Investigation of Two Combination HbF Induction Regimens, RN-1 and Hydroxyurea Versus RN-1 and Decitabine, in a Humanized Sickle Cell Mouse Model

Sickle cell disease (SCD) results from the substitution of glutamic acid for valine at the sixth amino acid of β-globin protein, leading to the formation of the abnormal hemoglobin S (HbS). Following deoxygenation in red blood cells (RBCs), HbS forms polymers causing the RBCs to become deformed and adherent leading to vaso-occlusive events resulting in splenic infarction, kidney failure, stroke, painful crises, chronic anemia, inflammation and decreased survival. Hydroxyurea, the only drug currently FDA approved for SCD, is not effective in some patients and, therefore, new combination regimens are  needed. RN-1, a new drug targeting the histone demethylase LSD-1, is a more effective inducer of γ-globin expression in sickle cell mice and HbF in baboons (P. anubis).  Decitabine(DAC), a DNMT1 inhibitor, is well known to induce high levels of HbF in non-human primates and SCD patients and is currently in clinical trials. We investigated the effect of two combination regimens: RN-1 in combination with hydroxyurea vs. RN-1 in combination with DAC. The knock-in humanized sickle cell mice (B6;129-Hba^tm1(HBA)TowHbb^{tm2(HBG1,HBB*)Tow/}J) were treated with 1) DMSO (vehicle control), 2) RN-1 (5mg/kg), 3) DAC(0.25mg/kg) and 4) RN-1(5mg/kg) + DAC(0.5mg/kg) for 10 days.  Analysis of F-retics and γ-globin mRNA performed on d10 showed F-retics were increased two fold  (p=0.01) in mice treated with RN-1(5mg/kg) or DAC(0.25mg/kg) compared to DMSO-treated.  γ-globin mRNA levels (γ/γ+β) were increased three fold in RN-1(5mg/kg) or DAC(0.25mg/kg) compared to DMSO.  The combination of RN-1(5mg/kg) and DAC(0.25mg/kg) did  increase F-retics and mRNA levels compared to mice treated with RN-1 alone(p=0.02).  Also, the RN-1 and DAC combination led to neutropenia and thrombocytopenia. We next examined the effect of RN-1(5mg/kg) and HU (100mg/kg) in combination. Knock-in humanized sickle cell mice (B6;129-Hbatm1(HBA)Tow Hbbtm2(HBG1,HBB)Tow/J) were treated with 1) DMSO (vehicle) 2) RN-1(5mg/kg)  and 3) RN-1(5mg/kg) + HU (100mg/kg). While RN-1 in combination with HU did not significantly increase F-cells or F-retics, there was a significant decrease in spleen weight [(DMSO: 1.17±0..06g; RN1:0.71± 0.1g; RN1+HU: 0.46±0.05g)(p=0.004:p=0.02)] and percentage of  sickle cells in a peripheral blood smear.[(DMSO: 8.4 ±1.3%; RN-1:4.1± 0.2%; RN1+HU: 1.57±0.6%)(p=0.006:p-0.014)].  We conclude that further studies to determine the effect of RN-1 in combination with HU or DAC in the sickle cell mouse model and cultures from sickle cell CD34+ cells are needed to evaluate its potential use in the treatment of sickle cell disease.   These results suggest that RN-1 in combination with hydroxyurea may be a useful therapeutic regimen.