 inhibition by bisulfite sequencing, including the -53 bp methylcytosine for HBG1 (Online Supplementary Figure S1). Consistent with the findings in the global methylcytosine assay, methylation of the -53 bp HBG1/ HBG2 cytosine residues was decreased for cells treated
treated cells (Figure 1C). Increases in globin gene expression correlated inversely with DNA methylation levels; at the cellular IC50 of the -53 bp methylcytosine assay, 0.33 mM GSK3482364 treatment caused more than a 2-fold increase in HBG1/HBG2 mRNA.
In order to further characterize the effect of GSK3482364 on cultured EPC, HbF protein expression was measured by enzyme-linked immunosorbent assay (ELISA). Day 7 EPC cultured in the presence of GSK3482364 for 5 additional days increased HbF 2-fold at 0.56 mM, and up to a maxi- mum of 300% of vehicle-treated levels (Figure 2A). In com- parison, decitabine treatment increased HbF 2-fold at 0.04 mM, and up to a maximum of 250% of vehicle. Notably, decitabine treatment reproducibly generated a bell-shaped curve response in the ELISA assay, consistent with cytotox- icity at high concentrations which was confirmed in paral- lel cell growth assays (Figure 2B). At high concentrations
ABC
Figure 2. Effect of GSK3482364 and decitabine on fetal hemoglobin, cell growth, and caspase activation in erythroid progenitor cells. Erythroid progenitor cells (EPC) were treated for 5 days with GSK3482364 (A) or decitabine (B) and then assayed for change in fetal hemoglobin (HbF) by enzyme-linked immunosorbent assay (green) or cell growth (blue); data represent the mean +/- standard deviation for n=20 assays. Concentrations resulting in 200% HbF and 50% growth inhibition are indicated with light blue and gray lines, respectively. (C) Caspase-Glo assay measure caspase 3/7 activation in EPC after 3 days treatment with GSK3482364 (black) or decitabine (red).
Figure 3. Effects of GSK3482364 and decitabine on fetal hemoglobin levels and reticulocyte differentiation after 18 days in cell culture. CD34+ bone marrow hematopoietic stem cells were cultured for 18 days in the presence of GSK3482364 or decitabine, with three phases of media exchange promoting erythroid dif- ferentiation into reticulocytes. Day 18 cells were stained with Syto16 (nucleic acids) and anti-fetal hemoglobin (anti-HbF) (APC) antibody. Erythroid progenitor cells are Syto16high events that retain nuclei, and reticulocytes are Syto16low events that lack nuclei.
of 0.33 mM (Figure 1C).
In order to determine the effect of decreased HBG1 and HBG2 promoter methylation on gene expression, mRNA from treated cells was measured by reverse transcritase qPCR (RT-qPCR) with an assay that detects both HBG1 and HBG2 mRNA (99% genetic identity). After 5 days of treatment, GSK3482364 caused a dose-dependent increase in HBG1/HBG2, raising levels 5.3-fold compared to vehicle
with GSK3482364 with an IC 50
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haematologica | 2021; 106(7)