Cly-124, a first-in-class DCN1 inhibitor partially suppresses CUL3 neddylation and induces fetal hemoglobin is a new potential treatment for sickle cell disease Free

Sickle cell disease (SCD) is an inherited blood disorder caused by a mutation in the beta-globin gene resulting in hemoglobin polymerization leading to hemolysis, vaso-occlusive crisis (VOC), and cumulative multi-organ damage. Human genetics and evidence from therapeutic interventions have demonstrated that increasing fetal hemoglobin (HbF) improves SCD complications and survival. Recently, an autologous cell-based gene therapy targeting the BCL11A enhancer was approved for VOC reduction in SCD through increase of HbF. Despite demonstrated efficacy, access to this therapy remains limited due to manufacturing complexity, toxicities of busulfan conditioning, and high cost, especially for individuals in low and middle income countries. Most compounds that increase HbF are limited by bone marrow toxicity and transcriptional non-specificity as exemplified by hydroxyurea, which exhibits dose-limiting myelosuppression, concerns about carcinogenicity, and highly variable patient response. Current unmet need in SCD remains high and would be served by an accessible, daily, oral therapy that safely increases HbF.

Using single cell transcriptomics and machine learning-based predictive algorithms we interrogated adult and fetal erythropoiesis, prioritizing compounds enriching fetal erythropoiesis gene signatures. Screening CD34-derived human erythroid cells with selected compounds identified neddylation inhibition associated with high levels of HbF induction. Pan-neddylation inhibitor MLN4924 or genetic knockout of ubiquitin conjugating enzyme E2 M (UBE2M) or Cullin 3 (CUL3) induced >25 % HbF (p<0.0001), but caused erythroid toxicity and lineage skewing. To discover a neddylation inhibitor that maintained potent HbF induction without cytotoxicity or broad biologic impact, we examined the regulation of CUL3 and identified defective in cullin neddylation protein 1 (DCN1), a UBC12-CUL3 scaffold, that enhances neddylation. Genetic knockout of DCN1 in human CD34 cells, induced HBG1/2 mRNA and HbF. Pharmacologic inhibition of DCN1 in vitro led to a dose-dependent 2.5-fold increase in HBG1/2 expression (EC50 = 17.5nM)25% HbF by HPLC, and 3-fold increase in globin gene ratio (p<0.001) suggesting a switching mechanism.

DCN1 inhibition selectively induces HBG1/2 expression through partial and self-limiting modulation of CUL3 neddylation (~60% maximum inhibition), without inducing erythroid cytotoxicity or impairing differentiation. Functional genomic profiling at the globin locus revealed increased chromatin accessibility with recruitment of transcription factor NFYA, a known HBG1/2 activator, and increased chromatin contacts between the locus control region (LCR) and regulatory non-coding RNA genes HBBP1 and BGLT3. DCN1 inhibition induced HbF with remarkable biologic specificity, differentially regulating 5 genes at 100 nM including HBG1, HBG2, and BGLT3.

We generated CLY-124, a potent DCN1 inhibitor with optimized pharmacology and pharmacokinetic properties. Efficacy of CLY-124 was studied in the humanized NBSGW mouse model, which recapitulates human erythropoiesis within mouse bone marrow. CLY-124 administration increased HBG1/2 gene expression up to 4-fold and ~30% HBG1/2 over total globin gene expression (p<0.0001), 2-fold higher than hydroxyurea and approaching levels reported for BCL11A enhancer knockout. Combination of a DCN1 inhibitor with hydroxyurea in vitro induced 25% HbFand demonstrated robust synergistic induction of HBG1/2 in vivo. Both CLY-124 monotherapy and synergistic combination of CLY-124 with hydroxyurea were well-tolerated in mice and did not demonstrate dose-limiting cytopenias or suppression of erythropoiesis.

DCN1 is a previously unexplored target for globin gene switching. CLY-124, is a first-in-class DCN1 inhibitor with potent HbF induction as monotherapy and in synergy with hydroxyurea, through a non-cytotoxic and non-epigenetic mechanism. Pre-clinical toxicology studies of CLY-124 demonstrated a safe profile with no hematological toxicity, major organ dysfunction, or laboratory abnormalities at clinically-relevant exposures. CLY-124 has begun dosing in a first-in-human study that will assess safety, PK, and HbF in healthy volunteers and participants with SCD.