Epithelial ovarian cancer is characterized by universal TP53 mutations, which result in G1/S checkpoint deficiencies. Therefore, it is hypothesized that the abrogation of the G2/M checkpoint with Wee1 inhibitor might preferentially sensitize TP53-defective ovarian cancer cells. Given the extremely high molecular diversity in ovarian cancer, one approach to improving the clinical efficacy is to identify drug combinations that either broaden the applicable spectrum or circumvent resistance. Here, through a high-throughput unbiased proteomic profiling (RPPA), we found the complementary activated mTOR pathway contributes greatly to Wee1 inhibitor resistance. A combination of Wee1 and mTOR inhibits synergistically inhibiting tumor growth in ovarian cancer cell lines and patient-derived xenograft that closely mimic the heterogeneity of patient tumors. Mechanistically, dual Wee1/mTOR inhibition induced massive DNA replication stress, leading to fork stalling and DNA damage. Moreover, we found that the addition of nucleotide metabolic substrate dNTPs alleviated replication stress, restored the cell cycle and reduced apoptosis to some extent, supporting dNTPs depletion is necessary for the synergy between Wee1 and mTOR inhibits. These results suggest that our study opening up a wider therapeutic window of Wee1 inhibitor for the treatment in epithelial ovarian cancers. AJCR Copyright © 2020.