PO.ET06.02 · 实验与分子治疗

基于DNA纤维的复制表型可区分接受阿泽司替（azenosertib）治疗的高级别子宫内膜癌患者来源类器官（PDO）中的WEE1抑制剂反应

DNA fiber-based replication phenotypes distinguish WEE1-inhibitor response in high-grade endometrial cancer patient-derived organoids (PDOs) treated with azenosertib

编号 243 展板 14 时间 4/19 02:00–05:00 区域 Section 11 主讲 Elena Ivanova, PhD

分会场 DNA Damage and Repair 1

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作者与单位 Authors & Affiliations

Elena Ivanova¹, Ke Cong¹, Shrabasti Roychoudhury¹, David Han¹, Magdalena Zielinska¹, Minh Ha¹, Abrielle Jens¹, Vaishnavi Anand¹, Bose S. Kochupurakkal¹, Courtney H. Qi¹, Arunika Shee¹, Maureen Mulready¹, Madison Zizzo¹, Alexis Rabbitt¹, Jennifer D. Curtis¹, Nabihah Tayob¹, Marisa R. Nucci², Cam A. Tran¹, Panagiotis A. Konstantinopoulos¹, Geoffrey I. Shapiro¹, Dipanjan Chowdhury¹, Cloud P. Paweletz¹, Ursula Matulonis¹, Joyce Liu¹

¹Dana-Farber Cancer Institute, Boston, MA,²Brigham and Women's Hospital, Boston, MA

摘要 Abstract

中文摘要

背景：高级别子宫内膜癌（EC），包括浆液性、癌肉瘤和透明细胞亚型，常携带TP53突变、复制应激（RS）和细胞周期失调，提示其对WEE1抑制敏感。我们开展了一项转化研究，以确定高级别EC PDO中与RS相关的药效动力学及反应的离体相关性。方法：来自高级别EC活检和手术样本的PDO在离体条件下接受阿泽司替处理（200 nM，24小时）。DNA纤维法测定：（i）复制叉速度（CldU+IdU作为进行中的复制叉），（ii）羟基脲（HU）所致RS下的复制叉稳定性，使用WEE1i+HU后的IdU:CldU比值，以及（iii）通过S1核酸酶法测定单链（ss）DNA缺口形成（WEE1i±S1）。结果：对四种PDO进行了分析：DF4850和DF4161（WEE1i敏感；IC50<100nM）以及DF042和DF4968（不敏感；IC50>1500nM）。在全部四种模型中，阿泽司替处理后复制叉速度下降，与其对RS的靶向效应一致。然而，复制叉降解和缺口形成与活力相一致。具体而言，在WEE1i+HU下，敏感模型（DF4850、DF4161）显示IdU:CldU比值显著降低，提示复制叉重启/稳定性受损。不敏感模型（DF042、DF4968）尽管接受了WEE1抑制仍能保护复制叉免于降解，且无变化。在S1缺口试验中，DF4850/DF4161在WEE1i+S1相比单独WEE1i时表现出更短的纤维片段，与ssDNA缺口增加一致，而DF042/DF4968再次显示无差异，表明其能够耐受WEE1i诱导的应激。因此，虽然复制叉减速是WEE1抑制的一致药效动力学效应，但HU敏感的复制叉不稳定性和S1敏感的缺口形成在敏感PDO模型中富集。结论：在高级别EC PDO中，阿泽司替一致地减缓复制叉，但HU下的复制叉稳定性和S1可检测的缺口形成可区分WEE1i敏感（DF4850、DF4161）与不敏感（DF042、DF4968）模型。这些以机制为基础的DNA纤维读出值得作为高级别EC中WEE1抑制剂的预测性和药效动力学生物标志物进行前瞻性评估。其他生物标志物分析，包括RS蛋白的免疫组化分析，正在进行中并将予以展示。

查看英文原文 English 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.

利益披露 Disclosure

E. Ivanova, None.. K. Cong, None.. S. Roychoudhury, None.. D. Han, None.. M. Zielinska, None.. M. Ha, None.. A. Jens, None.. V. Anand, None.. B. S. Kochupurakkal, None.. C. H. Qi, None.. A. Shee, None.. M. Mulready, None.. M. Zizzo, None.. A. Rabbitt, None.. J. D. Curtis, None.. N. Tayob, None.. M. R. Nucci, None.. C. A. Tran, None. P. A. Konstantinopoulos, Immunogen Other, Consulting/Advisory Board. GSK Other, Consulting/Advisory Board. Novartis Consulting/Advisory Board. Alkermes Consulting/Advisory Board. AstraZeneca Other, Consulting/Advisory Board. Bayer Other, Consulting/Advisory Board. Merck Other, Consulting/Advisory Board. Pfizer Other, Consulting/Advisory Board. Tesaro Other, Consulting/Advisory Board. Vertex Other, Consulting/Advisory Board. EMD Serono Other, Consulting/Advisory Board. Kadmon Other, Consulting/Advisory Board. BMS Other, Consulting/Advisory Board. IMV Other, Consulting/Advisory Board. Repare Other, Consulting/Advisory Board. Artios Other, Consulting/Advisory Board. Mersana Other, Consulting/Advisory Board. G. I. Shapiro, Merck KGaA/EMD-Serono ). Artios ). Lilly ). Pfizer ). Merck KGaA/EMD-Serono Other, Advisory Board. Circle Pharmaceuticals Other, Advisory Board. Concarlo Therapeutics Other, Advisory Board. Schrodinger Other, Advisory Board. FoRx Therapeutics Other, Advisory Board. MycRx Other, Advisory Board. Cyclacel Therapeutics Patent. C. P. Paweletz, XSphera Biosciences Stock, Other Business Ownership, Other, Consultant. Thermo Fisher Other, Received honoraria. Agilent Other, Received honoraria. Daiichi Sankyo ). Bicycle Therapeutics ). Transcenta ). Bicara Therapeutics ). AstraZeneca ). Janssen Pharmaceuticals ). Array Biopharma ). Takeda Oncology ). Bristol Myers Squibb ). TargImmune ). Mirati Therapeutics ). U. Matulonis, Symphogen Other, Data safety advisory board. Immunogen Other, Advisory board. Alkermes Other, Data safety advisory board. Allarity Other, Advisory board. Tango Other, Advisory board. Profound Bio Other, Advisory board. Esai Other, Advisory board. Eli Lilly Other, Advisory board. Novartis Other, Advisory board. Curelab Other, Advisory board. NextCure Other, Advisory board. Pfizer Other, Advisory board. J. Liu, AbbVie Other, Consulting/Advisory board. AstraZeneca Other, Consulting/Advisory board. BMS Other, Consulting/Advisory board. Clovis Oncology Other, Consulting/Advisory board. Daiichi Sankyo Other, Consulting/Advisory board. Eisai Other, Consulting/Advisory board. Genentech/Roche Other, Consulting/Advisory board. Genmab Other, Consulting/Advisory board. GSK Other, Consulting/Advisory board. LoxoLilly Other, Consulting/Advisory board. Merck Other, Consulting/Advisory board. SystImmune Other, Consulting/Advisory board. Regeneron Therapeutics Other, Consulting/Advisory board. Revolution Medicine Other, Consulting/Advisory board. Zentalis Pharmaceuticals Other, Consulting/Advisory board.

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