Metnase, PIKK, and RPA Roles in DNA Damage and Replication Stress Responses

描述(由申请人提供)：大多数癌症患者接受的放射和/或化疗会导致DNA损伤，从而阻止DNA复制。正常细胞和肿瘤细胞通过激活DNA修复、细胞周期停滞(检查点)系统来响应DNA损伤和相关的复制应激，当损伤严重时，程序化死亡途径，统称为DNA损伤反应(DDR)。DDR蛋白在肿瘤抑制和基因组稳定(癌症病因学)以及肿瘤对放化疗(癌症治疗)的反应中发挥关键作用。DDR通路决定细胞在DNA损伤后的命运，包括细胞存活、基因组稳定性，以及通过凋亡、自噬、坏死、衰老或有丝分裂灾难导致的细胞死亡/永久生长停滞。在S期，细胞特别容易受到DNA损伤，因为大多数DNA损伤阻碍了复制叉的复制，造成复制压力。这一建议集中在几个在DNA修复、检查点激活和从复制应激中恢复中发挥作用的蛋白质上。Metnase和DNA-PK最初都是因为它们在DNA双链断裂(DSB)非同源末端连接(NHEJ)修复中的作用而被研究的。最近的研究表明，Metnase和DNA-PK(以及复制蛋白A(RPA)、ATM/ATR、Chk1等)也在检查点激活和复制应激恢复中发挥作用。复制检查点可防止新的源触发并稳定停滞的复制分叉，以防止分叉崩溃，从而为修复和重新启动留出时间。持续的复制压力可能会导致叉子坍塌，产生标记为磷酸化的单端DSB H_2AX(γ-H_2AX)RPA在失速的叉子上积累在单链DNA上，RPA32亚单位被磷脂酰肌醇3-激酶相关蛋白激酶(PIKKs)DNA-PK、ATM和ATR在多个位置磷酸化，导致Chk1激活和复制停止。Metnase还调节Chk1的激活和复制抑制。我们的中心假设是Metnase、DNA-PK和RPA在DDR中发挥作用，影响遗传毒性应激后细胞的命运，包括细胞存活、基因组稳定性和死亡途径激活。我们将确定Metnase(Aim 1)和Pikk磷酸化的RPA(Aim 2)在复制应激反应中的作用，包括复制停止、分叉重新启动、基因组稳定性、细胞存活和细胞凋亡。在目标3中，我们将定义Metnase和Pikk/RPA通路之间的上位关系，并测试复制应激剂与DDR抑制剂的新组合，以增强对乳腺、肺、胰腺、结肠、头颈部和白血病肿瘤细胞的杀伤。更好地了解DDR因子如何调控细胞命运决定将推动新的癌症治疗方法的发展，以改善局部肿瘤控制，并降低治疗诱导肿瘤进展和继发肿瘤诱导的风险。

DESCRIPTION (provided by applicant): Most cancer patients receive radio- and/or chemotherapy that causes DNA damage, which blocks DNA replication. Normal and tumor cells respond to DNA damage and associated replication stress by activating DNA repair, cell cycle arrest (checkpoint) systems, and when damage is severe, programmed death pathways, collectively termed the DNA damage response (DDR). DDR proteins play crucial roles in tumor suppression and genome stabilization (cancer etiology) as well as tumor response to radio- and chemotherapy (cancer treatment). DDR pathways determine cell fates in response to DNA damage, including cell survival, genome stability, and cell death/permanent growth arrest via apoptosis, autophagy, necrosis, senescence, or mitotic catastrophe. Cells are particularly vulnerable to DNA damage during S phase because most DNA lesions stall replication forks, causing replication stress. This proposal focuses on several proteins with roles in DNA repair, checkpoint activation, and recovery from replication stress. Metnase and DNA-PK were both initially characterized for their roles in DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ). Recent studies demonstrate that Metnase and DNA-PK (along with replication protein A (RPA), ATM/ATR, Chk1, and others) also function in checkpoint activation and replication stress recovery. The replication checkpoint prevents new origin firing and stabilizes stalled replication forks to prevent fork collapse, allowing time for repair and for restart. Persistent replication stress can lead to fork collapse, producing one-ended DSBs marked by phosphorylated H2AX (γ-H2AX). RPA accumulates on single-stranded DNA at stalled forks and the RPA32 subunit is phosphorylated at multiple sites by phosphoinositide 3-kinase-related protein kinases (PIKKs) DNA-PK, ATM and ATR, leading to Chk1 activation and replication arrest. Metnase also regulates Chk1 activation and replication arrest. Our central hypothesis is that Metnase, DNA-PK, and RPA operate within the DDR to influence cell fate after genotoxic stress, including cell survival, genome stability, and death pathway activation. We will determine roles of Metnase (Aim 1) and PIKK phosphorylation of RPA (Aim 2) in replication stress responses including replication arrest, fork restart, genome stability, cell survival and cell death by apoptosis. In Aim 3 we will define epistatic relationships between Metnase and PIKK/RPA pathways, and test novel combinations of replication stress agents plus DDR inhibitors to enhance killing of breast, lung, pancreatic, colon, head and neck, and leukemic tumor cells. A better understanding of how DDR factors regulate cell fate decisions will drive the development of novel cancer therapies to improve local tumor control, and reduce the risk of therapy-induced tumor progression and secondary tumor induction.