Cancer Res Treat.  2019 Jul;51(3):1167-1179. 10.4143/crt.2018.526.

Therapeutic Targeting of the DNA Damage Response Using an ATR Inhibitor in Biliary Tract Cancer

• Nam AR
• Jin MH
• Park JE
• Bang JH
• Oh DY
• Bang YJ

• KMID: 2454308
• DOI: http://doi.org/10.4143/crt.2018.526

Abstract

PURPOSE
The DNA damage response (DDR) is a multi-complex network of signaling pathways involved in DNA damage repair, cell cycle checkpoints, and apoptosis. In the case of biliary tract cancer (BTC), the strategy of DDR targeting has not been evaluated, even though many patients have DNA repair pathway alterations. The purpose of this study was to test the DDR-targeting strategy in BTC using an ataxia-telangiectasia and Rad3-related (ATR) inhibitor.
MATERIALS AND METHODS
A total of nine human BTC cell lines were used for evaluating anti-tumor effect of AZD6738 (ATR inhibitor) alone or combination with cytotoxic chemotherapeutic agents through MTT assay, colony-forming assays, cell cycle analyses, and comet assays. We established SNU478-mouse model for in vivo experiments to confirm our findings.
RESULTS
Among nine human BTC cell lines, SNU478 and SNU869 were the most sensitive to AZD6738, and showed low expression of both ataxia-telangiectasia mutated (ATM) and p53. AZD6738 blocked p-Chk1 and p-glycoprotein and increased γH2AX, a marker of DNA damage, in sensitive cells. AZD6738 significantly increased apoptosis, G2/M arrest and p21, and decreased CDC2. Combinations of AZD6738 and cytotoxic chemotherapeutic agents exerted synergistic effects in colony-forming assays, cell cycle analyses, and comet assays. In our mouse models, AZD6738 monotherapy decreased tumor growth and the combination with cisplatin showed more potent effects on growth inhibition, decreased Ki-67, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling than monotherapy with each drug.
CONCLUSION
In BTC, DDR targeting strategy using ATR inhibitor demonstrated promising antitumor activity alone or in combination with cytotoxic chemotherapeutic agents. This supports further clinical development of DDR targeting strategy in BTC.

Keyword

DNA damage response; ATR inhibitor; Biliary tract neoplasms; ATM; p53

MeSH Terms

Animals
Apoptosis
Ataxia Telangiectasia
Biliary Tract Neoplasms*
Biliary Tract*
Cell Cycle
Cell Cycle Checkpoints
Cell Line
Cisplatin
Comet Assay
DNA Damage*
DNA Repair
DNA*
Humans
Mice
P-Glycoprotein
Cisplatin
DNA
P-Glycoprotein

Figure

• 

  Fig. 1. Anti-growth effect of AZD6738 in biliary tract cancer (BTC) cells. (A) Basal expression levels of ataxia-telangiectasia and Rad3-related (ATR), ataxia-telangiectasia mutated (ATM), and p53 in nine BTC cell lines analyzed by western blotting. The intensity was quantified using imageJ software. The intensity greater than “1” was classified as high expression, less than “0.6” was classified as low expression. (B) Anti-proliferative effects of AZD6738 in nine BTC cell lines evaluated by MTT assays (left) and colony-forming assays (CFA) (right). (C) Western blot analyses evaluating the effect of AZD6738 on signaling pathways in four BTC cell lines. SNU478, SNU869, SNU245, and SNU2670 cells were treated with increasing concentrations of AZD6738 (0, 0.1, 0.5, and 1 μM) for 5 days, after which protein extracts were immunoblotted with the indicated antibodies.

• 

  Fig. 2. AZD6738 monotherapy induces cell cycle arrest in sensitive cell lines. (A) Cell cycle analyses of SNU478, SNU869, SNU245, and SNU2670 cells were performed by flow cytometry after treatment with increasing concentrations of AZD6738 (0, 0.1, 0.5, and 1 μM) for 3 days. The data represent three independent experiments. **p < 0.01. (B) Western blot analysis demonstrating the effect of AZD6738 on the expression levels of apoptosis and cell cycle checkpoint molecules in SNU478, SNU869, SNU245, and SNU2670 cells. The cells were treated with increasing doses of AZD6738 (0, 0.1, 0.5, and 1 μM) for 5 days. C, control.

• 

  Fig. 3. The synergistic effect was observed with combinations of AZD6738 and cytotoxic chemotherapeutic agents. (A) Anti-proliferative effects of combination chemotherapy of AZD6738 and cytotoxic agents (cisplatin, gemcitabine, and 5-fluorouracil [5-FU]) evaluated by MTT assays. The combination indexes for the combinations of AZD6738 and the cytotoxic agents at the ED50 were calculated by the Chou and Talalay method in the SNU478, SNU869, SNU245, and SNU2670 cell lines (combination index [CI] > 1, antagonistic effect; CI=1, additive effect; CI < 1, synergistic effect). (B) Colony-forming assays were performed to demonstrate the combined effect of AZD6738 and cisplatin. The concentration of each drug is indicated in the graph. The data represent mean±SE of three independent experiments. **p < 0.01. (C) Western blot analyses were performed to evaluate the anti-proliferative effect of AZD6738 and cisplatin on biliary tract cancer cell lines. PARP, poly(ADP-ribose) polymerase.

