Effect of the NAMPT Activator P7C3-A20 on γ-Globin Expression in Baboon CD34+ Erythroid Cell Cultures
Abstract Increased levels of Fetal Hemoglobin (HbF) reduce the symptoms of sickle cell disease (SCD) and lengthen the life span of patients. New, more effective pharmacological agents that can be safely administered long term to increase HbF levels in SCD patients are highly sought. Expression of the γ-globin gene in adult erythroid cells is normally repressed by the recruitment of multi-protein co-repressor complexes to the γ-globin promoter by sequence-specific DNA binding proteins including BCL11A, LRF1 and TR2/TR4. Enzymes contained within these co-repressor complexes, such as DNMT1, LSD1, G9A, and HDACs, modify the chromatin surrounding the γ-globin promoter by catalyzing repressive epigenetic modifications to both histones and DNA. Small molecule pharmacological inhibitors of these enzymes are potent inducers of HbF in various in cell culture and animal models and in SCD patients, but the use of these drugs in patients has been hindered by their dose-dependent effects on hematopoietic differentiation. An alternative strategy to the use of these pharmacological inhibitors to increase HbF would be to employ pharmacological activators that increase the activity of proteins that positively promote γ-globin expression. Previous studies have shown that pharmacological activators of the Sirtuin 1 protein deacetylase increased γ-globin expression in cultured human CD34+ erythroid progenitor cell cultures (Dai et al; Am J Hematol 92:1177-1186, 2017). Because Sirtuin deacetylase activity is dependent upon nicotinamide adenine dinucleotide (NAD) as a co-factor, we tested the hypothesis that increased concentrations of nicotinamide, an NAD precursor, would also increase γ-globin expression. Baboon bone marrow derived CD34+ erythroid progenitor cells from 4 individual baboons were cultured on AFT024 monolayers for 14 days in the presence and absence of varying concentrations of nicotinamide. Globin chain expression was measured in cell lysates by high performance liquid chromatography (HPLC). Nicotinamide (500μM) appeared to increase γ-globin 2 fold (0.015±0.098 γ/γ+β) compared to untreated controls (0.072±0.04 γ/γ+β; n=4; p<0.08). Because the nicotinamide levels used in this experiments are higher than can be easily achieved by dietary supplementation, additional experiments were performed to test the effect of P7C3-A20, an allosteric activator of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD synthesis, on γ-globin expression. Addition of P7C3-A20 (2.5μM) to CD34+ erythroid progenitor cultures on d1, 4, 7, and 10 increased γ-globin 2.7 fold (0.247±0.10 γ/γ+β) compared to vehicle-treated controls (0.090±0.06 γ/γ+β; n=5; p<0.01). P7C3-A20 treatment did not affect cell viability or growth at concentration< 2.5μM and dose-response experiments showed increased γ-globin in cultures treated with submicromolar concentrations of the drug. Addition of P7C3-A20 to cultures on days 1 and 4 resulted in near maximal stimulation of γ-globin expression with lesser effects when the drug was added on later days (d4 and7 or d7 and 10) strongly suggesting that the drug targets cells at an early stage of differentiation. Additional experiments showed that the effect of P7C3-A20 (2.5μM) in combination with either the DNMT1 inhibitor decitabine (DAC) or the LSD1 inhibitor tranylcypromine (TCP) resulted in a greater than additive effects on γ-globin expression in the absence of cytotoxicity (Figure 1). In conclusion, the NAMPT activator P7C3-A20 increased γ-globin expression in baboon CD34+ erythroid progenitor cells with greater than additive effects in combination with DAC or TCP. P7C3-A20 has potent in vivo effects as a neuroprotective drug in mouse models and non-human primates. Therefore, the potential of this drug for in vivo HbF induction warrants further investigation. Figure 1 Figure 1. Disclosures Saunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
