title: Depletion of tyrosyl DNA phosphodiesterase 2 activity enhances etoposide-mediated double-strand break formation and cell killing.
abstract: DNA topoisomerase 2 (Top2) poisons, including common anticancer drugs etoposide and doxorubicin kill cancer cells by stabilizing covalent Top2-tyrosyl-DNA 5'-phosphodiester adducts and DNA double-strand breaks (DSBs). Proteolytic degradation of the covalently attached Top2 leaves a 5'-tyrosylated blocked termini which is removed by tyrosyl DNA phosphodiesterase 2 (TDP2), prior to DSB repair through non-homologous end joining (NHEJ). Thus, TDP2 confers resistance of tumor cells to Top2-poisons by repairing such covalent DNA-protein adducts, and its pharmacological inhibition could enhance the efficacy of Top2-poisons. We discovered NSC111041, a selective inhibitor of TDP2, by optimizing a high throughput screening (HTS) assay for TDP2's 5'-tyrosyl phosphodiesterase activity and subsequent validation studies. We found that NSC111041 inhibits TDP2's binding to DNA without getting intercalated into DNA and enhanced etoposide's cytotoxicity synergistically in TDP2-expressing cells but not in TDP2 depleted cells. Furthermore, NSC111041 enhanced formation of etoposide-induced γ-H2AX foci presumably by affecting DSB repair. Immuno-histochemical analysis showed higher TDP2 expression in a sub-set of different type of tumor tissues. These findings underscore the feasibility of clinical use of suitable TDP2 inhibitors in adjuvant therapy with Top2-poisons for a sub-set of cancer patients with high TDP2 expression.
authors.affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, United States; Department of Molecular Medicine, Institute of Health Sciences, Dokuz Eylul University, Inciralti, Izmir 35340, Turkey. Electronic address: ys452@georgetown.edu.
authors.forename: Yasemin Saygideger
authors.initials: YS
authors.lastname: Kont
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX-77030, United States; Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX-77555, United States. Electronic address: ardutta@utmb.edu.
authors.forename: Arijit
authors.initials: A
authors.lastname: Dutta
authors.affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, United States. Electronic address: apurva.mallisetty@gmail.com.
authors.forename: Apurva
authors.initials: A
authors.lastname: Mallisetty
authors.affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, United States. Electronic address: jm722@georgetown.edu.
authors.forename: Jeena
authors.initials: J
authors.lastname: Mathew
authors.affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, United States. Electronic address: tm484@georgetown.edu.
authors.forename: Tsion
authors.initials: T
authors.lastname: Minas
authors.affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, United States. Electronic address: cnk10@georgetown.edu.
authors.forename: Christina
authors.initials: C
authors.lastname: Kraus
authors.affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, United States. Electronic address: priyanka.dhopeshwarkar@ablinc.com.
authors.forename: Priyanka
authors.initials: P
authors.lastname: Dhopeshwarkar
authors.affiliation: Department of Pathology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, United States. Electronic address: kallakub@georgetown.edu.
authors.forename: Bhaskar
authors.initials: B
authors.lastname: Kallakury
authors.affiliation: Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX-77030, United States. Electronic address: smitra2@houstonmethodist.org.
authors.forename: Sankar
authors.initials: S
authors.lastname: Mitra
authors.affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, United States. Electronic address: au26@georgetown.edu.
authors.forename: Aykut
authors.initials: A
authors.lastname: Üren
authors.affiliation: Compass Laboratory Services, 1910 Nonconnah Rd, Memphis, United States. Electronic address: sadhikari@houstonmethodist.org.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Adhikari
mesh_terms: D000634:Aminoquinolines / Q000494:pharmacology*; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D018736:DNA Adducts / Q000037:antagonists & inhibitors / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000187:drug effects*; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism; D004273:DNA, Neoplasm / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004357:Drug Synergism; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D005047:Etoposide / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic; D057166:High-Throughput Screening Assays; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases; D000075223:Poly-ADP-Ribose Binding Proteins; D011485:Protein Binding; D059003:Topoisomerase Inhibitors / Q000494:pharmacology*; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D000634:Aminoquinolines; D000951:Antigens, Neoplasm; D018736:DNA Adducts; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D009687:Nuclear Proteins; D010726:Phosphodiesterase Inhibitors; D000075223:Poly-ADP-Ribose Binding Proteins; D059003:Topoisomerase Inhibitors; D014157:Transcription Factors; D005047:Etoposide; D010727:Phosphoric Diester Hydrolases; C474866:TDP2 protein, human; D004250:DNA Topoisomerases, Type II; C000618811:TOP2A protein, human
keywords: DNA repair; Enzyme assay; High throughput screening; Inhibitor; Molecular probe; NSC111041; TDP2