Development of the Novel Cytosine Analog Aza-T-Dcyd (AB1) As a Fetal Hemoglobin Inducer to Treat Sickle Cell Disease.

Introduction: Induction of fetal hemoglobin (HbF) expression in sickle cell disease (SCD) patients using small molecules is considered an effective therapeutic strategy to ameliorate severe clinical phenotypes. With this objective in mind, several drug molecules have been tested for HbF induction. At present, hydroxyurea (HU) is the only FDA-approved drug used to induce HbF in SCD patients, however, it is not effective in all patients. Studies have reported that DNA methyltransferase inhibitors such as decitabine stimulate γ-globin gene expression through DNA hypomethylation. However, decitabine has cytotoxic side effects and is most effective by intravenous administration. Thus, it is critical to develop alternative safe and orally active hypomethylating agents for inducing HbF. Our lab in collaboration with AkiraBio, conducted preclinical in vitro studies to assess a novel cytosine analog Aza-T-dCyd (AB1), for its ability to induce HbF in human K562 cells and erythroid progenitor cells derived from SCD patients.

Methods: K562 cells were cultured in Iscove's Modified Dulbecco's Eagle medium, 10% fetal bovine serum and penicillin/streptomycin. The human erythroid progenitors were generated from peripheral blood mononuclear cells isolated from two SCD patients under IRB exempt protocol as previously established in our lab (Li et al., Haematologica. 2018). The cultured K562 cells were treated either with AB1 alone at gradually increasing concentrations from 3nm to 200nm, decitabine (0.2µM), hydroxyurea (HU100µM) or combined AB1 with HU for 48 hours. The cultured sickle erythroid progenitors were treated with AB1 on day 8 and then harvested on day 10. The harvested cells were analyzed for cell viability and flow cytometry to measure HbF positive cells (F-cells). The γ-globin and β-globin mRNA levels were quantified by qRT-PCR and the protein levels of HbF and DNMT-1 were detected by Western blot technique.

Results: Initial studies to evaluate drug toxicity were completed. We observed higher cell viability of K562 cells treated with AB1 10nM and 30nM compared to untreated and HU 100µM (p=0.005) treated cells. Hence, subsequent experiments were conducted with the lower AB1 concentrations. By flow cytometry analysis, AB1 treatment significantly increased F-cells from 13.95% to 20.11% and 22.46% in 10nM and 30nM treated cells (p=0.0002) respectively. Moreover, mean fluorescence intensity analysis showed significant HbF expression in AB1 treated cells compared to untreated cells (p=0.00001). At the transcription level, AB1 increased γ-globin mRNA levels 1.4-fold compared to untreated cells. K562 cells treated with a combination of AB1 and HU at their maximum response concentrations (AB1 10 and 30nM; HU 100 µM) did not show additive effects on F-cells or γ-globin mRNA expression. Other combination concentrations were not assessed. In subsequent studies we investigated the HbF inducing capacity of AB1 in human erythroid progenitors generated from SCD patients (N=2). Normal cell viability was observed in sickle progenitors at 3nm, 6nM and 10nm AB1 treatment. The F-cell level increased from 16.21% to 27.68% at 10nm AB1 (p=0.02). By qRT-PCR assay, a significant 2-fold increase in γ-globin mRNA level was observed in AB1 treated erythroid cells, when normalized with total γ-globin and β-globin mRNA; β globin mRNA levels were unchanged. Finally, when we analyze the effects of AB1 on protein levels of HbF and DNMT-1 by Western blot analysis, 10nM and 30nM AB1 induced HbF and inhibited DNMT-1 expression the sickle erythroid progenitors.

Conclusion: Our study findings demonstrate that AB1 induced γ-globin transcription in K562 cells and human sickle erythroid progenitors. Studies to test the effects of AB1 in the Townes SCD mouse model are in progress.

Schaub:AkiraBio Inc.,: Consultancy, Current Employment, Current equity holder in private company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.