TY - JOUR
T1 - Depletion of tyrosyl DNA phosphodiesterase 2 activity enhances etoposide-mediated double-strand break formation and cell killing
AU - Kont, Yasemin Saygideger
AU - Dutta, Arijit
AU - Mallisetty, Apurva
AU - Mathew, Jeena
AU - Minas, Tsion
AU - Kraus, Christina
AU - Dhopeshwarkar, Priyanka
AU - Kallakury, Bhaskar
AU - Mitra, Sankar
AU - Üren, Aykut
AU - Adhikari, Sanjay
N1 - Funding Information:
We thank Dr. Rabindra Roy, Georgetown University Medical Center, for allowing his radio-active working facility and space for some of the experiments. We thank Dr. Venkata Yenugonda for helping us to contact Toronto research chemical, Canada for resynthesizing NSC111041. This project has been supported partly by pilots awards IRG-92-152-17 American Cancer Society, “American Cancer Society Institutional Research Grant” (SA), Nina Hyde Mechanism (SA) and NIH R03DA035193 (SA), and Federal funds (UL1TR000101) from the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, through the Clinical and Translational Science Awards Program (CTSA), a trademark of DHHS, part of the Roadmap Initiative, “Re-Engineering the Clinical Research Enterprise (SA), R01CA158910 (SM), P01CA92584 (SM). We thank LCCC’s Genomics & Epigenomics Shared Resource for High-throughput screening, surface plasmon resonance and biostatistics experiments. We also thank LCCC’s Histopathology & Tissue Shared Resource for human tissue samples, Proteomics & Metabolomics Shared Resource for high resolution mass spectrometry experiment, Tissue Culture Shared Resources for different cell lines (P30 CA51008). We thank Prof. Jeffrey Toretsky, Georgetown University for A4573, SKES, TC32 and TC71 cell lines. We thank Microscopy & Imaging Shared Resource of Houston Methodist Research Institutes. Finally, we thank Prof. Louis Weiner, Director, Lombardi Comprehensive Cancer Center for supporting this work.
Publisher Copyright:
© 2016 Elsevier B.V.
PY - 2016/7/1
Y1 - 2016/7/1
N2 - 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.
AB - 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.
KW - DNA repair
KW - Enzyme assay
KW - High throughput screening
KW - Inhibitor
KW - Molecular probe
KW - NSC111041
KW - TDP2
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U2 - 10.1016/j.dnarep.2016.04.009
DO - 10.1016/j.dnarep.2016.04.009
M3 - Article
C2 - 27235629
AN - SCOPUS:84969786580
SN - 1568-7864
VL - 43
SP - 38
EP - 47
JO - DNA Repair
JF - DNA Repair
ER -