Decitabine Targets the Erythroid Progenitor/Precursor Subpopulations for the γ-Globin Gene Demethylation in Baboon.

The DNA demethylating drug (5–Az–2′–deoxycytidine) elevates fetal hemoglobin (HbF) to therapeutic levels in patients with sickle cell disease. To further investigate the mechanism of action of this drug and the role of DNA methylation in γ–globin gene silencing, we have analyzed the level of methylation of five CpG sites in the 5′ region of the γ–globin gene in highly purified subpopulations of cells representing different stages of erythroid differentiation from baboon (P. Anubis) using bisulfite sequencing. Baboons were treated with three different doses of decitabine (0.52, 0.26, 0.17mg/kg/day) for 10 consecutive days and pre-treatment and post-treatment adult bone marrow (ABM) were analyzed. Fetal liver (FL;n=2) and ABM cells were purified by depletion of the erythroblast subpopulation using an anti-RBC antibody (Pharmingen) in combination with immunomagnetic columns (Miltenyi) and FACS purification of CD34+CD36−, CD34+CD36+ and CD34− CD36+ subpopulations. Clonal analysis of sorted subpopulations demonstrated enrichment of CFUe in the CD34−CD36+ subpopulation, BFUe in the CD34+CD36+ subpopulation and both BFUe and CFU-GM in the CD34+CD36− subpopulation, thus confirming that these sorted subpopulations were enriched for the cells representing different stages of erythroid differentiation. A progressive decrease in the level of γ-globin gene methylation, as the degree of differentiation increased, was observed in the subpopulations purified from FL (Table 1). In pre-treatment ABM the level of γ-globin gene methylation was significantly (P<0.05) reduced in erythroblasts when compared to the CD34+CD36− subpopulation. Decitabine treatment reduced the level of γ-globin gene methylation in a dose dependant manner to a similar extent in each subpopulation except the CD34+CD36− subpopulation that exhibited only minor reduction in the γ-globin gene methylation. These results demonstrate that decitabine treatment demethylates the γ-globin gene primarily in late erythroid progenitors (CD34+CD36+) and erythroid precursors (CD34−CD36+). Methylation of the γ-globin gene is not significantly reduced in the more primitive CD34+CD36- subpopulation after decitabine treatment. The greater sensitivity of the progenitor/precursor subpopulations may be due to increased cell cycle kinetics. The increased levels of DNA methytransferase in CD34+ cells may also contribute to the relative insensitivity of the most primitive subpopulation to decitabine. This analysis identifies the late progenitor/precursor subpopulation as the target subpopulation most sensitive to DNA demethylation by decitabine while the early progenitor/stem cell subpopulation is insensitive to the drug. Table 1: DNA methylation (%) of the γ-globin gene in purified cells of fetal liver and pre- and post-decitabine treated adult bone marrow samples Samples CD34+CD36− CD34+CD36+ CD34−CD36+ Erythroblasts Note: Decitabine doses for PA6973=0.52mg; PA6974=0.26mg; PA7002=0.17mg/kg/day Fetal liver (n=2) 95.4±3.96 66.25±4.17 27.3±1.41 3.7±5.23 ABM-pretreated (n=3) 96.23±0.48 87.21±5.96 79.59±13.42 74.87±8.87 BM-post treated PA6973 85.40 41.30 31.10 37.80 BM-post treated PA6974 94.83 61.90 50.79 52.46 BM-post treated PA7002 92.31 71.93 66.00 58.00