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

摘要 氧化应激、电离辐射和包括TOP2毒药在内的化疗药物 如依托泊苷等均可促进治疗所致衰老。目前的范例是DNA损伤 信号是细胞衰老的共同决定因素，无论是由端粒侵蚀还是由 染色体双链断裂。然而，我们最近的研究表明，脂质过氧化和 由此产生的反应性脂质物种(RLS)作为这一途径的关键介质。这项拟议的工作将 研究TOP2作为RLS促进加速衰老的关键靶点。在这里，我们将申请 检测TOP2半胱氨酸硫醇作为RLS潜在修饰位点的生化和分子工具 如4-羟基壬烯醛(4-HNE)。我们将确定RLS修改是否会导致稳定的形成 TOP2-DNA裂解复合体(Top2cc)，导致DNA双链断裂和细胞衰老。至 直接测试DNA损伤是否真的足以衰老，我们将应用Cas9和混杂 作为“纯”双链断裂的来源的gRNA。此外，将Cas9定向损害与RLS相结合将 测试这两个信号是在相同还是不同的路径上起作用。我们还将继续研究蛋白质组学- 广泛分析RLS的潜在靶点，超越TOP2，可能调节衰老。我们将延长这项工作 以小鼠同基因肿瘤为模型，评价RLS在TOP2体内中毒中的作用。我们还将使用基因 将依托泊苷或放射治疗后形成的衰老肿瘤细胞耗竭作为评估肿瘤细胞毒性的一个手段 治疗诱导肿瘤衰老对基因毒性治疗的反应。这项工作可能会建立一种新的 依托泊苷及相关化疗药物作为间接拓扑异构酶毒物的作用机制 抗衰老药物，可能对其临床使用产生影响，无论是单独使用还是与其他药物联合使用 探员们。

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.