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Repair of DNA damage induced by the novel nucleoside analogue CNDAG through homologous recombination

Abstract

Purpose

We postulate that the deoxyguanosine analogue CNDAG [9-(2-C-cyano-2-deoxy-1-β-d-arabino-pentofuranosyl)guanine] likely causes a single-strand break after incorporation into DNA, similar to the action of its cytosine congener CNDAC, and that subsequent DNA replication across the unrepaired nick would generate a double-strand break. This study aimed at identifying cellular responses and repair mechanisms for CNDAG prodrugs, 2-amino-9-(2-C-cyano-2-deoxy-1-β-d-arabino-pentofuranosyl)-6-methoxy purine (6-OMe) and 9-(2-C-cyano-2-deoxy-1-β-d-arabino-pentofuranosyl)-2,6-diaminopurine (6-NH2). Each compound is a substrate for adenosine deaminase, the action of which generates CNDAG.

Methods

Growth inhibition assay, clonogenic survival assay, immunoblotting, and cytogenetic analyses (chromosomal aberrations and sister chromatid exchanges) were used to investigate the impact of CNDAG on cell lines.

Results

The 6-NH2 derivative was selectively potent in T cell malignant cell lines. Both prodrugs caused increased phosphorylation of ATM and its downstream substrates Chk1, Chk2, SMC1, NBS1, and H2AX, indicating activation of ATM-dependent DNA damage response pathways. In contrast, there was no increase in phosphorylation of DNA-PKcs, which participates in repair of double-strand breaks by non-homologous end-joining. Deficiency in ATM, RAD51D, XRCC3, BRCA2, and XPF, but not DNA-PK or p53, conferred significant clonogenic sensitivity to CNDAG or the prodrugs. Moreover, hamster cells lacking XPF acquired remarkably more chromosomal aberrations after incubation for two cell cycle times with CNDAG 6-NH2, compared to the wild type. Furthermore, CNDAG 6-NH2 induced greater levels of sister chromatid exchanges in wild-type cells exposed for two cycles than those for one cycle, consistent with increased double-strand breaks after a second S phase.

Conclusion

CNDAG-induced double-strand breaks are repaired mainly through homologous recombination.

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References

  1. Ewald B, Sampath D, Plunkett W (2008) Nucleoside analogs: molecular mechanisms signaling cell death. Oncogene 27(50):6522–6537. https://doi.org/10.1038/onc.2008.316

    CAS  Article  PubMed  Google Scholar 

  2. Matsuda A, Nakajima Y, Azuma A, Tanaka M, Sasaki T (1991) Nucleosides and nucleotides. 100. 2'-C-cyano-2'-deoxy-1-beta-d-arabinofuranosyl-cytosine (CNDAC): design of a potential mechanism-based DNA-strand-breaking antineoplastic nucleoside. J Med Chem 34(9):2917–2919

    CAS  Article  Google Scholar 

  3. Azuma A, Nakajima Y, Nishizono N, Minakawa N, Suzuki M, Hanaoka K, Kobayashi T, Tanaka M, Sasaki T, Matsuda A (1993) Nucleosides and nucleotides. 122. 2'-C-cyano-2'-deoxy-1-beta-d-arabinofuranosylcytosine and its derivatives: a new class of nucleoside with a broad antitumor spectrum. J Med Chem 36(26):4183–4189

    CAS  Article  Google Scholar 

  4. Matsuda A (1995) 2'-C-Cyano-2'-deoxy-1-b-d-arabinofuranosyl-cytosine(CNDAC): a mechanism-based DNA-strandbreaking antitumor nucleoside. Nucleosides Nucleotides 14:461–471

    CAS  Article  Google Scholar 

  5. Azuma A, Huang P, Matsuda A, Plunkett W (2001) 2'-C-cyano-2'-deoxy-1-beta-d-arabino-pentofuranosylcytosine: a novel anticancer nucleoside analog that causes both DNA strand breaks and G(2) arrest. Mol Pharmacol 59(4):725–731

