Robert C.A.M. van Waardenburg

Tyrosyl-DNA phosphodiesterase I resolves both naturally and chemically induced DNA adducts and its potential as a therapeutic target

Abstract DNA is subject to a wide range of insults, resulting from endogenous and exogenous sources that need to be metabolized/resolved to maintain genome integrity. Tyrosyl-DNA phosphodiesterase I (Tdp1) is a eukaryotic DNA repair enzyme that catalyzes the removal of covalent 3′-DNA adducts. As a phospholipase D superfamily member Tdp1 utilizes two catalytic histidines each within a His-Lys-Asn motif. Tdp1 was discovered for its ability to hydrolyze the 3′-phospho-tyrosyl that in the cell covalently links DNA Topoisomerase I (Topo1) and DNA. Tdp1’s list of substrates has since grown and can be divided into two groups: protein-DNA adducts, such as camptothecin stabilized Topo1-DNA adducts, and modified nucleotides, including oxidized nucleotides and chain terminating nucleoside analogs. Since many of Tdp1’s substrates are generated by clinically relevant chemotherapeutics, Tdp1 became a therapeutic target for molecularly targeted small molecules. Tdp1’s unique catalytic cycle allows for two different targeting strategies: (1) the intuitive inhibition of Tdp1 catalysis to prevent Tdp1-mediated repair of chemotherapeutically induced DNA adducts, thereby enhancing their toxicity and (2) stabilization of the Tdp1–DNA covalent reaction intermediate, prevents resolution of Tdp1–DNA adduct and increases the half-life of this potentially toxic DNA adduct. This concept is best illustrated by a catalytic Tdp1 mutant that forms the molecular basis of the autosomal recessive neurodegenerative disease spinocerebellar ataxia with axonal neuropathy, and results in an increased stability of its Tdp1–DNA reaction intermediate. Here, we will discuss Tdp1 catalysis from a structure–function perspective, Tdp1 substrates and Tdp1 potential as a therapeutic target.

DNA topoisomerase-targeting chemotherapeutics: what’s new?

To resolve the topological problems that threaten the function and structural integrity of nuclear and mitochondrial genomes and RNA molecules, human cells encode six different DNA topoisomerases including type IB enzymes (TOP1 and TOP1mt), type IIA enzymes (TOP2α and TOP2β) and type IA enzymes (TOP3α and TOP3β). DNA entanglements and the supercoiling of DNA molecules are regulated by topoisomerases through the introduction of transient enzyme-linked DNA breaks. The covalent topoisomerase–DNA complexes are the cellular targets of a diverse group of cancer chemotherapeutics, which reversibly stabilize these reaction intermediates. Here we review the structure–function and catalytic mechanisms of each family of eukaryotic DNA topoisomerases and the topoisomerase-targeting agents currently approved for patient therapy or in clinical trials, and highlight novel developments and challenges in the clinical development of these agents.

Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage

Background: Tdp1 repair of adducted DNA involves a covalent enzyme-DNA intermediate, formed and resolved by Hisnuc and Hisgab residues. Stabilized Hisgab mutant-DNA complexes are cytotoxic. Results: DNA adduct cleavage by an adjacent His residue in HisnucAla mutants impair enzyme-DNA intermediate resolution. Conclusion: Alterations in active site geometry enhance the stability of cytotoxic Tdp1-DNA intermediates. Significance: These findings provide the rationale for developing chemotherapeutics that poison Tdp1. Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3′-DNA adducts, such as the 3′-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (Hisnuc) that attacks DNA adducts to form a covalent 3′-phosphohistidyl intermediate and a general acid/base His (Hisgab), which resolves the Tdp1-DNA linkage. A Hisnuc to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of Hisgab to Arg. However, here we report that expression of the yeast HisnucAla (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 Hisgab mutants, including H432N and the SCAN1-related H432R. Moreover, the HisnucAla mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the HisnucPhe mutant was catalytically inactive and suppressed Hisgab mutant-induced toxicity. These data suggest that the activity of another nucleophile when Hisnuc is replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to Hisnuc, can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate.

