Project Summary Management of gastrointestinal stromal tumor (GIST) treatment has been revolutionized by the identification of activating mutations in KIT and PDGFRA and clinical application of tyrosine kinase inhibitors (TKIs) in advanced disease. However, resistance to all approved lines of TKIs is a life-threatening clinical obstacle in metastatic GIST patients, who have polyclonal tumors comprised of a variety of resistance mutations in KIT/PDGFRA. Development of effective treatment strategies for refractory GIST requires identification of novel targets. Using kinome profiling and loss-of function assays, we found that GIST are exquisitely dependent on Wee1 activity. Wee1 has long been known as a critical G2/M cell cycle checkpoint gatekeeper controlling genomic integrity and regulating proliferation. However, recent studies have implicated Wee1, both directly and indirectly, in other cellular functions, including restoration of stalled replication forks and chromatin remodeling. Transcriptome profiling of TKI-sensitive GIST cell lines highlighted a role for oncogenic KIT/PDGFRA in DNA damage response (DDR) and replication stress. Our in vitro and in vivo studies revealed significant efficacy of adavosertib (Wee1 inhibitor) in combination with avapritinib (KIT/PDGFRA inhibitor) in TKI-sensitive KIT and PDGFRA-mutant GIST lines and in a patient-derived xenograft (PDX) model. Furthermore, our preliminary data in the TKI-refractory setting demonstrated therapeutic synergies between Wee1 inhibition and the topoisomerase II inhibitor, doxorubicin, at low nanomolar concentrations in a panel of TKI-resistant GIST cell lines, suggesting Wee1 inhibition sensitizes refractory GIST to doxorubicin, providing viable targets beyond KIT and PDGFRA. We hypothesize that oncogenic KIT/PDGFRA activates DDR proteins to inhibit replicative stress and DNA damage and maintain genomic stability in GIST leading to resistance to DNA damaging agents. Our preliminary findings provide a strong premise that Wee1 is a critical mediator of this process and that its inhibition has potential in the treatment of GIST tumors. Studies interrogating mechanisms of Wee1 and KIT/PDGFRA cooperativity in response to DNA damage and replication stress in GIST are warranted to understand this critical cell-protective mechanism in GIST, which may represent a novel therapeutic vulnerability. Ultimately, this proposal seeks to test the hypothesis that Wee1 inhibition will be a valuable addition to current treatments for GIST patients with TKI-sensitive or -resistant disease. We believe that the studies outlined in this proposal will provide novel insights into the role of Wee1 in maintaining genomic integrity as well as preclinical data to support clinical trials in GIST.