Abstract 1658: Proteolytic processing pathways for topoisomerase covalent complexes

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 can trigger cell death. Surviving Top2-induced damage likely requires the repair of the enzyme induced damage and has been hypothesized to include pathways that proteolyze the protein bound to DNA followed by nucleolytic removal of the remaining peptide that is bound to DNA by a phosphotyrosyl linkage. This model of repair suggests that inhibition of proteolysis would lead to elevated levels of intact Top2/DNA complexes, and increased sensitivity to Top2 poisons such as etoposide. We have tested this model using the proteasome inhibitor carfilzomib in a pediatric rhabdomyosarcoma cell line (Rh30). We found that carfilzomib enhanced cell killing by etoposide, and that co-treatment of Rh30 cells with carfilzomib led to increases in both Top2 alpha and Top2 beta covalent complexes. These results suggest that proteasomal degradation of both Top2 isoforms is important for repairing DNA damage arising from etoposide. We are also interested in identifying the determinants of Top2 degradation following treatment with etoposide, especially proteins that recognize the trapped enzyme as DNA damage. We used a yeast genetic model system to address this question. We showed that the proteasome inhibitor MG132 greatly enhanced the sensitivity of yeast cells to etoposide, and that co-treatment of MG132 and etoposide led to elevated levels of Top2/DNA complexes compared to etoposide alone. We found that deletion of the human RNF4 homolog Slx5/Slx8 (a SUMO dependent ubiquitin ligase) also leads to an increase in Top2 covalent complexes induced by etoposide. We found that deletion of the genes encoding either Slx5 or Slx8 along with the repair protein Tdp1 led to much higher levels of cell killing compared to single deletions. These results suggest that there are at least two independent pathways for repairing Top2 damage, one dependent on Slx5/Slx8 dependent protein degradation and a second pathway dependent on nucleolytic removal of Top2. We suggest that Slx5/Slx8 plays a role in targeting Top2 for degradation following etoposide treatment. Our results also provide a rationale for the combination of a proteasome inhibitor with etoposide as an approach of increasing the efficacy of etoposide. Citation Format: Yilun Sun, Karin C. Nitiss, John L. Nitiss. Proteolytic processing pathways for topoisomerase covalent complexes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1658. doi:10.1158/1538-7445.AM2015-1658