Targets of Reactive Lipid Species regulating DNA damage response and cell senescence

摘要 氧化应激、电离辐射和化疗剂，包括拓扑异构酶II（Top2）毒物 如依托泊苷都能促进治疗诱导的衰老。目前的模式是DNA损伤 信号转导是细胞衰老的共同决定因素，无论是由端粒侵蚀或 染色体双链断裂。然而，我们最近的研究表明脂质过氧化和 从而产生作为该途径的关键介质的反应性脂质物质（RLS）。这项工作将 研究Top2作为RLS促进加速衰老的关键靶标。在这里，我们将应用 生物化学和分子工具，以检查Top2半胱氨酸硫醇作为RLS修饰的潜在位点 如4-羟基壬烯醛（4-HNE）。我们将确定RLS修饰是否诱导形成稳定的 Top2-DNA切割复合物（Top2 cc），导致DNA双链断裂和细胞衰老。到 直接测试DNA损伤是否确实足以衰老，我们将应用Cas9和滥交 gRNA作为“纯”双链断裂的来源。此外，将Cas9定向损伤与RLS相结合， 提供了一个测试，这两个信号是否在相同或不同的途径中起作用。我们还将研究蛋白质组- 对可能调节衰老的Top2以外的RLS潜在靶点的广泛分析。我们将延长工作 使用小鼠中的同基因肿瘤来评估RLS在体内Top2中毒中的作用。我们还将使用遗传 消除依托泊苷或放疗后形成的衰老肿瘤细胞，作为评价 治疗诱导的衰老对肿瘤对遗传毒性治疗的反应。这项工作可能会建立一个新的 作为间接拓扑异构酶毒物的依托泊苷和相关化疗剂的作用机制， 促衰老药物，无论是单独使用还是与其他药物联合使用， 剂.

Abstract Oxidative stress, ionizing radiation and chemotherapy agents including topoisomerase II (Top2) poisons such as etoposide can all promote therapy-induced senescence. The current paradigm is that DNA damage signaling is the common determinant of cellular senescence, whether induced by telomere erosion or chromosomal double strand breaks. However, our recent studies have implicated lipid peroxidation and resulting production of reactive lipid species (RLS) as key mediators of this pathway. This proposed work will examine Top2 as the critical target of RLS that promotes accelerated senescence. Here, we will apply biochemical and molecular tools to examine Top2 cysteine thiols as potential sites for modification by RLS such as 4-hydroxynonenal (4-HNE). We will determine if RLS modifications induce formation of the stable Top2-DNA cleaved complex (Top2cc), resulting in DNA double strand breaks and cellular senescence. To directly test whether DNA damage is indeed sufficient for senescence, we will apply Cas9 and promiscuous gRNAs as a source of "pure" double strand breaks. Further, combining Cas9-directed damage with RLS will provide a test of whether the two signals act in the same or distinct pathways. We will also pursue proteome- wide analysis of potential targets of RLS beyond Top2 that may regulate senescence. We will extend the work to evaluate the role of RLS in Top2 poisoning in vivo, using syngeneic tumors in mice. We will also use genetic depletion of senescent tumor cells formed after etoposide or radiation as a means to evaluate the relevance of therapy-induced senescence to tumor response to genotoxic therapy. This work may establish a new mechanism of action for etoposide and related chemotherapy agents as indirect topoisomerase poisons and pro-senescent drugs, with potential for impacts on their clinical use, both alone and in combination with other agents.