Adavosertib targets Cell Cycle Regulator WEE1 and Inhibits Pediatric Neuroblastoma Growth

Abstract ID 93496 Poster Board 048 Purpose: High-risk neuroblastoma (NB) is a pediatric cancer that arises during the sympathetic nervous system's development. Despite recent advancements in treatment strategies involving intensive chemotherapies, radiation, and surgery, NB often relapses as a metastatic refractory tumor. Consequently, it is imperative to identify and develop innovative therapeutic methods to combat NB. The cell cycle regulator WEE1 regulates the activation of CDK1 and CDK2, inhibits the cell cycle's response to DNA damage, and facilitates cancer growth. In the present study, we hypothesized that direct targeting of WEE1 by using a selective inhibitor, adavosertib, will impede the NB cell cycle and inhibit NB proliferation and growth. Methods: We used the R2 Genomic Database platform to analyze multiple NB patient datasets, including 1235 primary NB patients. The impact of adavosertib on the growth of NB cells was assessed using cytotoxicity MTT assays, clonogenic formation assays, and 3D spheroidal assays. Apoptosis and cell cycle assays were conducted using specific kits and analyzed using an Attune Flow Cytometer. Standard methods for gene expression analysis and Western immunoblotting were used. We employed the NB xenograft model using athymic mice to determine the in vivo effects of adavosertib on NB in vivo growth. Results: Patient dataset analysis confirmed a strong inverse correlation between WEE1 expression and poor overall survival in NB cases. We also observed elevated WEE1 expression in higher NB cancer stages, showing a direct role of WEE1 in NB progression. Direct WEE1 inhibition using adavosertib significantly inhibits NB cell proliferation and colony formation in a dose-dependent manner in different NB cell lines, including both MYCN-amplified and MYC-amplified cell lines. Adavosertib significantly induces apoptosis and blocks cell cycle progression in NB cells in a dose-dependent manner compared to control treatment. Additionally, we found that WEE1 inhibition significantly inhibits the in vitro growth of NB 3D spheroids in a dose-dependent manner. Our gene expression and Western blot analysis revealed that advosertib inhibits the gene expression of different cell cycle regulators by directly inhibiting the phosphorylation of the WEE1 protein. Furthermore, our NB xenograft mouse model showed that adavoseritib significantly inhibits in vivo NB tumor growth and volume compared to control treatments. Conclusions: Overall, our results show that inhibition of WEE1 using adavosertib is a promising therapeutic strategy for NB. Further combining WEE1 inhibitors with current therapies holds the potential for developing effective targeted therapies for NB patients. Keywords: Neuroblastoma; adavosertib; cell cycle; WEE1; pediatric cancer