Abstract 4208: Azenosertib is a potent and selective WEE1 kinase inhibitor with broad antitumor activity across a range of solid tumors

Abstract Due to high proliferative rate, cancer cells experience significant stress during DNA replication and mitosis, leading to DNA damage and increased genomic instability. To mitigate this, cancer cells rely on checkpoint kinases like WEE1, which halt cell cycle progression and allow damaged DNA to be repaired before cells enter mitosis. Damage that is too extensive leads to apoptosis. Targeting WEE1 can exploit vulnerabilities created by replicative stress and genomic instability. Given these features are common in cancer, we hypothesized a WEE1-targeted therapeutic would have broad antitumor activity across various cancer types. Azenosertib (ZN-c3) is a novel, selective, orally bioavailable WEE1 inhibitor with optimized pharmacokinetic and pharmacodynamic properties. Preclinical studies demonstrate Azenosertib inhibits cancer cell proliferation through a WEE1-dependent mechanism, marked by a reduction of pY15-CDK1 levels and increases in DNA damage markers. Herein, we evaluated its antitumor activity in 54 mouse xenograft models representing 9 solid tumor subtypes and observed robust tumor growth inhibition (> 60% TGI) in 24 of these models, including breast, colorectal, lung, ovarian, pancreatic, prostate, soft tissue sarcomas, and endometrial cancers. We also explored various dosing schedules for Azenosertib, identifying regimens that maintain efficacy with minimal toxicity in preclinical models. To further understand the mechanisms underlying Azenosertib’s antitumor activity, we analyzed cell cycle changes in four cancer cell lines with variable sensitivity to Azenosertib. Treatment with Azenosertib caused accumulation of EdU-negative cells in the S-phase, reduced cells in the G1 phase, and promoted premature entry into mitosis. These effects suggest Azenosertib bypasses the G1/S and G2/M checkpoints, leading to immature S-phase and Mitosis entry, consistent with WEE1 inhibition of CDK1 and CDK2. Further analysis of DNA repair revealed an increase in γH2AX-positive cells, primarily in G1, EdU-negative S, and G2/M, and the co-localization of mitotic marker pHH3 and DNA damage marker γH2AX. Notably, WEE1 plays a critical role in safeguarding the integrity of the cell cycle, and this regulation is disrupted by Azenosertib. As a result, cells with unresolved DNA damage are driven into premature mitosis, exacerbating DNA damage and leading to mitotic catastrophe. Azenosertib is currently being investigated as a single agent in a dose escalation Phase I clinical trial (NCT04158336) evaluating safety, tolerability, and efficacy in cancer patients with previously treated, advanced solid tumors. Early clinical data from heavily pretreated patients with different types of cancer show promising signs of activity. Our findings highlight the potential of WEE1 inhibition as a broad therapeutic strategy, for gynecological cancers and various malignant indications. Citation Format: Wen Liu, Jianhui Ma, Jiali Li, Daehwan Kim, Sangyub Kim, Alexandra Levy, Kimberly Blackwell, Alejandro Recio-Boiles, Jennifer M. Segar, Shirai Sen, Deborah Doroshow, Danielle Jandial, Olivier Harismendy, Stephan K. Grant, Ahmed Samatar, Mark R. Lackner, Kimberlee M. Fischer. Azenosertib is a potent and selective WEE1 kinase inhibitor with broad antitumor activity across a range of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4208.