Development of a ZBTB7A and Wiz Dual Degrading, HbF-Activating CELMoD™ for the Treatment of Sickle Cell Disease
Authors: Rychak Emily , Lui Goldie Yuan Lam , Fimbres Cristhian , Kang Christine Kim , Sun Younguk , Peng Sophie , Deng Jingjing , Franc Sarah , Ciccimaro Eugene , Peacock Kimberly , Tran Minerva , Christoforou Andy , Zhu Jinyi , Polido Geraldine , Pagarigan Barbra , Johnson Scott , Wang Faye , Katavolos Paula , Gaffney Bonny , Garcia Estela , Landry France , Paddison Fletcher , Shipkova Petia , Powers Hannah , Tamò Giorgio , Dietrich August , Carrancio Soraya , Ramakrishnan Radha , Augustine Karen , Narla Rama Krishna , Cummins Thomas , Rolfe Mark , Bence Neil , Lopez-Girona Antonia 
Published: December 2024

The reactivation of fetal hemoglobin (HbF, α2γ2) is a recognized disease modifier for sickle cell disease (SCD) where treatments are limited and there remains a significant unmet need. Achieving a concentration of 30% total HbF, distributed homogeneously in at least 70% of HbF-expressing (F) red blood cells (equating to 10 pg per F cell), has been shown to inhibit the polymerization of sickle hemoglobin (HbS, α2βS2) under deoxygenated conditions and significantly ameliorate SCD-related symptoms (Steinberg MH et al., Blood, 2014). Several transcriptional repressors of HbF have been identified, including B-cell lymphoma/leukemia 11A (BCL11A), zinc finger and BTB domain containing 7A (ZBTB7A) and widely interspaced zinc finger protein (WIZ), which until now, were considered undruggable. Here, we describe the development of BMS-986470, a novel, orally bioavailable HbF-activating cereblon (CRBN) E3 ligase modulator (CELMoD™) agent that targets ZBTB7A and WIZ for degradation and is currently under clinical development for SCD (NCT06481306).

To identify HbF-activating CELMoD compounds, a phenotypic screen was conducted using primary erythroblasts derived from healthy CD34+ cells. Compounds with dual degrading activity targeting ZBTB7A and WIZ emerged as the most effective γ-globin inducers, resulting in the highest levels of HbF. BMS-986470 was subsequently designed following an extensive structure-activity-relationship optimization campaign to define the optimal degradation of ZBTB7A and WIZ for maximal γ-globin induction. Global proteomic profiling of BMS-986470 demonstrated that ZBTB7A and WIZ were the predominant proteins regulated in the proteome, and predictive modeling confirmed that they were the major substrates contributing to HbF induction. Degrons in both proteins were identified, and ternary complex structures of ZBTB7A:BMS-986470:CRBN/DDB1 and WIZ:BMS-986470:CRBN/DDB1 were resolved at high resolution, explaining the selectivity and potency of BMS-986470 against both substrates.

Genetically engineered HUDEP-2 cells were established to validate the advantage of ZBTB7A and WIZ dual degradation over the degradation of WIZ alone at inducing HbF. Knockout of either ZBTB7A or WIZ alone significantly induced γ-globin expression and the dual silencing of both transcription factors synergistically enhanced the proportion of F-cells to &gt;95% and total HbF tetramer to &gt;80%. Introducing single point mutations into the degrons of either ZBTB7A or WIZ protein alone reduced BMS-986470-mediated γ-globin induction, and expression of double point mutations in both ZBTB7A and WIZ significantly abrogated γ-globin induction. BMS-986470 was also a potent degrader of ZBTB7A and WIZ in primary erythroblasts derived from healthy donor and SCD patient samples, achieving levels of &gt;90 % F-cells and &gt;40 % total HbF, without affecting erythroblast viability or erythroid differentiation. In these preclinical models, HbF induction by BMS-986470 was significantly higher than the standard of care, hydroxyurea.

Utilizing a murine model of human erythropoiesis, BMS-986470 did not affect human erythroblast differentiation and demonstrated a significant dose-dependent decrease in hZBTB7A and hWIZ protein, along with an increase in F-cells and γ-globin expression. Notably, mice treated at the minimal efficacious dose achieved up to a 3-fold increase in total HbF levels as compared to the vehicle control. BMS-986470 also significantly induced F-cells and γ-globin protein in the peripheral blood of Townes mice expressing human CRBN, and reduced sickling under hypoxic conditions, ex vivo. In naïve healthy cynomolgus monkeys treated daily with BMS-986470 for 16 days, with a 2-week observation period, significant, dose dependent degradation of WIZ and ZBTB7A was evident, along with an increase in circulating immature erythrocytes, and increased HBG1/2 transcript, γ-globin protein and erythroid progenitor markers in the peripheral blood and bone marrow. BMS-986470 was well-tolerated at all doses tested.

In summary, we disclose the discovery and preclinical characterization of BMS-986470, a potential first-in-class, dual degrader of ZBTB7A and WIZ, with robust γ -globin induction activity leading to HbF levels predicted to significantly ameliorate SCD pathology. BMS-986470 is currently under clinical investigation (NCT06481306) for patients with SCD.