Increased Fetal Hemoglobin Levels Following Decitabine Treatment of Normal Baboons (P. anubis) are Achieved by Transcriptional Activation of the γ-Globin Gene.
Authors: Akpan Imo , Banzon Virryan , Ibanez Vinzon , Vaitkus Kestis , DeSimone Joseph , Lavelle Donald 
Published: October 2019

Abstract 4066

Poster Board III-1001

Increased fetal hemoglobin (HbF) levels can alleviate symptoms associated with sickle cell disease (SCD) and β-thalassemia. The development of pharmacological inducers of HbF is a desirable goal expected to impact the clinical course of these diseases. Pharmacological inhibitors of DNA methyltransferase, 5-azacytidine and decitabine (2'-deoxy-5-azacytidine), induce high levels of HbF in vivo in the baboon model and in patients with SCD and β-thalassemia Bisulfite sequence analysis has shown that increased HbF is associated with a decrease in DNA methylation of 5 CpG residues within the 5' γ-globin gene region in both baboons and SCD patients treated with decitabine. Chromatin immunoprecipitation experiments have also shown increased association of RNA polymerase II, histone acetylation, and histone H3 (lys4) trimethyl with the γ-globin gene in bone marrow erythroid cells following decitabine treatment of experimental baboons. These results are consistent with the hypothesis that γ-globin expression is repressed by DNA methylation and that DNMT inhibitors increase HbF by reducing DNA methylation of the γ-globin promoter, thus allowing increased transcription of the γ-globin gene. The mechanism of action of these drugs remains controversial, however. A recent model based on experiments conducted in a primary human erythroid cell culture system suggested that DNMT inhibitors increase HbF by post-transcriptional or translational mechanisms mediated by stress signal transduction pathways (Mabaera et al, Exp Hematol 36:1057, 2008). Therefore we have conducted experiments in the baboon model to determine to what extent transcriptional versus translational and post-transcriptional mechanisms are responsible for increased HbF following decitabine treatment in vivo. Three normal baboons were treated with decitabine (0.5mg/kg/d) for ten days. The baboons were not phlebotomized to test whether the ability of decitabine to increase HbF levels required erythropoietic stress. Levels of γ-globin chain synthesis (γ/γ+β ratio) in pre-treatment and post-treatment bone marrow (BM) aspirates were determined by biosynthetic radiolabelling with [3H] leucine followed by separation of globin chains by HPLC. The effect of decitabine treatment on the level of γ-globin transcripts was determined by real time PCR using the ΔΔCT method. Results showed that the γ/γ+β chain ratios in pre-treatment BM aspirates of 3 normal baboons were 0.01, 0.01, and 0.1 and increased to 0.51, 0.24, and 0.49, respectively, in post-treatment BM. Decitabine treatment increased the level of γ-globin transcripts 55, 45, and 5.9 fold in these three baboons (see table).

Animal Pre-treatment γ-globin chain synthesis (γ/γ+β) Post-treatment γ-globin chain synthesis (γ/γ+β) Fold change γ-globin chain synthesis Fold change γ-globin mRNA 7482 0.01 0.51 51 55 7470 0.01 0.24 24 45 7472 0.10 0.49 4.9 5.9

These results show that decitabine treatment induces similar fold increases in γ-globin chain synthesis and γ-globin transcripts. Therefore we conclude that decitabine does not increase HbF levels through a translational mechanism. Previous results from our laboratory showed that association of RNA polymerase II and histone H3 (lys4) trimethyl with the γ-globin gene were increased following decitabine treatment to levels similar to those associated with the β-globin gene. Therefore we conclude that decitabine increases HbF mainly by increasing γ-globin gene transcription rather than through post-transcriptional or translational effects. Because our experiments were conducted in normal unbled baboons, we also conclude that the ability of decitabine to increase HbF levels does not require erythropoietic stress.

Disclosures:

No relevant conflicts of interest to declare.