c Protein extracts were probed with anti-cyclin B1, CDC2 and pCDC2 antibodies. generated or analyzed during this study are included in this article and its additional files. Abstract Background Targeting poly ADP-ribose polymerase (PARP) has been recently identified as a promising option against gastric cancer (GC). However, PARP inhibitors alone achieve limited efficacy. Combination strategies, especially with homologous recombination (HR) Tasquinimod impairment, are of great hope to optimize PARP inhibitors efficacy and Trp53inp1 expand target populations but remains largely unknown. Herein, we investigated whether a WEE1/ Polo-like kinase 1 (PLK1) dual inhibitor AZD1775 reported to impair HR augmented anticancer activity of a PARP inhibitor olaparib and its underlying mechanisms. Methods GC cell lines and in vivo xenografts were employed to determine antitumor activity of PARP inhibitor combined with WEE1/PLK1 dual inhibitor AZD1775. Western blot, genetic Tasquinimod knockdown by siRNA, flow cytometry, Immunohistochemistry were performed to explore the underlying mechanisms. Results AZD1775 dually targeting WEE1/PLK1 enhanced effects of olaparib on growth inhibition and apoptotic induction in GC cells. Mechanistic investigations elucidate that WEE1/PLK1 blockade downregulated several HR-related proteins and caused an accumulation in H2AX. As confirmed in both Tasquinimod GC cell lines and mice bearing GC xenografts, these effects were enhanced by AZD1775-olaparib combination compared to olaparib alone, suggesting that disrupting HR-mediated DNA damage repairs (DDR) by WEE1/PLK1 blockade might be responsible for improved GC Tasquinimod cells response to PARP inhibitors. Given the DNA damage checkpoint as a primary target of WEE1 inhibition, our data also demonstrate that AZD1775 abrogated olaparib-activated DNA damage checkpoint through CDC2 de-phosphorylation, followed by mitotic progression with unrepaired DNA damage (marked by increased pHH3-stained and H2AX-stained cells, respectively). Conclusions PARP inhibitor olaparib combined with WEE1/PLK1 dual inhibitor AZD1775 elicited potentiated anticancer activity through disrupting DDR signaling and the DNA damage checkpoint. It sheds light on the combination strategy of WEE1/PLK1 dual inhibitors with PARP inhibitors in the treatment of GC, even in HR-proficient patients. Electronic supplementary material The online version of this article (10.1186/s13046-018-0790-7) contains supplementary material, which is available to authorized users. strong class=”kwd-title” Keywords: PARP inhibitor, WEE1/PLK1 dual inhibitor, Combination, Gastric cancer, HR deficiency, DNA damage checkpoint Background Gastric cancer (GC) is one of the most common malignancies and a leading cause of cancer-related mortality in China [1]. Although emerging targeted strategies have brought new hope to antitumor therapy, options for advanced GC with high heterogeneity are still few, only three drugs (trastuzumab, ramucirumab and apatinib) have been currently approved, and the prognosis of advanced GC remains poor. Hence, development of novel strategies against advanced GC is urgently needed. Poly ADP-ribose polymerase (PARP) inhibitors that competitively combine and trap PARP to disrupt (SSB) single-strand DNA breaks repairs and elicit anticancer activity emerge as a promising strategy for GC [2C4]. However, PARP inhibitors alone exert limited efficacy in the treatment of cancers and how to optimize PARP inhibitors eligible populations and effectiveness remain poorly understood. Of interest, SSB can be converted into double-strand DNA breaks (DSB), which results in Tasquinimod treatment failure of targeting PARP when homologous recombination (HR) is functional [2, 3]. Thus, defects in HR has been identified as a predictor for PARP inhibitors sensitivity. For instance, PARP inhibitors olaparib and rucaparib have been approved to treat BRCA-defective ovarian or prostate cancer patients [5] while GC patients harboring low-ATM gains greater survival benefit than high-ATM patients when treated with olaparib plus paclitaxel [4]. Cancers deficient in alternative HR-related factors like RAD51, 53BP1, ARID1A and CCDC6 are also proved sensitive to PARP inhibitors [3, 6, 7]. Based on these, compromising HR functions.