Combinatorial Effect of Pharmacological Inhibitors of Lsd-1 and DNMT on γ-Globin Synthesis in Cultured Baboon Bone Marrow Erythroid Progenitors

Abstract 249

Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). New therapies to increase HbF levels in patients are urgently needed. Previous studies from our laboratory have shown that decitabine, an inhibitor of DNA methyltransferase (DNMT), increases HbF to therapeutic levels in non-human experimental primates and SCD patients, thus demonstrating that drugs targeting repressive epigenetic modifications can be effective HbF-inducing agents. Both DNMT1 and Lysine Specific Demethylase-1, (LSD-1), a mono- and dimethyl- histone H3 K4 demethylating enzyme, are components of the DRED multiprotein complex, a repressor of γ-globin expression (Cui et al Mol Cell Biol 31:3298–3311, 2011). Experiments were performed to test the effect of the pharmacological LSD-1 inhibitor, tranylcypromine (TC), alone and in combination with the DNA methyltransferase inhibitor decitabine (DAC), on γ-globin expression. Baboon bone marrow (BM) CD34+ erythroid progenitors were isolated from BM aspirates of normal baboons and grown in a co-culture system with the AFT024 murine fetal liver cell line in Iscove's media containing 30% fetal bovine serum, 200ng/ml SCF, 2U/ml epo, and 1μM dexamethasone. TC (0.5 to 5.0μM) was added to cultures on d7 and d10, while decitabine (0.1μM) was added on d7. On d14 erythroid cells were purified by immunomagnetic column chromatograpy using a baboon-specific anti-red blood cell antibody (BD Bioscience). Effects on globin expression were measured by biosynthetic radiolabelling of nascent globin chain synthesis in the presence of [³H] leucine followed by HPLC separation of individual globin chains. DNA was isolated and effect of the drugs on DNA methylation of the γ-globin promoter determined by bisulfite sequence analysis. Effects on erythroid differentiation were investigated by examination of Wright's stained cytospin preparations. In three independent cultures TC induced dose-dependent increases in γ-globin chain synthesis (γ/γ+β). In addition TC and DAC in combination increased γ-globin chain synthesis in an additive manner (see Table).

Bisulfite sequence analysis showed that increased γ-globin chain synthesis in TC-treated cultures was not associated with increased DNA hypomethylaton of the γ-globin promoter. Examination of Wright's stained cytospin preparations of d14 cultures showed increased numbers of basophilic and polychromatic erythroblasts in cultures exposed to TC compared to untreated controls suggesting that TC reduced the rate of erythroid differentiation. In contrast, numbers of basophilic and polychromatic cells were reduced and orthochromatic cells were increased in cultures treated with TC and DAC compared to TC alone. We conclude that TC increased γ-globin chain synthesis in a dose-dependent manner in baboon erythroid progenitor cell cultures while TC and DAC, in combination, produced additive effects on γ-globin chain synthesis with minimal effects on erythroid differentiation. These results suggest that combinatorial use of epigenetic modifiers may be superior to single agents and support further studies to determine whether TC and other LSD-1 pharmacological inhibitors can effectively be used in combination with DNMT inhibition to increase HbF levels in vivo.