Miller, S. A.; Monti, A.; Zhou, E. H.; Lin, C. H. S.; Lynch, O.; Mukherjee, S.; Hei, C.; Nicewonger, R.; Diner, C.; Macias-Garcia, A.; Marino, K.; Li, N.; Ujjinamatada, R.; Salegaonkar, A.; Kuppasani, S.; Rogers-Grazado, M.; Hayford, C.; Lovering, F.; Tang, Q.; Ye, Y.; Zhang, W.; Fulton, T.; O'Brien, J. J.; Sun, X.; Thorarensen, A.; Cortes, M.; Trenor, C. C.; Krishnamoorthy, S.

Few genetic loci are as well-characterized as the globin gene locus, and the substitution of healthy {gamma}-globin (HbF) for missing or mutated {beta}-globin (HbB) is an established therapeutic strategy for {beta}-hemoglobinopathies including sickle cell disease (SCD) and {beta}-thalassemia. Although substantial progress has been made in understanding HbF derepression and globin switching, many current therapeutic strategies involving small molecules increase HbF through broad epigenetic perturbations or cytotoxic stress, raising concerns about dose-limiting cytopenias and off-target effects. By coupling single-cell transcriptomics, genetic perturbations, and functional genomics, we identified an unknown role of neddylation in the regulation of fetal hemoglobin (HbF). Partial impairment of neddylation of cullin ubiquitin ligase 3 (CUL3) through defective in cullin neddylation 1 (DCN1) inhibition leads to highly selective chromatin changes, histone demethylation, and globin locus binding of known activators of HbF transcription. Further, DCN1 inhibition drives globin switching and HbF increases in vitro and in vivo with minimal off-target transcriptional effects and no evidence of cytotoxicity or stress erythropoiesis. To therapeutically target this axis, we report the discovery and characterization of CLY-124, a first-in-class, covalent DCN1 inhibitor with favorable pharmacology properties. In a humanized mouse model, CLY-124 showed a dose-dependent increase in HbF as monotherapy and in synergy with hydroxyurea (HU), a current standard of care. Collectively, these findings highlight the power of single-cell transcriptomics to elucidate undiscovered biologic insights with therapeutic potential, and the promise of DCN-1 inhibitors like CLY-124 to address {beta}-hemoglobinopathies. With an appropriate nonclinical safety profile, a first-in human study of safety, pharmacokinetics and HbF assessments in healthy volunteers and participants with SCD is ongoing for CLY-124.