    CAS  Article  Google Scholar 

  6. Liu X, Wang Y, Benaissa S, Matsuda A, Kantarjian H, Estrov Z, Plunkett W (2010) Homologous recombination as a resistance mechanism to replication-induced double-strand breaks caused by the antileukemia agent CNDAC. Blood 116(10):1737–1746. https://doi.org/10.1182/blood-2009-05-220376(blood-2009-05-220376 [pii])

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Ohtawa M, Ichikawa S, Teishikata Y, Fujimuro M, Yokosawa H, Matsuda A (2007) 9-(2-C-Cyano-2-deoxy-beta-D-arabino-pentofuranosyl)guanine, a potential antitumor agent against B-lymphoma infected with Kaposi's sarcoma-associated herpesvirus. J Med Chem 50(9):2007–2010. https://doi.org/10.1021/jm070032y

    CAS  Article  PubMed  Google Scholar 

  8. Ichikawa S, Otawa M, Teishikata Y, Yamada K, Fujimuro M, Yokosawa H, Matsuda A (2009) 9-(2-C-Cyano-2-deoxy-beta-d-arabino-pentofuranosyl)guanine, a potential antitumor agent against B-lymphoma infected with Kaposi's sarcoma-associated herpesvirus. Nucleic Acids Symp Ser (Oxf) 53:95–96. https://doi.org/10.1093/nass/nrp048(nrp048 [pii])

    CAS  Article  Google Scholar 

  9. Kisor DF (2005) Nelarabine: a nucleoside analog with efficacy in T cell and other leukemias. Ann Pharmacother 39(6):1056–1063. https://doi.org/10.1345/aph.1E453

    CAS  Article  PubMed  Google Scholar 

  10. Gandhi V, Plunkett W (2006) Clofarabine and nelarabine: two new purine nucleoside analogs. Curr Opin Oncol 18(6):584–590. https://doi.org/10.1097/01.cco.0000245326.65152.af

    CAS  Article  PubMed  Google Scholar 

  11. Ziv Y, Bar-Shira A, Pecker I, Russell P, Jorgensen TJ, Tsarfati I, Shiloh Y (1997) Recombinant ATM protein complements the cellular A–T phenotype. Oncogene 15(2):159–167. https://doi.org/10.1038/sj.onc.1201319

    CAS  Article  PubMed  Google Scholar 

  12. Anderson CW, Allalunis-Turner MJ (2000) Human TP53 from the malignant glioma-derived cell lines M059J and M059K has a cancer-associated mutation in exon 8. Radiat Res 154(4):473–476

    CAS  Article  Google Scholar 

  13. Anderson CW, Dunn JJ, Freimuth PI, Galloway AM, Allalunis-Turner MJ (2001) Frameshift mutation in PRKDC, the gene for DNA-PKcs, in the DNA repair-defective, human, glioma-derived cell line M059J. Radiat Res 156(1):2–9

    CAS  Article  Google Scholar 

  14. Ross GM, Eady JJ, Mithal NP, Bush C, Steel GG, Jeggo PA, McMillan TJ (1995) DNA strand break rejoining defect in xrs-6 is complemented by transfection with the human Ku80 gene. Cancer Res 55(6):1235–1238

    CAS  PubMed  Google Scholar 

  15. Liu X, Guo Y, Li Y, Jiang Y, Chubb S, Azuma A, Huang P, Matsuda A, Hittelman W, Plunkett W (2005) Molecular basis for G2 arrest induced by 2'-C-cyano-2'-deoxy-1-beta-d-arabino-pentofuranosylcytosine and consequences of checkpoint abrogation. Cancer Res 65(15):6874–6881. https://doi.org/10.1158/0008-5472.CAN-05-0288(65/15/6874 [pii])

