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.