Monomethylfumarate (MMF), a Novel Inducer Of Fetal Hemoglobin In Human Erythroid Progenitors and Retinal Pigment Epithelial Cells
Authors: Makala Levi , Promsote Wanwisa , Li Biaoru , Ganapathy Vadivel , Pamela Martin , Pace Betty S. 
Published: October 2019

Abstract
Retinopathy is a common complication of sickle cell disease (SCD) leading to blindness in 10% of patients; however there is a critical lack of effective strategies for prevention and treatment. Pharmacologic induction of fetal hemoglobin (HbF) remains the best approach to reduce morbidity and treat complications of SCD. Hydroxyurea (HU) is the only FDA-approved drug for this purpose; however a significant number of SCD patients do not respond to therapy. Moreover, little is known regarding the efficacy of HU or other HbF-inducing drugs in the retina. The pleiotropic actions of fumaric acid esters in a broad spectrum of tissues, high tolerability and oral bioavailability, and recent FDA approval of Tecfidera (BG-12; Biogen Idec) for use in multiple sclerosis, make these agents attractive for rapid extrapolation to clinical trials in SCD. Pharmacokinetic studies indicate that following oral intake of dimethyl fumarate (DMF; BG-12), it is rapidly hydrolyzed and converted into MMF. Therefore, we investigated the ability of DMF and MMF to induce γ-globin expression in KU812 and human primary erythroid cells (Table 1). Increased γ-globin promoter activity was observed by dual luciferase assay in KU812 stable lines with maximal 4-fold and 10-fold induction at 200μM DMF and 1000μM MMF respectively. These findings were confirmed in human primary erythroid cells generated in liquid culture from adult CD34+ stem cells; 4- to 8-fold induction of the γ/β-globin mRNA levels was observed. Subsequent FACS analysis demonstrated a 32-fold increase in HbF-positive cells by MMF, a value significantly higher than that produced by HU. Western blot confirmed an increase in HbF protein with both compounds.
Table 1 Fetal hemoglobin induction by DMF and MMF in human erythroid progenitors. Methods DMF (200μM)(Fold change) MMF (100μM)(Fold change) HU (100μM)(Fold change) KU812 reporter lines γF/βR luciferase activity Dual luciferase assay 10.6±0.3* 4.18±0.5* ND Primary Erythroid Cells γ/β mRNA RT-qPCR 4.13±0.1* 7.9±1.5* 6.6±1.8* %HbF positive cells FACS 25.9±0.4* 30.4±0.5* 15.9±0.3* HbF protein levels Western blot 2.1 3.2 2.5 γF/βR, γFirefly/βRenilla luciferase activity; *p<0.05; ND, not done
Although sickle retinopathy is thought to be largely a vascular disease, there is clinical evidence of early, non-vascular involvement of photoreceptor cell dysfunction. Given their isolation from a vascular supply and high oxygen demand, photoreceptors depend solely upon retinal pigmented epithelial (RPE) cells for metabolic support including oxygen delivery by hemoglobin synthesized locally in RPE cells. Hence, RPE dysfunction produced by sickle hemoglobin polymerization might compromise RPE and visual function. We demonstrated the ability of DMF (BG-12) to induce HbF synthesis in human erythroid progenitors. Subsequently, we studied whether the same occurs in RPE, with specific interest in MMF, the main bioactive ingredient. MMF (1000μM) induced γ-globin mRNA 4 to 6-fold in the human RPE cell line, ARPE-19; immunofluorescence analysis revealed a corresponding 30% increase (by ImageJ analysis) in HbF protein expression. These data were confirmed in primary RPE cells isolated from HbAA and HbSS expressing Townes humanized knock-in SCD mouse eyes. As observed in human primary erythroid cells, the induction of human γ-globin mRNA and HbF protein by MMF was significantly higher (p<0.01) than that by HU. We also observed increased retinal γ-globin expression and HbF production in the eyes of HbAA and HbSS mice (n=6/group) sacrificed 24 h post- intravitreal delivery of 1000μM MMF, extending our findings to in vivo conditions. Our work demonstrates for the first time γ-globin activation and HbF production in RPE cells. The physiological importance of hemoglobin production in RPE is yet to be determined; however it is possible that sickle hemoglobin expression in RPE may contribute to retinal dysfunction. The finding that DMF and MMF induce HbF is novel and supports the possible re-purposing of BG-12 for SCD treatment. Additionally, we previously identified a transporter protein, SLC5A8, which facilitates the entry of these agents across the blood-retinal barrier into retina (Ananth et al. Invest Ophthalmol Vis Sci. 2013; 54:1592-602) supporting the applicability of BG-12 to the prevention and treatment of systemic and retinal complications of SCD.

Disclosures:
No relevant conflicts of interest to declare.