    CAS  Article  PubMed  Google Scholar 

  16. Wang Y, Liu X, Matsuda A, Plunkett W (2008) Repair of 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine-induced DNA single-strand breaks by transcription-coupled nucleotide excision repair. Cancer Res 68(10):3881–3889. https://doi.org/10.1158/0008-5472.CAN-07-6885[pii]

    CAS  Article  PubMed  Google Scholar 

  17. Beucher A, Birraux J, Tchouandong L, Barton O, Shibata A, Conrad S, Goodarzi AA, Krempler A, Jeggo PA, Lobrich M (2009) ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2. EMBO J 28(21):3413–3427. https://doi.org/10.1038/emboj.2009.276

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Morrison C, Sonoda E, Takao N, Shinohara A, Yamamoto K, Takeda S (2000) The controlling role of ATM in homologous recombinational repair of DNA damage. EMBO J 19(3):463–471

    CAS  Article  Google Scholar 

  19. Lees-Miller SP, Godbout R, Chan DW, Weinfeld M, Day RS 3rd, Barron GM, Allalunis-Turner J (1995) Absence of p350 subunit of DNA-activated protein kinase from a radiosensitive human cell line. Science 267(5201):1183–1185

    CAS  Article  Google Scholar 

  20. Feldmann E, Schmiemann V, Goedecke W, Reichenberger S, Pfeiffer P (2000) DNA double-strand break repair in cell-free extracts from Ku80-deficient cells: implications for Ku serving as an alignment factor in non-homologous DNA end joining. Nucleic Acids Res 28(13):2585–2596

    CAS  Article  Google Scholar 

  21. Thompson LH, Schild D (2001) Homologous recombinational repair of DNA ensures mammalian chromosome stability. Mutat Res 477(1–2):131–153 (S0027510701001154 [pii])

    CAS  Article  Google Scholar 

  22. Pierce AJ, Johnson RD, Thompson LH, Jasin M (1999) XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev 13(20):2633–2638

    CAS  Article  Google Scholar 

  23. Brown ET, Robinson-Benion C, Holt JT (2008) Radiation enhances caspase 3 cleavage of Rad51 in BRCA2-defective cells. Radiat Res 169(5):595–601. https://doi.org/10.1667/RR1129.1(RR1129 [pii])

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Orelli BJ, Bishop DK (2001) BRCA2 and homologous recombination. Breast Cancer Res 3(5):294–298

    CAS  Article  Google Scholar 

  25. Wilson DM 3rd, Thompson LH (2007) Molecular mechanisms of sister-chromatid exchange. Mutat Res 616(1–2):11–23. https://doi.org/10.1016/j.mrfmmm.2006.11.017(S0027-5107(06)00317-4 [pii])

    CAS  Article  PubMed  Google Scholar 

  26. Cohen MH, Johnson JR, Justice R, Pazdur R (2008) FDA drug approval summary: nelarabine (Arranon) for the treatment of T cell lymphoblastic leukemia/lymphoma. Oncologist 13(6):709–714. https://doi.org/10.1634/theoncologist.2006-0017

    CAS  Article  PubMed  Google Scholar 

  27. Gandhi V, Keating MJ, Bate G, Kirkpatrick P (2006) Nelarabine. Nat Rev Drug Discov 5(1):17–18. https://doi.org/10.1038/nrd1933

    CAS  Article  PubMed  Google Scholar 

  28. Gandhi V, Mineishi S, Huang P, Yang Y, Chubb S, Chapman AJ, Nowak BJ, Hertel LW, Plunkett W (1995) Difluorodeoxyguanosine: cytotoxicity, metabolism, and actions on DNA synthesis in human leukemia cells. Semin Oncol 22(4 Suppl 11):61–67

    CAS  PubMed  Google Scholar 

  29. Shewach DS, Daddona PE, Ashcraft E, Mitchell BS (1985) Metabolism and selective cytotoxicity of 9-beta-d-arabinofuranosylguanine in human lymphoblasts. Cancer Res 45(3):1008–1014

