Effect of the LSD-1 Inhibitor RN-1 on g-globin and Global gene expression during Erythroid Differentiation in Baboons
Increased levels of Fetal Hemoglobin reduce the severity of symptoms of patients with sickle cell disease and decrease the risk of death.  An affordable, small molecule drug that stimulates HbF expression in vivo would be ideally suited to treat the large numbers of SCD patients that exist worldwide.  Our previous work showed that administration of the LSD1 (KDM1A) inhibitor RN-1 to normal baboons increased Fetal Hemoglobin (HbF) and was tolerated over a prolonged treatment period.  HbF elevations were associated with changes in epigenetic modifications that included increased levels of H3K4 di-and tri-methyl lysine at the g-globin promoter.  While dramatic effects of the loss of LSD1 on hematopoietic differentiation have been observed in murine LSD1 gene deletion and silencing models, the effect of pharmacological inhibition of LSD1 in vivo on hematopoietic differentiation is unknown. The goal of these experiments was to investigate the in vivo mechanism of action of the LSD1 inhibitor RN-1 by 1) determining its effect on g-globin expression in highly purified subpopulations of bone marrow erythroid cells enriched for varying stages of erythroid differentiation isolated directly from baboons treated with RN-1 and also by 2) investigating the effect of RN1 on the global transcriptome in a highly purified population of proerythroblasts.   Our results show that RN-1 administered to baboons targets an early event during erythroid differentiation responsible for ?-globin repression and increases the expression of a limited number of genes including genes involved in erythroid differentiation such as GATA2, GFi-1B, and LYN. Overall design: The effect of RN-1 on global gene expression was investigated by RNAseq analysis of CD105+CD34-CD117+bRBC+ FACS-purified BM cells (proerythroblasts) of untreated control baboons (n=2) and baboons treated with both short-term (0.25mg/kg/d; 3d; n=2) and long-term schedules (0.25mg/kg/d; 5d/wk; >265d; n=2).   The untreated control and short-term treatment samples were obtained from the same two individual baboons, PA897 and PA8548. The long-term treatment samples were obtained from two different baboons (PA8695, LTT1, PA8698 LTT2). ).  Gene expression differences between these long-term treatment samples were compared directly with the two control samples from PA8697 (CTRL 1) and PA8548 (CTRL 2).