Reticulocyte mitochondrial retention increases reactive oxygen species and oxygen consumption in mouse models of sickle cell disease and phlebotomy-induced anemia.

Sickle cell disease (SCD) is caused by a mutation of the ß-globin gene that results in the production of hemoglobin S (HbS). People with SCD experience anemia, severe acute pain episodes, persistent chronic pain, multiorgan damage, and a reduced life span. The pathophysiology of SCD caused by the polymerization of HbS on deoxygenation results in red cell deformability and the generation of reactive oxygen species (ROS). These 2 factors lead to red cell fragility and hemolysis. Reticulocytosis is an independent predictor of disease morbidity and mortality in SCD. We previously established that humans and mice with SCD exhibit abnormal mitochondrial retention in erythrocytes increasing ROS-associated hemolysis. Here, we investigated the hypothesis that mitochondrial retention and increased ROS are a consequence of stress erythropoiesis. Our results show clearly that stress erythropoiesis in phlebotomized, anemic AA mice results in mitochondrial retention and increased ROS in reticulocytes. We observed that elevated mitochondrial retention in reticulocytes also alters oxygen consumption and potentially contributes to increased HbS polymerization and red blood cell hemolysis. Therefore, these events occurring due to stress erythropoiesis contribute significantly to the pathology of SCD and suggest new therapeutic targets.

Combinatorial targeting of epigenome-modifying enzymes with decitabine and RN-1 synergistically increases HbF.

Increased fetal hemoglobin (HbF) levels reduce the symptoms of sickle cell disease (SCD) and increase the lifespan of patients. Because curative strategies for bone marrow transplantation and gene therapy technologies remain unavailable to a large number of patients, the development of a safe and effective pharmacological therapy that increases HbF offers the greatest potential for disease intervention. Although hydroxyurea increases HbF, a substantial proportion of patients fail to demonstrate an adequate response. Pharmacological inhibitors of DNA methyltransferase (DNMT1) and lysine-specific demethylase 1A (LSD1), 2 epigenome-modifying enzymes associated with the multiprotein corepressor complex recruited to the repressed γ-globin gene, are powerful in vivo inducers of HbF. The hematological side effects of these inhibitors limit feasible clinical exposures. We evaluated whether administering these drugs in combination could reduce the dose and/or time of exposure to any single agent to minimize adverse effects, while achieving additive or synergistic increases in HbF. The DNMT1 inhibitor decitabine (0.5 mg/kg per day) and the LSD1 inhibitor RN-1 (0.25 mg/kg per day) administered in combination 2 days per week produced synergistic increases in F-cells, F-reticulocytes, and γ-globin messenger RNA in healthy baboons. Large increases in HbF and F-cells were observed in healthy, nonanemic, and anemic (phlebotomized) baboons. Combinatorial therapy targeting epigenome-modifying enzymes could thus be a useful strategy for producing larger increases in HbF to modify the clinical course of SCD.