Abstract 4727: Role of SUMOylating enzymes in repair of Topoisomerase II mediated DNA damage
Authors: Ramesh Bhargavi , Sun Yilun , Nitiss John L. , Anand Jay , Nitiss Karin C. 
Published: August 2019

Topoisomerase 2 (Top2) enzymes regulate DNA topology for efficient DNA replication and transcription by introducing transient double strand breaks (DSBs) in the DNA. To generate the DSBs, Top2 forms a covalent intermediate with the DNA termed as the Top2 DNA covalent complex (Top2cc). Top2 is a target of clinically used anticancer agents such as etoposide and doxorubicin. Mammalian cells have two Top2 isozymes, Top2α and Top2β. Top2α is primarily expressed in proliferating cells and essential for cell division. Top2β is expressed in all cells and has been shown to play key roles in transcription. Top2 targeting agents generate anticancer activity by trapping both Top2αcc and Top2βcc. Factors that affect repair of Top2cc are likely to be a key determinant of the clinical efficacy of Top2 targeting agents. Identifying factors that play a role in repair of Top2cc offers a potential strategy for increasing clinical efficacy of Top2 targeting agents. Previous work has demonstrated that an important pathway for removal of trapped Top2 from DNA is initiated through proteolytic degradation of Top2 by the proteasome. Other proteases may also participate in degradation of the protein covalently bound to DNA. Proteasomal inhibition increased Top2cc, especially Top2β, and enhanced the cytotoxicity of Top2 targeting drugs. To study steps involved in proteasomal degradation of Top2cc, we developed a method to purify genomic DNA under conditions that preserve proteins covalently bound to DNA. Levels of trapped topoisomerases can be measured using Top2 isoform specific antibodies. Alternately, after micrococcal nuclease degradation of DNA, the proteins covalently bound to DNA can be characterized for post-translational modifications (PTMs) using antibodies specific for the modifying agents. Etoposide treatment induced SUMOylation of both Top2α and Top2β covalently bound to DNA. Blocking SUMOylation of Top2 by siRNA knockdown of the SUMO E2 conjugating enzyme UBC9 increased levels of both Top2α and Top2β covalent complexes, indicating that SUMOylation is likely involved in the repair of both Top2α and Top2β covalent complexes. We have extended this approach to identify SUMO ligases that directly act on Top2 isoforms. We found that depletion of the SUMO E3 PIAS4 resulted in elevated levels of Top2αcc with minimal effect on levels of Top2βcc. This result suggest that PIAS4 participates in a repair pathway that is specific for repairing Top2α covalent complexes. We have constructed PIAS4 knockouts using CRISPR/Cas9 and are currently examining the roles of other SUMO E3 ligases that may participate in the repair of Top2 damage. Our current results suggest that some pathways for repair of Top2 covalent complexes are likely isoform specific. This allows targeting of specific Top2 isoforms without the need to identify agents that discriminate between Top2 isoforms, and may help alleviate some of the undesirable side effects of Top2 targeting agents.

Citation Format: Bhargavi Ramesh, Yilun Sun, John L. Nitiss, Jay Anand, Karin C. Nitiss. Role of SUMOylating enzymes in repair of Topoisomerase II mediated DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4727.