Elucidating the critical role of Wee1 in GIST

项目摘要 胃肠道间质瘤（GIST）治疗的管理已经发生了革命性的变化， KIT和PDGFRA的激活突变以及酪氨酸激酶抑制剂（TKI）在晚期乳腺癌中的临床应用 疾病然而，对所有已批准的TKI系列的耐药性是转移性肿瘤患者中危及生命的临床障碍。 GIST患者，其具有由KIT/PDGFRA中的多种抗性突变组成的多克隆肿瘤。 难治性GIST的有效治疗策略的发展需要确定新的目标。使用 通过激酶组分析和功能丧失测定，我们发现GIST精确地依赖于Wee 1活性。 Wee 1长期以来被认为是控制基因组完整性的关键G2/M细胞周期检查点看门人， 调节增殖。然而，最近的研究表明，Wee 1直接或间接地参与了 细胞功能，包括恢复停滞的复制叉和染色质重塑。转录组 对TKI敏感的GIST细胞系的分析强调了致癌KIT/PDGFRA在DNA损伤反应中的作用 (DDR)和复制压力。我们的体外和体内研究显示，阿达沃塞替（Wee 1 抑制剂）与avapritinib（KIT/PDGFRA抑制剂）联合治疗TKI敏感性KIT和PDGFRA突变型GIST 细胞系和患者来源的异种移植物（PDX）模型中。此外，我们在TKI难治性 结果证明了Wee 1抑制和拓扑异构酶II抑制剂之间的治疗协同作用， 在一组TKI耐药GIST细胞系中，低纳摩尔浓度的多柔比星表明Wee 1 抑制使难治性GIST对阿霉素敏感，提供了KIT和PDGFRA以外的可行靶点。我们 假设致癌KIT/PDGFRA激活DDR蛋白以抑制复制应激和DNA损伤 并维持GIST中的基因组稳定性，从而导致对DNA损伤剂的抗性。我们的初步研究结果 提供了一个强有力的前提，即Wee 1是这一过程的关键介质，并且其抑制作用具有潜在的抑制作用。 GIST肿瘤的治疗。Wee 1与KIT/PDGFRA协同作用的机制探讨 对GIST中DNA损伤和复制应激的反应是理解这种关键的细胞保护作用的必要条件。 GIST的机制，这可能代表了一种新的治疗漏洞。最终，该提案旨在 检验Wee 1抑制将是GIST患者当前治疗的有价值补充的假设， TKI敏感或耐药疾病。我们相信，这项提案中概述的研究将提供新的见解， Wee 1在维持基因组完整性以及临床前数据以支持GIST临床试验中的作用。

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.