    CAS  PubMed  Google Scholar 

  30. Shewach DS, Mitchell BS (1989) Differential metabolism of 9-beta-d-arabinofuranosylguanine in human leukemic cells. Cancer Res 49(23):6498–6502

    CAS  PubMed  Google Scholar 

  31. Gumy-Pause F, Wacker P, Sappino AP (2004) ATM gene and lymphoid malignancies. Leukemia 18(2):238–242. https://doi.org/10.1038/sj.leu.2403221

    CAS  Article  PubMed  Google Scholar 

  32. Liberzon E, Avigad S, Yaniv I, Stark B, Avrahami G, Goshen Y, Zaizov R (2004) Molecular variants of the ATM gene in Hodgkin's disease in children. Br J Cancer 90(2):522–525. https://doi.org/10.1038/sj.bjc.6601522

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Takeuchi S, Koike M, Park S, Seriu T, Bartram CR, Taub HE, Williamson IK, Grewal J, Taguchi H, Koeffler HP (1998) The ATM gene and susceptibility to childhood T cell acute lymphoblastic leukaemia. Br J Haematol 103(2):536–538

    CAS  Article  Google Scholar 

  34. Sakashita A, Hattori T, Miller CW, Suzushima H, Asou N, Takatsuki K, Koeffler HP (1992) Mutations of the p53 gene in adult T cell leukemia. Blood 79(2):477–480

    CAS  Article  Google Scholar 

  35. Staub M, Eriksson S (2006) The role of deoxycytidine kinase in DNA synthesis and nucleoside analog activation. Chapter 2:29–52

    Google Scholar 

  36. Sabini E, Ort S, Monnerjahn C, Konrad M, Lavie A (2003) Structure of human dCK suggests strategies to improve anticancer and antiviral therapy. Nat Struct Biol 10(7):513–519. https://doi.org/10.1038/nsb942

    CAS  Article  PubMed  Google Scholar 

  37. Wang J, Choudhury D, Chattopadhyaya J, Eriksson S (1999) Stereoisomeric selectivity of human deoxyribonucleoside kinases. Biochemistry 38(51):16993–16999. https://doi.org/10.1021/bi9908843

    CAS  Article  PubMed  Google Scholar 

  38. Zhu C, Johansson M, Permert J, Karlsson A (1998) Phosphorylation of anticancer nucleoside analogs by human mitochondrial deoxyguanosine kinase. Biochem Pharmacol 56(8):1035–1040. https://doi.org/10.1016/s0006-2952(98)00150-6

    CAS  Article  PubMed  Google Scholar 

  39. Zhu C, Johansson M, Permert J, Karlsson A (1998) Enhanced cytotoxicity of nucleoside analogs by overexpression of mitochondrial deoxyguanosine kinase in cancer cell lines. J Biol Chem 273(24):14707–14711. https://doi.org/10.1074/jbc.273.24.14707

    CAS  Article  PubMed  Google Scholar 

  40. Rodriguez CO Jr, Stellrecht CM, Gandhi V (2003) Mechanisms for T cell selective cytotoxicity of arabinosylguanine. Blood 102(5):1842–1848. https://doi.org/10.1182/blood-2003-01-0317(2003-01-0317 [pii])

    CAS  Article  PubMed  Google Scholar 

  41. Liu X, Jiang Y, Nowak B, Hargis S, Plunkett W (2016) Mechanism-based drug combinations with the DNA strand-breaking nucleoside analog CNDAC. Mol Cancer Ther 15(10):2302–2313. https://doi.org/10.1158/1535-7163.MCT-15-0801

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Liu X, Jiang Y, Nowak B, Qiang B, Cheng N, Chen Y, Plunkett W (2018) Targeting BRCA1/2 deficient ovarian cancer with CNDAC-based drug combinations. Cancer Chemother Pharmacol 81(2):255–267. https://doi.org/10.1007/s00280-017-3483-6

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

The authors are grateful to Drs. Asha Multani and Jin Ma (T. C. Hsu Molecular Cytogenetics Core at MDACC) for their technical support in cytogenetic studies (SCE).

