Abstract Cells have developed extensive networks and pathways to detect and repair DNA at the sites of damage. The DNA damage response (DDR) involves a complex cascade of events and many proteins containing BRCA1 C-terminal (BRCT) modular domains are involved in this process. These domains are known to bind phosphopeptides and can occur as singleton or as tandem domains (tBRCT). Previously in our lab, 7 tBRCT containing proteins were used to perform a systematic analysis of protein-protein interactions (PPIN) in the DDR network. 18 kinases were found to be interactors in this network. We hypothesized that the kinases contained in this DDR network would be targets for sensitization to standard chemotherapy. We extracted the 18 kinases and conducted systematic pharmacological and siRNA based screens to evaluate the effect of inhibition of these kinases in combination with platinum-based agents. These screens revealed that inhibition of WEE1, a kinase known to be a negative regulator of mitosis has synergistic effects in combination with cisplatin in various lung cancer cell lines. In the tBRCT network, WEE1 interacted with PAXIP1, a tBRCT-containing protein that forms foci at sites of DNA damage and is required for cells to progress to mitosis. Therefore, the PAXIP1-WEE1 interaction in combination with results from our inhibitor screen prompted us to evaluate the role of PAXIP1 in WEE1 inhibitor treatment of lung cancer cell lines. AZD1775 (formerly, MK-1775) is a WEE1 inhibitor currently in clinical trials for various tumor types. We evaluated the levels of WEE1 expression in 15 lung cancer cell lines and observed that cell lines with high WEE1 levels have a better response to AZD1775 than cell lines that express low levels of WEE1. Furthermore, PAXIP1 levels are directly related to the cellular response to AZD1775 alone or in combination with cisplatin. Overexpressing PAXIP1 in lung cancer cell lines leads to an increased mitotic index upon WEE1 inhibition at the G2/M checkpoint. Overexpression of PAXIP1 also leads to increased levels of apoptosis when cells are treated with AZD1775 compared to cells with low levels of PAXIP1. We are currently evaluating the expression levels of PAXIP1 and WEE1 in lung tumors using tissue microarrays and exploring the mechanisms by which PAXIP1 regulates WEE1. Overall, our results indicate that PAXIP1 modulates the efficacy of WEE1 inhibition by AZD1775 and could be a potential biomarker of AZD1775 response. Citation Format: Ankita Jhuraney, Nicholas T. Woods, Fumi Kinose, Douglas W. Cress, Jhanelle E. Gray, Eric B. Haura, Uwe Rix, Alvaro N. Monteiro. Overexpression of PAXIP1 potentiates WEE1 inhibitor action in lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2414. doi:10.1158/1538-7445.AM2014-2414