In vitro and in vivo induction of fetal hemoglobin with two potent and orally bioavailable LSD1 inhibitors

Pharmacological small molecules that target fetal hemoglobin (HbF) repressors serve as potent, cost-effective, and accessible therapeutic strategies to β-globinopathies such as sickle cell disease (SCD). LSD1 inhibition has been shown to induce HbF levels both in vitro and in vivo. However, all potent LSD1 inhibitors in HbF induction in vivo are covalent irreversible compounds, which can cause some adverse effects. In this study, we utilized structure-aided drug design based on the scaffold of a reversible LSD1 inhibitor GSK-690, and developed potent new reversible LSD1 inhibitors that induce robust γ-globin expression in human primary erythroid differentiation culture. Moreover, in a transgenic mouse model of SCD, oral administration of the novel LSD1 inhibitors induces significant elevation of HbF levels and alleviates the disease pathologies resulted from SCD. In addition, combined treatment of an BRD4 degrader, BD-9136 with the LSD1 inhibitors represses the induction of RUNX1 and PU.1, therefore rescues the yield of erythroid cells caused by LSD1 inhibition. Our data indicate that our novel LSD1 inhibitors can effectively induce HbF levels and reduce disease pathologies in SCD mice, and are well-tolerated by oral administration. We anticipate that these new compounds will offer new therapeutic possibilities for treating SCD. To examine the chromatin occupancy of BRD4 in adult erythroid cells, CUT&RUN was performed in adult erythroid cells differentiated from CD34+ hematopoietic stem and progenitor cells (HSPCs) isolated from peripheral blood (PB) (purchased from Fred Hutchinson Cancer Center). PB-CD34+ HSPCs were differentiated in a standard 3-phase erythroid differentiation culture for 11 days before collection for CUT&RUN. CUT&RUN experiments were carried out using either BRD4 antibody or IgG antibody (negative control) to examine the BRD4 chromatin occupancy. All experiments were carried out in independent duplicates from 2 different CD34+ cell donors.