Project description:Identifying and overcoming a mechanism of resistance to Wee1 kinase inhibitor AZD1775 in high grade serous ovarian cancer cells (OVCAR8)
Project description:Identifying and overcoming a mechanism of resistance to Wee1 kinase inhibitor AZD1775 in high grade serous ovarian cancer cells (ES-2)
Project description:PurposeEsophageal cancer is a deadly malignancy with a 5-year survival rate of only 5% to 20%, which has remained unchanged for decades. Esophageal cancer possesses a high frequency of TP53 mutations leading to dysfunctional G1 cell-cycle checkpoint, which likely makes esophageal cancer cells highly reliant upon G2-M checkpoint for adaptation to DNA replication stress and DNA damage after radiation. We aim to explore whether targeting Wee1 kinase to abolish G2-M checkpoint sensitizes esophageal cancer cells to radiotherapy.Experimental designCell viability was assessed by cytotoxicity and colony-forming assays, cell-cycle distribution was analyzed by flow cytometry, and mitotic catastrophe was assessed by immunofluorescence staining. Human esophageal cancer xenografts were generated to explore the radiosensitizing effect of AZD1775 in vivo.ResultsThe IC50 concentrations of AZD1775 on esophageal cancer cell lines were between 300 and 600 nmol/L. AZD1775 (100 nmol/L) as monotherapy did not alter the viability of esophageal cancer cells, but significantly radiosensitized esophageal cancer cells. AZD1775 significantly abrogated radiation-induced G2-M phase arrest and attenuation of p-CDK1-Y15. Moreover, AZD1775 increased radiation-induced mitotic catastrophe, which was accompanied by increased γH2AX levels, and subsequently reduced survival after radiation. Importantly, AZD1775 in combination with radiotherapy resulted in marked tumor regression of esophageal cancer tumor xenografts.ConclusionsAbrogation of G2-M checkpoint by targeting Wee1 kinase with AZD1775 sensitizes esophageal cancer cells to radiotherapy in vitro and in mouse xenografts. Our findings suggest that inhibition of Wee1 by AZD1775 is an effective strategy for radiosensitization in esophageal cancer and warrants clinical testing.
Project description:High grade serous ovarian cancer (HGSOC) is a major cause of female cancer mortality. The approval of poly (ADP-ribose) polymerase (PARP) inhibitors for clinical use has greatly improved treatment options for patients with homologous recombination repair (HRR)-deficient HGSOC, although the development of PARP inhibitor resistance in some patients is revealing limitations to outcome. A proportion of patients with HRR-proficient cancers also benefit from PARP inhibitor therapy. Our aim is to compare mechanisms of resistance to the PARP inhibitor olaparib in these two main molecular categories of HGSOC and investigate a way to overcome resistance that we considered particularly suited to a cancer like HGSOC, where there is a very high incidence of TP53 gene mutation, making HGSOC cells heavily reliant on the G2 checkpoint for repair of DNA damage and survival. We identified alterations in multiple factors involved in resistance to PARP inhibition in both HRR-proficient and -deficient cancers. The most frequent change was a major reduction in levels of poly (ADP-ribose) glycohydrolase (PARG), which would be expected to preserve a residual PARP1-initiated DNA damage response to DNA single-strand breaks. Other changes seen would be expected to boost levels of HRR of DNA double-strand breaks. Growth of all olaparib-resistant clones isolated could be controlled by WEE1 kinase inhibitor AZD1775, which inactivates the G2 checkpoint. Our work suggests that use of the WEE1 kinase inhibitor could be a realistic therapeutic option for patients that develop resistance to olaparib.
Project description:WEE1 kinase is a key regulator of the G2/M transition. The WEE1 kinase inhibitor AZD1775 (WEE1i) induces origin firing in replicating cells. We show that WEE1i induces CDK1-dependent RIF1 phosphorylation and CDK2- and CDC7-dependent activation of the replicative helicase. WEE1 suppresses CDK1 and CDK2 kinase activities to regulate the G1/S transition after the origin licensing is complete. We identify a role for WEE1 in cell cycle regulation and important effects of AZD1775, which is in clinical trials.
