Mechanisms of PIDDosome Signaling, a p53-Independent Apoptotic Response to DNA Damage

项目摘要 P53基因突变及其伴随的凋亡通路损害肿瘤细胞对辐射和 在许多人类恶性肿瘤中进行化疗。结合遗传学、功能基因组学和蛋白质组学 在过去的6年里，我们在哺乳动物细胞和斑马鱼胚胎中发现了一种新的凋亡途径。 通过激活PIDDosome(PIDD-RAIDD-RIDD)，完全绕过P53依赖的通路 Caspase-2)复合体(Sidi等人，Cell 2008；Ando等人，Mol Cell 2012；Thompson等人，Mol Cell 2015； Ando等人，J Cell Biol 2017)。不同于线粒体凋亡体(cytc-Apaf1-caspase-9)和死亡 受体复合体(Fas-FADD-caspase-8)，PIDDosome不需要p53来激活或 功能。PIDDosome组装可以通过抑制其负调控因子Chk1激酶来激活。AS 这样，Chk1抑制剂恢复了p53突变斑马鱼胚胎、MEF和人类癌症的辐射敏感性 细胞系。PIDDosome对破坏DNA的化疗也有反应 拓扑异构酶抑制剂。总之，PIDDosome途径定义了一个新的凋亡轴和 一种有前景的克服癌症治疗耐药性的靶向策略。 然而，我们对PIDDosome的分子理解仍然非常有限。为了扩大我们的 了解该途径，并确定新的诊断工具和其中的药物靶点，这项提议将 重点研究DNA损伤在脊椎动物细胞中触发PIDDosome组装的机制。 到目前为止，我们已经证明，DNA损伤通过(I)ATM/ATR介导的方式触发PIDDosome的形成 PIDD的磷酸化，使RAIDD能够招募到平台上(Mol Cell 2012)；以及 PIDD与核磷蛋白(NPM1)的结合，为PIDDosome组装(JCB)提供支架 2017年)。在目标1中，我们将阐明一种新发现的PIDD相互作用因子DNA的机制 修复蛋白FANCI，将PIDD招募到DNA交联链上，并通过ATM在这些位置使其磷酸化 损伤。值得注意的是，这些实验可能确定FANCI是第一个通过生物化学描述的 脊椎动物的“修复/死亡”开关。在目标2中，我们将阐明NPM1和两个 新发现的核仁PIDD结合蛋白NOLC1和NCL协调协调 PIDDosome的形成对IR的响应。这些实验可能最终勾勒出主要的细胞凋亡 核仁DNA损伤反应中的分支。最后，利用异种移植瘤模型 辐射抗性(Liu等人，NAT Cell Biol，原则上接受)，我们将首次评估 PIDDosome靶向作为克服TP53突变癌症放射抵抗的策略的潜力。 总之，这些研究将PIDDosome整合到细胞对DNA修复的反应中 失效、复制应激和核仁应激。因此，我们的建议非常适合揭示这一角色 PIDDosome在癌症病因学中的作用，是细胞凋亡领域争论最激烈的问题之一。

Project Summary Mutations in p53 and attendant apoptotic pathways impair tumor cell responses to radiation and chemotherapy in many human malignancies. Combining genetics, functional genomics and proteomics in mammalian cells and zebrafish embryos these past 6 years, we identified a novel apoptotic pathway that bypasses p53-dependent pathways altogether via activation of the PIDDosome (PIDD-RAIDD- caspase-2) complex (Sidi et al., Cell 2008; Ando et al., Mol Cell 2012; Thompson et al., Mol Cell 2015; Ando et al., J Cell Biol 2017). Unlike the mitochondrial apoptosome (cytc-Apaf1-caspase-9) and death receptor complex (FAS-FADD-caspase-8), the PIDDosome does not require p53 for activation or function. PIDDosome assembly can be activated by inhibiting its negative regulator, Chk1 kinase. As such, Chk1 inhibitors restore radiosensitivity in p53 mutant zebrafish embryos, MEF, and human cancer cell lines. The PIDDosome is also responsive to DNA damaging chemotherapies such as topoisomerase inhibitors. Altogether, the PIDDosome pathway defines both a novel apoptotic axis and a promising targeted strategy for overcoming treatment resistance in cancer. However, our molecular understanding of the PIDDosome remains very limited. To expand our knowledge of the pathway and identify novel diagnostic tools and drug targets therein, this proposal will focus on the mechanisms by which DNA damage triggers PIDDosome assembly in vertebrate cells. Thus far, we have shown that DNA damage triggers PIDDosome formation via (i) ATM/ATR-mediated phosphorylation of PIDD, which enables RAIDD recruitment to the platform (Mol Cell 2012); and (ii) the binding of PIDD to nucleophosmin (NPM1), which provides a scaffold for PIDDosome assembly (JCB 2017). In Aim 1, we will elucidate the mechanism by which a newly identified PIDD interactor, the DNA repair protein FANCI, recruits PIDD to DNA crosslinks and enables its phosphorylation by ATM at these lesions. Notably, these experiments may identify FANCI as the first biochemically described “repair/death” switch in vertebrates. In Aim 2, we will elucidate the mechanism by which NPM1 and two newly identified nucleolar PIDD-binding proteins, NOLC1 and NCL, coordinately orchestrate PIDDosome formation in response to IR. These experiments may ultimately outline the major apoptotic branch in the nucleolar DNA damage response. Finally, using xenograft models of intrinsic tumor radioresistance (Liu et al., Nat Cell Biol, accepted in principle), we will assess for the first time the potential of PIDDosome targeting as a strategy to overcome radioresistance in TP53 mutant cancers. Altogether, these studies integrate the PIDDosome in the cellular responses to DNA repair failure, replication stress and nucleolar stress. Our proposal is thus ideally positioned to reveal the role of the PIDDosome in cancer etiology, one of the most hotly debated questions in the field of apoptosis.