Next Generation Clinical-Stage HbF Inducers Exhibit Multiple Molecular Actions In Vitro and In Vivo and Offer Personalized Therapeutic Regimens
Authors: Pace Betty S. , Sangerman Jose I , Boosalis Michael S , Wolf Roman , Dai Yan , Chui David H.K. , Makala Levi , Li Biadru , Faller Douglas V , Perrine Susan P 
Published: October 2019

Abstract
High-level expression of fetal (Υ) globin reduces clinical complications in sickle cell disease and this is achieved with hydroxyurea (HU) in young children. However, non-cytotoxic high-potency therapeutics, particularly which can be utilized in combination with HU, are needed for most adolescent and adult patients who have continued serious clinical events. We have identified additional pharmaceutical candidates which induce HbF without cytotoxicity, using a Υ-globin gene promoter linked to GFP for robotic high-throughput screening, and screening five diverse chemical libraries. From a library of US and EU drugs which are approved for treatment of other medical conditions, a small panel of approved therapeutics were found to induce Υ globin expression, and have benign safety profiles, are orally active, and are suitable for long-term use. Three orally active candidates were evaluated in anemic baboons, and two, DLT and PB-04, induced Υ-globin mRNA by 15- to 33-fold over baseline levels. In 3/3 beta-globin locus YAC transgenic mice, one candidate (PB-04; 20 mg/kg) given by intra-peritoneal (IP) injections (for experimental feasibility) 3 times/ week for 5 wks significantly increased F-cells from 0.1 to 9%, 0.4 to 18%, and 0.13 to 12% respectively; and mean fluorescence intensity (MFI) increased by 10- to 33-fold. Responses were observed within one week. In hydroxyurea treated mice (100 mg/kg; IP, 5 days/ wk) F-cells increased from 0.3 to 2.3% on average (p<0.05), and MFI increased by 6- to 7-fold, while water vehicle did not increase F-cells significantly. PB-04 has been used clinically for decades as an excipient solely to prolong the half-life of another pharmaceutical, and is suitable for repurposing. In ChIP assays in K562 cells, PB-04 treatment demonstrated dual actions of displacing HDAC3 by 20-fold and LSD-1 by 3-fold from the Υ globin gene promoter.
To investigate potential effects of genetic modifiers of HbF on responses to different HbF inducer classes, erythroid progenitors from 40 sickle and beta thalassemic subjects were sub-genotyped for 3 major quantitative trait loci (QTL) (Bcl-11A, HMIP, Xmn-I) and cultured +/- 7 HbF inducers which are in clinical trials or approved. Most HbF inducers, including decitabine and butyrate used as positive controls, are active in 50-70% of progenitors, with differential Υ-globin mRNA responses observed. Only 10% of progenitors did not respond to any inducing agent. Most progenitors with the Xmn-1 variant responded with higher Υ globin transcription to all inducers. Sodium dimethylbutyrate (HQK-1001) and decitabine, produced 6-fold overall mean induction. PB-04 produced 9-fold mean induction above untreated control levels from the same subject. HDAC inhibitors (Butyrate, MS-275) which suppress Bcl-11A expression, demonstrate higher activity in progenitors from subjects without an underlying SNP in Bcl-11A. Another HDAC inhibitor, SB939, produced responses in 80% of progenitors. 25-30% of subjects’ progenitors exhibit high induction, 12-to 40-fold above untreated controls, to dimethylbutyrate, PB-04, decitabine, and an HDAC1/2 inhibitor 14F, suggesting a “high responder genotype” of which only half had a recognized favorable QTL. Taken together, these in vitro and in vivo studies identify a mini-pipeline of clinical-stage HbF-inducing therapeutics, with both epigenetic and targeted molecular actions, which can be investigated clinically to develop tailored therapeutics and therapeutic combinations for high-level induction of HbF in subgenotyped hemoglobinopathy patients.
Seven new and established inducers have been evaluated in erythroid progenitors cultured from sub-genotyped hemoglobinopathy patients, 3 new drugs (PB-04, DLT, RSV) and two HDAC inhibitors (MS-275 and SB939), induced 3- to 40-fold higher Υ-globin mRNA above untreated control levels.

Disclosures:
Faller: Phoenicia BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Perrine:Phoenicia BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Research Funding.