Abstract 3628: Tyrosyl-DNA phosphodiesterase I as a therapeutic target: lessons from yeast functional studies

Tyrosyl-DNA phosphodiesterase I (Tdp1) resolves various 3′phospho-adducts within DNA breaks induced by numerous chemotherapeutics. This includes Tdp1 ability to repair DNA topoisomerase I (Topo1)-DNA covalent complexes reversibly stabilized by camptothecins (CPTs) such as the FDA approved CPT derivatives topotecan and irinotecan. Tdp1 uses a two-step catalytic cycle that requires the formation of a Tdp1-DNA covalent intermediate through its nucleophilic histidine (Hisnuc) resulting in dissociation of the adduct, while the general acid/base histidine (Hisgab) mediates Tdp1 dissociation. A Tdp1Hisgab to Arg mutant stabilizes the Tdp1-DNA intermediate and is associated with autosomal recessive ataxia SCAN1. Alternative substitutions of Hisgab transforms yeast Tdp1 into a potent Topo1-depended toxin via stabilization of enzyme-DNA intermediates. We propose that stabilization of the Tdp1-DNA complex is a potential novel therapeutic anti-cancer strategy. As proof-of-concept, we analyzed two different catalytic human Tdp1 mutants in HEK293 cells without additional stress. Expression of hTdp1HisnucAla and hTdp1HisgabAsn mutants induced cytotoxicity, which correlates with stabilization of their enzyme-DNA intermediates. Serendipitously, we discovered that Tdp19s N-terminal residues are critical for Tdp1HisnucAla catalytic activity but not for wild type Tdp1. In addition, analyzing the effect of the N-terminal domain of other Tdp1 catalytic mutants revealed that these residues influence the formation of covalent Tdp1-DNA intermediates in vitro, which is conserved from yeast to human. To better understand the interaction between Tdp1 and its substrates, we examined the cellular role of the poorly conserved N-terminal domain (∼80aa yTdp1 and 140aa hTdp1). This domain is not essential for catalytic activity per se, however, we observed that it is critical for Tdp1 cellular function in yeast and human cells. Comparison of the full-length and N-terminal truncated Tdp1 mutants showed similar cellular distribution, but a converse toxicity. This suggests that the N-terminal domain is a critical determinant of Tdp1 cellular function and this function is conserved from yeast to human. Thus, understanding the mechanism of interaction between Tdp1 and Topo1-DNA intermediate is important for the development of Tdp1 as a therapeutic target. Overall, these results support our concept that stabilization of Tdp1-DNA covalent intermediates converting this DNA repair enzyme into a cellular toxin is a potential novel anti-cancer therapeutic strategy, which is different from the intuitive strategy of inhibiting (preventing) Tdp1 catalytic activity. This work is in part supported by the ADDA. Citation Format: Selma M. Cuya, Ashley C. Conoway, Robert C.A.M. van Waardenburg. Tyrosyl-DNA phosphodiesterase I as a therapeutic target: lessons from yeast functional studies. [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 3628. doi:10.1158/1538-7445.AM2015-3628