DNA Damage Checkpoint Recovery and Cancer

描述（由申请人提供）：DNA损伤后的细胞命运由细胞DNA损伤反应（DDR）机制决定，该机制包括DNA修复、细胞周期检查点和程序性细胞死亡。尽管大量的研究致力于揭示DDR的激活网络，但对于细胞如何使DDR失活并恢复细胞周期进程，这一过程被称为检查点恢复，我们知之甚少。重要的是，新出现的证据表明，癌细胞可能在肿瘤发生过程中利用恢复机制绕过DDR，放射或化疗后的检查点恢复会导致化疗耐药和肿瘤复发，这表明检查点恢复是改善癌症治疗和患者生存的理想靶点。该项目的长期目标是描述检查点恢复的机制，并研究检查点恢复对癌症进展和治疗的影响。我们最近描述了第一个研究检查点恢复的体外系统，并使用该系统鉴定了Greatwall （Gwl）激酶是检查点恢复的重要调节因子。有趣的是，我们的初步研究发现了新的依赖磷酸酶的检查点恢复激酶调控；我们的研究结果表明Gwl和Plk1的协同激活，并将Gwl磷酸化和相互作用描述为检查点恢复的早期事件。此外，在肿瘤细胞系和小鼠肿瘤模型中的初步研究表明，Gwl是增强化疗和阻断肿瘤复发的有效靶点。在本提案中，我们将解决关于Gwl在检查点恢复中的调节机制及其在癌症治疗和肿瘤复发中的作用的基本问题。目的1将研究DNA损伤如何调节Gwl和Plk1。我们的研究将利用无细胞系统和位点特异性磷酸酶测定来研究Gwl和Plk1的DNA损伤诱导和磷酸酶依赖性调节。目标2将询问Gwl和Plk1如何从检查点逮捕中重新激活。我们将描述新的Gwl/Plk1相互作用和磷酸化作为启动恢复的潜在分子开关。在Aim 3中，我们将描述Gwl上调如何促进顺铂治疗后的细胞恢复，从而促进肿瘤复发。然后，我们将直接评估靶向Gwl或其在检查点恢复期间的初始激活的新治疗理念

DESCRIPTION (provided by applicant): Cell fate after DNA damage is determined by the cellular DNA damage response (DDR) mechanism that encompasses DNA repair, cell cycle checkpoints, and programmed cell death. Although extensive efforts were dedicated to revealing the activation network of the DDR, little is known about how the cell deactivates the DDR and resumes cell cycle progression, a process termed checkpoint recovery. Importantly, emerging evidence suggests that cancer cells may utilize the recovery mechanism to bypass the DDR during tumorigenesis, and that checkpoint recovery from radio- or chemotherapy causes chemoresistance and tumor recurrence, suggesting checkpoint recovery as an ideal target to improve cancer therapy and patient survival. The long-term goal of this project is to delineate the mechanism of checkpoint recovery and to investigate the implication of checkpoint recovery for cancer progression and therapeutics. We recently characterized the first in vitro system to study checkpoint recovery, and used this system to identify Greatwall (Gwl) kinase as an essential regulator of checkpoint recovery. Interestingly, our preliminary studies discovered novel phosphatases-dependent regulation of checkpoint recovery kinases; our results suggested coordinated activation of Gwl and Plk1, and characterized Gwl phosphorylation and interaction as early events of checkpoint recovery. Moreover, preliminary studies in cancer cell lines and mouse tumor models suggested Gwl as an effective target to enhance chemotherapy and block tumor recurrence. In this proposal we will address fundamental questions regarding the regulatory mechanism of Gwl in checkpoint recovery and its role in cancer therapy and tumor recurrence. Aim 1 will investigate how Gwl and Plk1 are regulated by DNA damage. Our study will take advantage of the cell-free system and a site-specific phosphatase assay to investigate DNA damage-induced, and phosphatase-dependent regulation of Gwl and Plk1. Aim 2 will ask how Gwl and Plk1 re-activate from checkpoint arrest. We will characterize novel Gwl/Plk1 interaction and phosphorylation as potential molecular switches that initiate recovery. In Aim 3, we will characterize how Gwl up-regulation promotes cell recovery from cisplatin treatment and thereby facilitating tumor recurrence. We will then directly evaluate new therapeutic ideas in which targeting Gwl or its initial activation during checkpoint recovery leads to more effective and specific cancer therapy.