8526 The WEE1 Kinase Inhibitor Adavosertib - A Promising Therapeutic Enhancer Of Cytotoxic Therapies In Advanced Adrenocortical Carcinoma

Background: Adrenocortical cancer (ACC) is a rare malignant tumor associated with poor prognosis and limited therapeutic options in advanced cases. Inhibition of WEE1 with adavosertib has emerged as novel strategy for cancer therapy. WEE1 regulates cell cycle checkpoints by enabling DNA damage repair before mitotic entry. Therefore, adavosertib could enhance existing DNA damage-based therapies currently used in ACC care. Here, we evaluated the expression of WEE1 in ACC tissues and cell lines and investigate the effect of adavosertib alone or in combination with cisplatin or gemcitabine both in vitro and in vivo. Methods: WEE1 expression was evaluated in a large cohort of adrenocortical tissues by qRT-PCR (n=19 normal adrenal glands/NAG; n=20 adenomas/ACA; n=52 ACC) and by immunohistochemistry (n=114 ACC with known TP53 mutation status), as well as in five ACC cell lines at both RNA and protein levels. Adavosertib effect, alone or in combination, was assessed in vitro by cell viability assay, flow cytometry, and Western blot, and in vivo in NCI-H295R-xenografted nude mice. Results:WEE1 levels were significantly higher in ACC compared to NAG (0.032 vs 0.012, p=0.04) and low levels were associated with better overall survival in ACC independently of established prognostic factors (HR=0.32, 95%CI 0.12-0.86, p=0.02). TP53-mutated ACC (n=34) showed significantly stronger nuclear protein staining than wild-type tissues (median score 97 vs 72, p <0.001). In NCI-H295R, JIL-2266 and CU-ACC2 cells adavosertib effectively induced cytotoxicity with IC50 of 1.17, 1.35 and 0.4 µM, respectively, whereas CU-ACC1 and MUC-1 were less sensitive. The sensitivity to adavosertib partially correlated with baseline WEE1 expression in cells. Inhibition of WEE1 markedly induced apoptosis and cell death with enrichment of cells in the S phase in all cell lines. We further showed that adavosertib decreased CDK1 phosphorylation and increased phosphorylated H2AX and cleaved PARP. Adavosertib showed an additive-synergic effect with both cisplatin and gemcitabine in all cell lines, including the resistant ones. Consistent with these findings, combination therapy caused significant tumor growth suppression in NCI-H295R-xenografts (median tumor volume after 24 days of treatment, mm³: 303 in adavosertib + cisplatin and 276 mm3 in adavosertib + gemcitabine vs 585 in vehicle group, p=0.48 and 0.01, respectively), without appreciable adverse effects. Conclusion: Our study demonstrates that WEE1 is highly expressed in ACC tissues and targetable in ACC models. Adavosertib exerts a cytotoxic effect enhancing in vitro and in vivo the efficacy of cisplatin and gemcitabine, representing a new promising pharmacological approach in ACC over standard therapies