JQ1 Induces DNA Damage and Apoptosis, and Inhibits Tumor Growth in a Patient-Derived Xenograft Model of Cholangiocarcinoma

Cholangiocarcinoma (CCA) is a fatal disease with a 5-year survival of <30%. For a majority of patients, chemotherapy is the only therapeutic option, and virtually all patients relapse. Gemcitabine is the first-line agent for treatment of CCA. Patients treated with gemcitabine monotherapy survive ∼8 months. Combining this agent with cisplatin increases survival by ∼3 months, but neither regimen produces durable remissions. The molecular etiology of this disease is poorly understood. To facilitate molecular characterization and development of effective therapies for CCA, we established a panel of patient-derived xenograft (PDX) models of CCA. We used two of these models to investigate the antitumor efficacy and mechanism of action of the bromodomain inhibitor JQ1, an agent that has not been evaluated for the treatment of CCA. The data show that JQ1 suppressed the growth of the CCA PDX model CCA2 and demonstrate that growth suppression was concomitant with inhibition of c-Myc protein expression. A second model (CCA1) was JQ1-insensitive, with tumor progression and c-Myc expression unaffected by exposure to this agent. Also selective to CCA2 tumors, JQ1 induced DNA damage and apoptosis and downregulated multiple c-Myc transcriptional targets that regulate cell-cycle progression and DNA repair. These findings suggest that c-Myc inhibition and several of its transcriptional targets may contribute to the mechanism of action of JQ1 in this tumor type. We conclude that BET inhibitors such as JQ1 warrant further investigation for the treatment of CCA. Mol Cancer Ther; 17(1); 107–18. ©2017 AACR.

Dysregulated human Tyrosyl-DNA phosphodiesterase I acts as cellular toxin

Tyrosyl-DNA phosphodiesterase I (TDP1) hydrolyzes the drug-stabilized 3’phospho-tyrosyl bond formed between DNA topoisomerase I (TOPO1) and DNA. TDP1-mediated hydrolysis uses a nucleophilic histidine (Hisnuc) and a general acid/base histidine (Hisgab). A Tdp1Hisgab to Arg mutant identified in patients with the autosomal recessive neurodegenerative disease SCAN1 causes stabilization of the TDP1-DNA intermediate. Based on our previously reported Hisgab-substitutions inducing yeast toxicity (Gajewski et al. J. Mol. Biol. 415, 741-758, 2012), we propose that converting TDP1 into a cellular poison by stabilizing the covalent enzyme-DNA intermediate is a novel therapeutic strategy for cancer treatment. Here, we analyzed the toxic effects of two TDP1 catalytic mutants in HEK293 cells. Expression of human Tdp1HisnucAla and Tdp1HisgabAsn mutants results in stabilization of the covalent TDP1-DNA intermediate and induces cytotoxicity. Moreover, these mutants display reduced in vitro catalytic activity compared to wild type. Co-treatment of Tdp1mutant with topotecan shows more than additive cytotoxicity. Overall, these results support the hypothesis that stabilization of the TDP1-DNA covalent intermediate is a potential anti-cancer therapeutic strategy.

Sphingolipid metabolism and drug resistance in ovarian cancer

Despite progress in understanding molecular aberrations that contribute to the development and progression of ovarian cancer, virtually all patients succumb to drug resistant disease at relapse. Emerging data implicate bioactive sphingolipids and regulation of sphingolipid metabolism as components of response to chemotherapy or development of resistance. Increases in cytosolic ceramide induce apoptosis in response to therapy with multiple classes of chemotherapeutic agents. Aberrations in sphingolipid metabolism that accelerate the catabolism of ceramide or that prevent the production and accumulation of ceramide contribute to resistance to standard of care platinum- and taxane-based agents. The aim of this review is to highlight current literature and research investigating the influence of the sphingolipids and enzymes that comprise the sphingosine-1-phosphate pathway on the progression of ovarian cancer. The focus of the review is on the utility of sphingolipid-centric therapeutics as a mechanism to circumvent drug resistance in this tumor type.

