MTOR Inhibition Overcomes Primary And Acquired Resistance To Wee1 Inhibition By Augmenting Replication Stress In Epithelial Ovarian Cancers

Objectives: Epithelial ovarian cancer is characterized by universal TP53 mutations,which result in G1/S checkpoint deficiencies.Therefore,it is hypothesized that abrogation of the G2/M checkpoint with Wee1 inhibitor might preferentially sensitize p53-defective ovarian cancer cells.Ultimately,drug resistance cannot be avoided.Our research is devoted to exploring the mechanism of resistance of Wee1 inhibition and propose solutions.Methods: Here,through a high-throughput unbiased proteomic profiling(RPPA),we found the complementally activated mTOR pathway contribute greatly to Wee1 inhibitor resistance.Then,we verified the results by western blotting.The effect of Wee1 combined with mTOR on the proliferation of ovarian cancer cells was analyzed by CCK8,clone formation.Apoptosis and cell cycle were analyzed by flow cytometry.DNA damage was studied by immunofluorescence.DNA fiber was used to analyze the replication pressure of tumor cells.PDX in vivo model was used to verify the effect in vivo.Results: In our study,high-throughput protein array(RPPA)analysis showed that mTOR pathway was complementary activated in ovarian cancer cells treated with Wee1 inhibitor,and western blot was used to verify the complementary activation of mTOR in ovarian cancer cells after short-term exposure and long-term exposure to Wee1 inhibitor AZD1775.We analyzed the effect of Wee1 inhibition combined with mTOR inhibition on ovarian cancer cells by CCK8,clone formation.The changes of apoptosis and cell cycle after combined inhibition of Wee1 and mTOR were analyzed by flow cytometry.The DNA damage of cells was studied by immunofluorescence,the replication pressure of tumor cells after combined inhibition of Wee1 and mTOR was analyzed by DNA fiber,and the effect of combined inhibition of Wee1 and mTOR was saved by reverse addition of d NTPs.The in vivo model of PDX,which can best reflect the molecular characteristics and heterogeneity of patients,was used to verify the effect of combined inhibition of Wee1 and mTOR on ovarian cancer in vivo.The mTOR pathway,DNA damage and replication of pressure marker RPA32 in tumor tissue samples of PDX were analyzed by immunohistochemistry,which was consistent with the conclusion of in vitro experiment.Conclusions: Our study confirmed that dual Wee1/mTOR inhibition induced a large number of DNA replication stress,resulting in replication fork stall and DNA damage,and the depletion of nucleotide metabolic substrates is a necessary condition for the synergistic action of Wee1i and mTORi.Our study has opened up a broader therapeutic window for Wee1 inhibitors in the treatment of epithelial ovarian cancer.