Abstract B144: Discovery and preclinical evaluation of novel oral WEE1 degraders
Authors: Xing Weiqiang , Tao Meixin , Huang Fan , Gu Liuge , Cao Min , Wang Chunhao , Diao Jianxiong , Du Chunming , Bai Xinyu , Chen Mingchen , Zheng Xin , Lei Ying , Xie Yang , Guo Taylor B. 
Published: December 2023

Background: WEE1 is a key component of cell cycle checkpoints as it inhibits cell cycle progression through phosphorylation of CDK1. Inhibition of WEE1 could induce premature mitotic entry of cells with damaged DNA, leading to mitotic catastrophe and cell death. WEE1 small molecule inhibitors (SMIs) have demonstrated clinical benefits particularly in gynecological malignancies. However, limited antitumor activity and multifaceted toxicity profile from SMIs warranted the discovery of novel WEE1-targeting modalities to afford better therapeutic index towards a greater patient population. Here, we report the discovery of a novel oral, heterobifunctional WEE1 degrader NXD01 and its preclinical evaluation.

Methods: We discovered NXD01 through our AI-empowered, prediction-guided compound optimization platform neoDegrader. We quantified NXD01-mediated WEE1 protein degradation by western blot and evaluated the anti-proliferative activity of NXD01 by CellTiter-Glo assay in multiple human cancer cell lines. We further assessed downstream effects of WEE1 degradation on phosphorylation of its target CDK1, and markers of cell cycle and apoptosis by western blot and flow cytometry. We studied the in vivo antitumor activity of NXD01 by oral gavage in the SK-UT-1 (human uterine leiomyosarcoma cell) xenograft model, followed by PK/PD analysis of the tumor tissues.

Results: In SK-UT-1 cells, NXD01 potently degraded WEE1 protein at a DC50 of 1.4 nM with no obvious hook effect and achieved complete degradation within 4 h of treatment (DC90 = 4.5 nM). Marked reduction in CDK1-Y15 phosphorylation and increased γ-H2AX levels (a marker for DNA damage) and PARP cleavage (apoptosis) were all detected for NXD01 at 100 nM, one-tenth the effective concentration for the WEE1 SMI azenosertib (ZN-c3). Treatment of NXD01 (but not ZN-c3) at 100 nM greatly increased the proportion of cells in the G2/M phase indicating mitotic arrest. These results translated into a more potent anti-proliferative effect by NXD01 compared to ZN-c3 in vitro: the IC50 was 1.3 nM vs 37 nM for MOLT4, a T cell leukemia line, as well as across 10 gynecological cancer cell lines (NXD01 IC50: 10-210 nM; ZN-c3 IC50: 100-975 nM). NXD01 exhibited good exposure after oral administration in mice (dose normalized AUC0-t: 765 mg∙h/mL, t1/2: 3 h, F%: 15.7%). NXD01 showed strong in vivo antitumor activity in the SK-UT-1 xenograft model, achieving 96% tumor growth inhibition (TGI) at 30 mg/kg BID, as compared to 81% TGI for ZN-c3 at 60 mg/kg QD. Analysis of tumor samples suggested that 85% of in-tumor WEE1 protein had been degraded by NXD01 at 12 h post dose.

Conclusion: We have discovered a structurally-novel, orally bioavailable, cereblon-mediated, heterobifunctional degrader of WEE1 protein NXD01. NXD01 displayed stronger in vitro and in vivo antitumor activity than currently the most advanced WEE1 small molecule inhibitor azenosertib, making NXD01 a differentiated candidate with strong potential for clinical development.

Citation Format: Weiqiang Xing, Meixin Tao, Fan Huang, Liuge Gu, Min Cao, Chunhao Wang, Jianxiong Diao, Chunming Du, Xinyu Bai, Mingchen Chen, Xin Zheng, Ying Lei, Yang Xie, Taylor B. Guo. Discovery and preclinical evaluation of novel oral WEE1 degraders [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B144.