Abstract 2761: Cellular consequences of human tyrosyl-DNA phosphodiesterase I dysregulation

Tyrosyl-DNA phosphodiesterase I (Tdp1) is a highly conserved eukaryotic DNA repair enzyme that catalyzes the resolution of 3’ and 5’ phospho-DNA adducts. Tdp1 has been implicated in the repair of DNA topoisomerase I (Topo1)-DNA covalent complexes reversibly stabilized by camptothecins (CPTs) such as the FDA approved CPT derivatives topotecan and irinotecan. Tdp1 utilizes a two-step catalytic cycle that centers on the formation of an obligatory Tdp1-DNA covalent complex (Tdp1-cc) through its nucleophilic histidine (Hisnuc), resulting in dissociation of the adduct, while its general acid/base histidine (Hisgab) mediates Tdp1 dissociation. A Tdp1Hisgab to Arg (H493R) mutant stabilizes the Tdp1-cc and is associated with autosomal recessive ataxia SCAN1. Alternative substitutions of Hisgab or substitutions of the Hisnuc transforms yeast Tdp1 into a potent toxin via stabilization of Tdp1-cc. We propose that stabilization of this Tdp1-DNA covalent complex is a potential novel therapeutic anti-cancer strategy. As proof-of-concept, we analyzed two catalytic Hisgab (H493R or H493N) mutants and one Hisnuc (H263A) mutant of hTdp1 in HEK293 cells. Doxycycline-induced expression of Tdp1H263A, Tdp1H493R, and Tdp1H493N mutant enzymes induced Tdp1-dependent cytotoxicity without additional genotoxic stress. Utilizing two different immuno-assays, we validated that the observed Tdp1-dependent toxicity correlates with stabilization of their enzyme-DNA covalent complex. Moreover, all of these Tdp1 catalytic mutants show reduced catalytic activity compared to wild type hTdp1, but they do not all show a stabilized Tdp1-cc in this in vitro assay. This indicates a significant difference between in vitro Tdp1 activity and cellular Tdp1 activity, which is most likely due to the difference in substrate; a small oligonucleotide with a 3’phospho-tyrosyl modification versus covalent complex of full length Topo1 with genomic DNA. However, these results confirm our previous yeast studies: Stabilization of the Tdp1-cc converts a DNA repair enzyme into a cellular toxin, which constitutes a potential novel therapeutic strategy to treat cancer. In addition, we are comparing schedule dependent ‘drug’-combinations of our toxic Tdp1 mutant expression with topotecan, etoposide (targets Topo2-cc) and cisplatin to evaluate the potential therapeutic value of this novel Tdp1 targeted strategy. This work is in part supported by the ADDA, UAB ACS-IRG, and DOD OCRP WX81WH-15-1-0198. Citation Format: Selma M. Cuya, Kellie M. Regal, Robert C.A.M. Van Waardenburg. Cellular consequences of human tyrosyl-DNA phosphodiesterase I dysregulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2761.

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

Abstract 118: The cytoplasmic domain of ICAM-2 interacts with α-actinin to confer a non-metastatic phenotype in neuroblastoma cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, accounting for 15% of all childhood cancer deaths. Despite recent improvements in outcome for children with early stage disease, the majority of children with high-risk neuroblastoma survive less than 5 years. Nearly all newly diagnosed patients achieve apparent remission, but patients with high-risk disease relapse and die from metastatic progression within 2-5 years. Our long-range goal is to develop therapies that prevent metastatic relapse. We recently made the novel observation that intercellular adhesion molecule-2 (ICAM-2) prevents development of disseminated tumors in a murine model of metastatic neuroblastoma. Interestingly, ICAM-2 suppressed metastatic but not tumorigenic potential in preclinical models, supporting a novel mechanism of regulating metastatic disease. Aside from this observation, little is known about the function of ICAM-2 in tumor cells. Importantly, and consistent with data generated using neuroblastoma cell lines, we also showed that primary human neuroblastoma cells expressing ICAM-2 are those recognized clinically to have limited metastatic potential. Because the actin cytoskeletal network is a primary regulator of cell motility and metastatic potential, we hypothesized that ICAM-2 affected the phenotype of neuroblastoma cells by interacting with the actin cytoskeletal linker protein α-actinin. We used in silico modeling to examine the likelihood that the cytoplasmic domain of ICAM-2 binds directly to α-actinin. We then used site-directed mutagenesis to express variants of ICAM-2 with mutated α-actinin binding domains, and compared the impact of ICAM-2 wild type (WT) and each variant on neuroblastoma cell motility. We used dual immunofluorescence and co-immunoprecipitation approaches to demonstrate co-localization of ICAM-2 WT and α-actinin in situ; and we evaluated ICAM-2 WT and variants for their ability to produce disseminated tumors in in vivo models. In vitro and in vivo characteristics of cells expressing ICAM-2 variants with modified α-actinin binding domains differed from cells expressing ICAM-2 WT and also from cells that express no detectable ICAM-2. Unlike the WT protein, ICAM-2 variants did not completely suppress development of disseminated neuroblastoma tumors in vivo. We concluded that the interaction of ICAM-2 with α-actinin was critical to conferring the ICAM-2-mediated non-metastatic phenotype in NB cells. This work was supported by the University of Alabama at Birmingham-Comprehensive Cancer Center-New Faculty Development Award (P30 CA013148). Citation Format: Joseph M. Feduska, Stephen G. Aller, Stuart Cramer, Robert C.A.M. van Waardenburg, Karina J. Yoon. The cytoplasmic domain of ICAM-2 interacts with α-actinin to confer a non-metastatic phenotype in neuroblastoma cells. [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 118. doi:10.1158/1538-7445.AM2014-118

