Multiple Oral Therapeutics Suppress Repressors (LSD-1, HDACs, and BCL11A) of Gamma Globin Gene Expression

Induction of gamma globin expression to certain target levels reduces clinical severity in sickle cell disease, globin imbalance, and anemia in the beta thalassemias. However, achieving high level expression in diverse patients with variable baseline HbF levels is clinically challenging. To determine whether different therapeutics have complimentary molecular actions that could be combined for higher efficacy, we evaluated a panel of oral fetal globin-inducing therapeutics in clinical use or trials for effects on transcriptional suppressors of HBG, including components of the NuRD complex, LSD1 and HDACs 1-3, and the downstream repressor BCL11A, in erythroid progenitors cultured from sickle cell and beta thalassemia patients. Using chromatin immunoprecipitation assays, immunoblot, and qRT-PCR, we investigated two pan-HDAC inhibitors, MS-275 and SB939, a short chain fatty acid derivative without global histone deacetylation effects, sodium dimethylbutyrate (ST20), and PB-04, a PK-enhancing drug identified through high throughput screening (HTS), which has a long-standing benign clinical safety profile. These therapeutics induce fetal globin expression by 1.5 to 12-fold in different individuals’ progentiors over untreated controls and are active in 60-90% of patients’ progenitors. BCL11A mRNA and protein were suppressed by 3-7 fold and 5-10 fold, respectively, over untreated cells from the same subject by all agents tested. PB-04 treatment decreased LSD1 mRNA by 5-fold. MS-275 and ST20 suppressed KLF-1 mRNA by 3- and 2.5-fold respectively. ChIP analyses demonstrated that PB-04 reduced LSD1 and HDAC-3 occupancy on the gamma globin gene promoter by 4-fold and 3-fold, respectively, coincident with gamma globin induction; ST20 decreased HDAC-2 and LSD1 binding by 6- and 2-fold respectively. Further, histone marks associated with gene activation were enriched at the gamma-globin promoter by treatment with PB-04, including acetylated Histone H3K9 (AcH3K9), the target of HDAC3, and dimethylated H3K4 (H3K4me2), a target of LSD1. Collectively, these results identify oral clinical stage therapeutics which disrupt two major mechanisms of transcriptional repression. The findings provide a basis for targeting therapies in individuals and combining pharmacologic agents which induce HbF expression through complimentary molecular mechanisms to provide high level induction.