Abstract A025: Inducing mitotic catastrophe in high replicative stress castration resistant prostate cancer through inhibition of WEE1

Abstract Introduction: The cell cycle is a tightly regulated process essential for controlled cell growth and division. In cancer, aberrant proliferation leads to the accumulation of DNA damage and genomic instability, forcing malignant cells to rely on alternative checkpoint mechanisms to maintain survival. These checkpoints are also commonly dysfunctional in cancer. In particular, disruptions in the G1 checkpoint, often seen in aggressive tumors, shift dependency toward the G2/M checkpoint, where WEE1 kinase plays a critical role in regulating CDK1 activity and preventing premature mitotic entry. Metastatic castration-resistant prostate cancer (mCRPC) frequently harbors mutations in tumor suppressors and G1 checkpoint regulators, such as RB1 and TP53. These mutations increase replicative stress, making these tumors not only more dependent on the G2/M checkpoint but also potentially more sensitive to WEE1 inhibition (WEE1i). Given this, we hypothesize that WEE1 inhibitors will be more effective in mCRPC models with inherently elevated replicative stress, as disruption of the G2/M checkpoint will force premature mitotic entry, leading to mitotic catastrophe and cell death. Methods: We determined the half-maximal inhibitory concentrations (IC50) of two WEE1 inhibitors, AZD1775 (adavosertib) and ZN-c3 (azenosertib), in a panel of LNCaP-derived prostate cancer cell lines: LNCaP (parental), LNCaP-AR (androgen receptor amplification), and LNCaP-DKO (RB1 and TP53 double knockout). IC50 values were established using escalating drug doses and IncuCyte live-cell imaging to measure proliferation. Based on IC50 results, a uniform sub-lethal dose was selected for further analyses. Immunoblotting was performed to assess downstream targets of WEE1i, including markers of DNA damage, cell cycle progression, and apoptosis. Propidium iodide staining and flow cytometry were used to assess shifts in cell cycle phase distributions following WEE1i. Results: As expected, the LNCaP-AR and LNCaP-DKO models, both characterized by elevated replicative stress, exhibited greater sensitivity to WEE1i compared to the parental line. Immunoblot analysis revealed increased levels of phospho-CDK1 (Y15), accumulation of the DNA damage marker γ-H2AX (S139), and evidence of elevated mitosis in AR and DKO cells through pHH3 (S10), following short-term treatment with either WEE1 inhibitor. Flow cytometry showed a reduction in G1 and S-phase populations and accumulation in G2/M phase in LNCaP-AR and DKO cells. Conclusion: Our findings demonstrate that mCRPC models with G1 checkpoint mutations and inherently high replicative stress are more susceptible to WEE1i, revealing a potential predictive biomarker that can be leveraged for therapeutic efficacy. Citation Format: Patricia G. Madel, Arunangsu Dey, Christopher Henning, Yuhan Zhang, Anthony Williams, Russell Szmulewitz. Inducing mitotic catastrophe in high replicative stress castration resistant prostate cancer through inhibition of WEE1 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A025.