Project description:PurposeWee1 kinase inhibitors are effective radiosensitizers in cells lacking a G1 checkpoint. In this study we examined the potential effect of Wee1 kinase inhibition on inducing replication stress in hepatocellular carcinoma (HCC).Methods and materialsFive independent datasets from the Oncomine database comparing gene expression in HCC compared to normal tissue were combined and specific markers associated with Wee1 sensitivity were analyzed. We then performed a series of in vitro experiments to study the effect of Wee1 inhibition on irradiated HCC cell lines with varying p53 mutational status. Clonogenic survival assays and flow cytometry using anti-γH2AX and phospho-histone H3 antibodies with propidium iodide were performed to study the effect of AZD1775 on survival, cell cycle, and DNA repair. Additionally, nucleoside enriched medium was used to examine the effect of altering nucleotide pools on Wee1 targeted radiation sensitization.ResultsOur analysis of the Oncomine database found high levels of CDK1 and other cell cycle regulators indicative of Wee1 sensitivity in HCC. In our in vitro experiments, treatment with AZD1775 radiosensitized and chemosensitized Hep3B, Huh7, and HepG2 cell lines and was associated with delayed resolution of γH2AX foci and the induction of pan-nuclear γH2AX staining. Wee1 inhibition attenuated radiation-induced G2 arrest in the Hep3B (TP53 null) and Huh7 (TP53 mutant) cell lines but not in the TP53 wild-type cell line HepG2. Supplementation with nucleosides reversed the radiation-sensitizing effect of AZD1775 and reduced the amount of cells with pan-nuclear γH2AX staining after radiation.ConclusionsRadiation sensitization with Wee1 inhibition occurs in cells regardless of their p53 mutational status. In this study we show for the first time that replication stress via the overconsumption of nucleotides plays an important role in AZD1775-induced radiation sensitization.
Project description:ObjectiveA major challenge in the treatment of platinum-resistant high-grade serous ovarian cancer (HGSOC) is lack of effective therapies. Much of ongoing research on drug candidates relies on HGSOC cell lines that are poorly documented. The goal of this study was to screen for effective, state-of-the-art drug candidates using primary HGSOC cells. In addition, our aim was to dissect the inhibitory activities of Wee1 inhibitor adavosertib on primary and conventional HGSOC cell lines.MethodsA comprehensive drug sensitivity and resistance testing (DSRT) on 306 drug compounds was performed on three patient-derived genetically unique HGSOC cell lines and two commonly used ovarian cancer cell lines. The effect of adavosertib on the cell lines was tested in several assays, including cell-cycle analysis, apoptosis induction, proliferation, wound healing, DNA damage, and effect on nuclear integrity.ResultsSeveral compounds exerted cytotoxic activity toward all cell lines, when tested in both adherent and spheroid conditions. In further cytotoxicity tests, adavosertib exerted the most consistent cytotoxic activity. Adavosertib affected cell-cycle control in patient-derived and conventional HGSOC cells, inducing G2/M accumulation and reducing cyclin B1 levels. It induced apoptosis and inhibited proliferation and migration in all cell lines. Furthermore, the DNA damage marker γH2AX and the number of abnormal cell nuclei were clearly increased following adavosertib treatment. Based on the homologous recombination (HR) signature and functional HR assays of the cell lines, the effects of adavosertib were independent of the cells' HR status.ConclusionOur study indicates that Wee1 inhibitor adavosertib affects several critical functions related to proliferation, cell cycle and division, apoptosis, and invasion. Importantly, the effects are consistent in all tested cell lines, including primary HGSOC cells, and independent of the HR status of the cells. Wee1 inhibition may thus provide treatment opportunities especially for patients, whose cancer has acquired resistance to platinum-based chemotherapy or PARP inhibitors.
