Abstract 842: DNA opilymerase β participates in the repair of DNA damage from topoisomerase II

Abstract

Abstract DNA topoisomerase II (Top2) is the target of several important anti-cancer agents, including doxorubicin and etoposide. Clinically active Top2 agents, termed Top2 poisons, act by blocking the enzyme reaction at a step where the protein is covalently bound to DNA. This leads to the accumulation of Top2/DNA complexes that act as DNA damaging agents that interfere with DNA metabolism and trigger cell death. The mechanisms leading to tumor specific killing by Top2 poisons remain poorly understood, and have been hypothesized to result in part from DNA repair defects that arise during tumorigenesis. If this hypothesis is correct, then targeting repair pathways important for topoisomerase mediated cell killing could be used to enhance the activity of Top2 poisons. Similar reasoning has led to extensive trials of agents such as PARP inhibitors, and other DNA repair inhibitors. The DNA repair pathways that have been examined for sensitization to Top2 poisons include homologous recombination, non-homologous end-joining, proteasome mediated degradation, and nucleolytic activities that may participate in the removal of protein/DNA adducts (such as Tdp1 and Tdp2). We have been studying the repair of damage from Top2 poisons, and have optimized a quantitative assay for Top2/DNA covalent complexes in cultured mammalian cells. This assay, termed the ICE assay, detects Top2 covalent complexes by DNA purification followed by antibody detection of protein adducts. Using this assay, we have demonstrated a role of the MRN complex in processing Top2/DNA adducts, using both MRN knockdowns and small molecule inhibitors of the Mre11 nuclease activities. We also confirmed the importance of proteolytic pathways in repairing Top2 damage. A surprising candidate protein for participation in repair of damage from Top2 poisons is DNA polymerase β (pol β), an “X” family polymerase critical for base excision repair. Recent results have implicated pol β in processing of protein/DNA adducts (1). We found that the pol β inhibitor pamoic acid (4-[(3-Carboxy-2-hydroxynaphthalen-1-yl)methyl]-3-hydroxynaphthalene-2-carboxylic acid) and etoposide led to a large increase in Top2/DNA covalent complexes compared to etoposide alone. We are currently validating these results using other pol β inhibitors and gene knockdown approaches. We speculate that the dRP lyase activity may play a role in processing DNA damage arising from Top2 poisons. Literature cited: 1. Lawson, M.H., et al. (2011) Two Novel Determinants of Etoposide Resistance in Small Cell Lung Cancer. Cancer Res, 71, 4877-4887. Citation Format: Yilun Sun, Sule Bertram, John L. Nitiss. DNA opilymerase β participates in the repair of DNA damage from topoisomerase II. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 842. doi:10.1158/1538-7445.AM2014-842