DNA Damage Checkpoint Recovery and Cancer

描述(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。虽然人们致力于揭示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，人们知之甚少。重要的是，新的证据表明，在肿瘤发生过程中，癌细胞可能利用恢复机制绕过DDR，而从放化疗中恢复检查点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。我们最近鉴定了第一个研究检查点恢复的体外系统，并使用这个系统确定长城(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于磷酸酶的检查点恢复激酶的调节；我们的结果表明Gw1和Plk1的协同激活，并将Gw1的磷酸化和相互作用表征为检查点恢复的早期事件。此外，对癌细胞系和小鼠肿瘤模型的初步研究表明，GWL是加强化疗和阻止肿瘤复发的有效靶点。在这项提案中，我们将解决有关Gwl在检查点恢复中的调节机制以及它在癌症治疗和肿瘤复发中的作用的基本问题。目的1研究Gw1和Plk1是如何受DNA损伤调控的。我们的研究将利用无细胞系统和位点特异性磷酸酶检测来研究DNA损伤诱导的Gw1和Plk1的磷酸酶依赖的调节。目标2将询问Gwl和Plk1如何从检查站逮捕中重新激活。我们将表征新的Gwl/Plk1相互作用和磷酸化作为启动恢复的潜在分子开关。在目标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，我们将直接评估新的治疗思路，其中靶向Gwl或其在检查点恢复期间的初始激活 更有效和更具体的癌症治疗。

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.