Introduction: The reactivation of HBG1/2 gene expression is a validated therapeutic strategy for treating β-hemoglobinopathies, such as sickle cell disease (SCD) and β-thalassemia. Currently, hydroxyurea (HU) is the only approved drug for HbF induction, underscoring the need for new agents. HBG1/2 repression is mediated by epigenetic regulatory complexes, including NuRD and CoREST, which contain HDAC1/2. Inhibition of these enzymes increases HbF by promoting an open chromatin conformation near the HBG1/2 promoters. Proteolysis Targeting Chimeras (PROTACs) that degrade such epigenetic regulators represent a promising pharmacological approach. In this study, we evaluated a novel HDAC1/2 degrader for its ability to induce HbF while assessing its effects on cell viability, proliferation, and erythroid differentiation.

Methods: We tested a novel HDAC1/2 degrader, ARP-47, during in vitro erythroid differentiation of human CD34+ cells from healthy donor's peripheral blood. Cells were treated with the compound (1–400 nM) and percentage of F-cells, erythroid differentiation (%CD71+CD235+), viability, and live cell counts were analyzed via flow cytometry. We quantified HBG1/2, HBB, HDAC1, and HDAC2 mRNA by qPCR, and measured γ-globin, HDAC1, and HDAC2 protein levels by Western blot. Erythroid progenitor distribution was assessed via cytospin preparations.

Results: Treatment with ARP-47 increased the percentage of F-cells at all doses, reaching a plateau at 10 nM, where the increase was statistically significant compared to vehicle (DMSO = 55.63% ± 10.11 vs. ARP-47 10 nM = 72.75% ± 12.07, p = 0.0073, n = 4). At 10nM, erythroid differentiation was unaffected (DMSO = 78.68% ± 7.69 vs. ARP-47 10 nM = 71.95% ± 8.34, p = 0.157, n = 4), as were viability (DMSO = 91.55% ± 3.95 vs. ARP-47 10 nM = 91.93% ± 3.175, p = 0.5472, n = 4) and live cell counts (DMSO = 33.58 × 10⁴ ± 1.2 × 10⁴ vs. ARP-47 10 nM = 31.85 × 10⁴ ± 2.876 × 10⁴, p = 0.2609, n = 4). qPCR revealed a 2.5-fold increase in HBG1/2 mRNA (DMSO = 0.655 ± 0.143 a.u. vs. ARP-47 10 nM = 1.686 ± 0.601 a.u., p = 0.023, n = 4), while HBB remained unchanged (DMSO = 0.964 ± 0.202 a.u. vs. ARP-47 10 nM = 0.984 ± 0.33 a.u., p = 0.921, n = 4), significantly increasing the HBG/(HBG+HBB) ratio (DMSO = 0.405 ± 0.069 a.u. vs. ARP-47 10 nM = 0.631 ± 0.055 a.u., p = 0.005, n = 4). HDAC1 and HDAC2 mRNA levels were unaffected (HDAC1: p = 0.5326; HDAC2: p = 0.9167). Western blot analysis showed a 2-fold increase in γ-globin protein (DMSO = 0.599 ± 0.192 a.u. vs. ARP-47 10 nM = 1.101 ± 0.183 a.u., p = 0.0121, n = 3), consistent with the increase in the percentage of F-cells. HDAC1 and HDAC2 levels decreased by approximately 30% (p = 0.0252) and 40% (p = 0.0236), respectively. Cytospin analysis showed no changes in erythroid progenitor distribution.Conclusion: ARP-47, a novel HDAC1/2 degrader, effectively induces HbF in CD34+ erythroid cells while preserving viability, cell counting, and erythroid differentiation. These findings demonstrate that selective HDAC1/2 degradation represents a distinct and potentially safer therapeutic approach for β-hemoglobinopathies, supporting further preclinical development of ARP-47 as a first-in-class candidate.

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2025
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