Abstract 3327: N-terminal domain of Tyrosyl-DNA phosphodiesterase I (Tdp1) is critical for its cellular function.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Tyrosyl-DNA phosphodiesterase I (Tdp1) is a highly conserved eukaryotic DNA repair enzyme that catalyzes the resolution of 3’ and 5’ phospho-DNA adducts. Tdp1 has been implicated in the repair of DNA topoisomerase I (Top1)-DNA covalent complexes reversibly stabilized by camptothecins (CPTs) such as the FDA approved CPT derivatives topotecan and irinotecan. Tdp1 contains two HKD-motifs that provide two catalytic histidines that function as a nucleophile and an acid-base residue. A mutation of the acid-base His to Arg (H493R) in human Tdp1 is associated with the rare recessive ataxia SCAN1. hTdp1H493R and the analogous yeast mutant (Tdp1H432R) enhances cell sensitivity to CPT. In addition, the toxicity induced by this mutant is caused by the formation of a more stable Tdp1-DNA covalent intermediate, a rare characteristic for a DNA repair enzyme. However, this His to Arg substitution induces a minor toxic phenotype compared to other substitutions, such as the His432 to Asn substitution, which induces a Top1 dependent cellular lethality. A band depletion assay suggests that in vivo/cell Tdp1His432Asn remains in complex with Top1 on the DNA, which was not observed in a biochemical in vitro assay. Biochemical studies revealed that Tdp1 catalysis is independent of the N-terminal domain. Among Tdp1 proteins, the N-terminal domain is poorly conserved in sequence and size (∼80aa for yeast and 140aa for human Tdp1). We investigated the role of the N-terminal domain for Tdp1 activity in the cell. The N-terminal truncated proteins showed similar cellular distribution as the full-length proteins. Interestingly, the N-terminal truncated proteins did not display the toxicity that was observed with the full-length Tdp1 mutant proteins. This suggests that the N-terminal domain is a critical determinate of Tdp1 cellular function. Preliminary results from our human cell line model shows similar results implying that the function of the N-terminal domain is conserved among Tdp1 proteins although it is poorly conserved. Further studies are necessary to ensure that these constructs are properly distributed. Moreover, the N-terminal domain of hTdp1 is post-translational modified, while our preliminary results suggest that this domain is important for protein-protein interaction and Tdp1 recruitment to its substrates. Understanding Tdp1 substrate and protein-interactions are important in the development of Tdp1 as therapeutic target. This work is in part supported by the ADDA. Citation Format: Selma M. Cuya, Keith C. Wanzeck, Evan Q. Comeaux, Robert C. van Waardenburg. N-terminal domain of Tyrosyl-DNA phosphodiesterase I (Tdp1) is critical for its cellular function. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3327. doi:10.1158/1538-7445.AM2013-3327