P36 - Adavosertib-mediated wee1 inhibition as a strategy to overcome cisplatin resistance in non-seminoma testicular cancer: insights from preclinical models

Background/Introduction Testicular germ cell tumors (TGCTs) are the most common solid malignancy in young adult males with non-seminomatous representing a clinically aggressive subtype. Although cisplatin (CP)-based chemotherapy is highly effective, with cure rates exceeding 95%, a subset of patients develop resistance. The identification of therapeutic strategies to overcome CP-resistance remains an urgent clinical need. Purpose In this study, we evaluated the expression and pharmacological targeting of WEE1, a key regulator of the G2/M checkpoint and replication stress response, in non- seminoma cell models using the WEE1 inhibitor adavosertib (AZD1775). Methods Two non-seminoma cell lines, NCCIT and NT2/D1, and their CP-resistant subclones (NCCIT-R and NT2/D1-R) were used. The effects of adavosertib and CP, alone or in combination, on cell viability, cell-cycle progression, apoptosis, and DNA damage markers was assessed. Results WEE1 was expressed in non-seminoma cell models. Adavosertib reduced cell viability in a dose-dependent manner across all cell models in both 2D and 3D cultures. IC50 values were consistently in the low micromolar range with minimal variation (NCCIT: 0.550 μM; NCCIT-R: 0.630 μM; NT2/D1: 0.415 μM; NT2/D1-R: 0.630 μM). Adavosertib altered cell-cycle distribution, increasing S-phase population. Western blot analysis showed reduced CDK1 phosphorylation, indicating elevated CDK1 activity, with increased H3 phosphorylation, a mitotic marker. These findings suggest WEE1 inhibition disrupts cell cycle checkpoints, promotes replication stress, and drives cells with unrepaired DNA damage into premature mitosis, resulting in mitotic catastrophe. Caspase 3/7 activity was also increased after treatment, specifically at 24 hours, together with elevated markers of DNA damage (pH2Ax) and apoptosis (cleaved-PARP and cleaved-Caspase 3). In combination treatments, adavosertib enhanced CP efficacy across all models, including resistant lines. Notably, in NCCIT-R cells, CP IC50 dropped from 11.65 to 1.99 μM when combined with adavosertib, indicating a sixfold increase in CP-sensitivity. Conclusion(s) Overall, our findings demonstrate that WEE1 is expressed in non- seminoma models and is targetable. Pharmacological inhibition of WEE1 by adavosertib impairs cell viability, disrupts cell-cycle control, induces apoptosis, and restores CP-sensitivity in resistant models. These results provide compelling preclinical evidence supporting the combination of WEE1 inhibition with standard chemotherapy as a promising strategy to overcome CP-resistance in non-seminoma testicular cancer.