Abstract Background: High-grade endometrial cancers (EC), including serous, carcinosarcoma, and clear cell subtypes, frequently harbor TP53 mutations, replication stress (RS), and cell-cycle dysregulation, suggesting vulnerability to WEE1 inhibition. We conducted a translational study to define RS-linked pharmacodynamics and ex vivo correlates of response in high-grade EC PDOs. Methods: PDOs from high-grade EC biopsies and surgical samples were treated ex vivo with azenosertib (200 nM, 24 h). DNA-fiber assays measured: (i) fork speed (CldU+IdU as ongoing replication forks), (ii) replication fork stability under RS caused by hydroxyurea (HU), using the IdU:CldU ratio after WEE1i+HU, and (iii) single-stranded (ss)DNA gap formation via the S1 nuclease assay (WEE1i±S1). Results: Four PDOs were profiled: DF4850 and DF4161 (WEE1i-sensitive; IC50s<100nM) and DF042 and DF4968 (insensitive; IC50s>1500nM). Across all four models, fork speed decreased after azenosertib, consistent with on-target effects on RS. However, fork degradation and gap formation aligned with viability. Specifically, under WEE1i+HU, the sensitive models (DF4850, DF4161) showed a significant reduction in the IdU:CldU ratio, indicating impaired fork restart/stability. The insensitive models (DF042, DF4968) were able to protect forks from degradation despite WEE1 inhibition and showed no change. In the S1 gap assay, DF4850/DF4161 exhibited shorter fiber tracts with WEE1i+S1 vs WEE1i alone, consistent with increased ssDNA gaps, whereas DF042/DF4968 again showed no difference, indicating the ability to tolerate WEE1i-induced stress. Thus, while fork slowing is a uniform pharmacodynamic effect of WEE1 inhibition, HU-sensitive fork instability and S1-sensitive gap formation are enriched in sensitive PDO models. Conclusions: In high-grade EC PDOs, azenosertib uniformly slows replication forks, but fork stability under HU and S1-detectable gap formation distinguish WEE1i-sensitive (DF4850, DF4161) from insensitive (DF042, DF4968) models. These mechanism-anchored DNA-fiber readouts merit prospective evaluation as predictive and pharmacodynamic biomarkers for WEE1 inhibitors in high-grade EC. Additional biomarker analyses, including immunohistochemical analyses of RS proteins, are ongoing and will be presented. Citation Format: Elena Ivanova, Ke Cong, Shrabasti Roychoudhury, David Han, Magdalena Zielinska, Minh Ha, Abrielle Jens, Vaishnavi Anand, Bose S. Kochupurakkal, Courtney H. Qi, Arunika Shee, Maureen Mulready, Madison Zizzo, Alexis Rabbitt, Jennifer D. Curtis, Nabihah Tayob, Marisa R. Nucci, Cam A. Tran, Panagiotis A. Konstantinopoulos, Geoffrey I. Shapiro, Dipanjan Chowdhury, Cloud P. Paweletz, Ursula Matulonis, Joyce Liu. DNA fiber-based replication phenotypes distinguish WEE1-inhibitor response in high-grade endometrial cancer patient-derived organoids (PDOs) treated with azenosertib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 243.
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