Pharmacological WEE1 inhibition as a strategy to overcome cisplatin resistance in non-seminoma testicular cancer: insights from preclinical models

Testicular germ cell tumors 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, a subset of patients develop resistance. This study explored a potential new treatment by targeting WEE1, a key cell-cycle regulator, using the drug adavosertib in non-seminoma cell models, aiming to overcome CP resistance. 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. 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 in low micromolar range (NCCIT: 0.550 mu M; NCCIT-R: 0.630 mu M; NT2/D1: 0.415 mu M; NT2/D1-R: 0.630 mu M). Adavosertib altered cell-cycle distribution, increasing S-phase population. Western blot analysis revealed that inhibiting WEE1 increases CDK1 activity and mitotic marker pH3, indicating disrupted cell cycle control. This leads to replication stress and forces cells with DNA damage into early mitosis, causing mitotic catastrophe. The treatment also triggered higher levels of DNA damage and cell death, as shown by increased caspase activity and apoptosis markers (cleaved-PARP and cleaved-Caspase 3). In combination treatments, adavosertib enhanced CP efficacy across all models, including resistant lines. Overall, our 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.