Funding

This work was supported in part by grants R01 CA28596 (W. Plunkett), P50 CA100632 (W. Plunkett), and Cancer Center Support Grant P30 CA16672 from the National Cancer Institute, Department of Health and Human Services.

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Authors and Affiliations

Authors

Contributions

XL and WP: conception and design. XL and YJ: development of methodology. XL, YJ, BN, SI, MO, and AM: acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.). XL, YJ, and WP: analysis and interpretation of data (e.g., statistical analysis, biostatistics, and computational analysis). XL, YJ, and WP: writing, review, and/or revision of the manuscript. XL, YJ, and BN: administrative, technical, or material support (i.e., reporting or organizing data, and constructing databases). WP: study supervision.

Corresponding author

Correspondence to William Plunkett.

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280_2020_4035_MOESM1_ESM.jpg

Supplemental Figure 1. Chromosomal aberrations induced by CNDAG 6-NH2. Representative images of mitotic spreads of AA8 and UV41 cells untreated and treated with 8 µM CNDAG 6-NH2 for 15 hours and 30 hours, respectively. Arrows indicate chromosomal breaks or gaps

280_2020_4035_MOESM2_ESM.jpg

Supplemental Figure 2. Cell cycle progression of CCRF-CEM cells after treatment with CNDAC or CNDAG derivatives. Suspension cells were exposed to 0.5 μM CNDAC, 50 μM CNDAG 6-OMe, and 50 μM CNDAG 6- NH2, respectively, for 24 hr. Cells were harvested, fixed and subjected to analysis by flow cytometry. (A) DNA content profiles are presented for all samples. (B) A stacked-bar plot is shown to present the percentage of all cell cycle phases in the whole populations

280_2020_4035_MOESM3_ESM.jpg

Supplemental Figure 3. Lack of p53 does not sensitize HCT116 cells to CNDAG. HCT116 cells with wild-type p53 (■) and p53 knocked-out (o) were washed into drugfree medium after a 24-hour exposure to CNDAG 6-NH2 at a range of concentrations and allowed to form colonies in 7–8 days. All points, mean ± SD of triplicate plates

280_2020_4035_MOESM4_ESM.jpg

Supplemental Figure 4. Sister chromatid exchanges in AA8 cells treated with mitomycin C (MMC). A representative image of metaphase chromosome spreads is shown.

280_2020_4035_MOESM5_ESM.jpg

Supplemental Figure 5. Proposed model for roles of DNA repair proteins in cellular response to CNDAG. CNDAG prodrugs are metabolized into the active form CNDAG under the action of adenosine deaminase (ADA). Deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) are responsible for phosphorylating CNDAG. CNDAG triphosphates are incorporated into DNA and subsequently induce SSBs through β-elimination during the first S phase. The transcription-coupled nucleotide excision repair pathway (TC-NER) recognizes SSBs and contributes to cell survival (Wang, Liu, Matsuda, and Plunkett, 2008). Unrepaired SSBs are converted into DSBs mainly by replication in the second S phase. DSBs are lethal if left unrepaired. The homologous recombination (HR) pathway, which is activated by ATM, and involves RAD51, XRCC3 and BRCA2, is a major contributor to this survival function. The non-homologous end-joining (NHEJ) pathway is dispensable for cell survival of CNDAC-induced damage.

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Liu, X., Jiang, Y., Nowak, B. et al. Repair of DNA damage induced by the novel nucleoside analogue CNDAG through homologous recombination. Cancer Chemother Pharmacol 85, 661–672 (2020). https://doi.org/10.1007/s00280-020-04035-x

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Keywords

  • Nucleoside analogue
  • Homologous recombination
  • ATM signaling pathway
  • XPF
  • T-ALL