PO.ET06.02 · 实验与分子治疗
DNA fiber-based replication phenotypes distinguish WEE1-inhibitor response in high-grade endometrial cancer patient-derived organoids (PDOs) treated with azenosertib
作者与单位
摘要 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.