• 

  Fig. 4. The potential impact of AZD6738 on DNA damage. (A) Comet assays were performed on SNU478, SNU869, SNU245, and SNU2670 cells treated with AZD6738 (0.5 μM), cisplatin (0.5 μM), or their combination for 5 days. Scale bars=100 μm. (B) Comparisons of comet tail length and tail intensity between cells treated with AZD6738 (0.5 μM), cisplatin (0.5 μM), or their combination for 5 days. The data represent mean±standard error of three independent experiments. **p < 0.01, ***p < 0.001.

• 

  Fig. 5. Anti-tumor growth of AZD6738 alone or combination therapy. (A) In vivo efficacy of AZD6738 (25 mg/kg), cisplatin (4 mg/kg), or their combination in SNU478 xenograft model mice. **p < 0.01. T, start of treatment; CT, day of cisplatin treatment. (B) The tumors were harvested and analyzed by immunohistochemistry. Ki-67, TUNEL expression, and Chk1 phosphorylation were evaluated in the SNU478 xenograft model mice. Scale bars=50 μm. (C) Western blot assays were performed on the excised tumors from the xenograft model mice to elucidate the effects of AZD6738 and/or cisplatin.

Reference

References

1. Banales JM, Cardinale V, Carpino G, Marzioni M, Andersen JB, Invernizzi P, et al. Expert consensus document: Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA). Nat Rev Gastroenterol Hepatol. 2016; 13:261–80.

2. O'Connor MJ. Targeting the DNA damage response in cancer. Mol Cell. 2015; 60:547–60.

3. Pearl LH, Schierz AC, Ward SE, Al-Lazikani B, Pearl FM. Therapeutic opportunities within the DNA damage response. Nat Rev Cancer. 2015; 15:166–80.

4. Curtin NJ. DNA repair dysregulation from cancer driver to therapeutic target. Nat Rev Cancer. 2012; 12:801–17.

5. Branzei D, Foiani M. Regulation of DNA repair throughout the cell cycle. Nat Rev Mol Cell Biol. 2008; 9:297–308.

6. Zou L. Single- and double-stranded DNA: building a trigger of ATR-mediated DNA damage response. Genes Dev. 2007; 21:879–85.

7. Buisson R, Boisvert JL, Benes CH, Zou L. Distinct but concerted roles of ATR, DNA-PK, and Chk1 in countering replication stress during S phase. Mol Cell. 2015; 59:1011–24.

8. Eykelenboom JK, Harte EC, Canavan L, Pastor-Peidro A, Calvo-Asensio I, Llorens-Agost M, et al. ATR activates the S-M checkpoint during unperturbed growth to ensure sufficient replication prior to mitotic onset. Cell Rep. 2013; 5:1095–107.

9. Weber AM, Ryan AJ. ATM and ATR as therapeutic targets in cancer. Pharmacol Ther. 2015; 149:124–38.

10. Karnitz LM, Zou L. Molecular pathways: targeting ATR in cancer therapy. Clin Cancer Res. 2015; 21:4780–5.

11. Foote KM, Lau A, Nissink JW. Drugging ATR: progress in the development of specific inhibitors for the treatment of cancer. Future Med Chem. 2015; 7:873–91.

12. Thanan R, Pairojkul C, Pinlaor S, Khuntikeo N, Wongkham C, Sripa B, et al. Inflammation-related DNA damage and expression of CD133 and Oct3/4 in cholangiocarcinoma patients with poor prognosis. Free Radic Biol Med. 2013; 65:1464–72.

13. Moy AP, Shahid M, Ferrone CR, Borger DR, Zhu AX, Ting D, et al. Microsatellite instability in gallbladder carcinoma. Virchows Arch. 2015; 466:393–402.

14. Nakamura H, Arai Y, Totoki Y, Shirota T, Elzawahry A, Kato M, et al. Genomic spectra of biliary tract cancer. Nat Genet. 2015; 47:1003–10.

15. Churi CR, Shroff R, Wang Y, Rashid A, Kang HC, Weatherly J, et al. Mutation profiling in cholangiocarcinoma: prognostic and therapeutic implications. PLoS One. 2014; 9:e115383.

16. Bouwman P, Jonkers J. The effects of deregulated DNA damage signalling on cancer chemotherapy response and resistance. Nat Rev Cancer. 2012; 12:587–98.

17. Muller PA, Vousden KH. Mutant p53 in cancer: new functions and therapeutic opportunities. Cancer Cell. 2014; 25:304–17.

18. Reaper PM, Griffiths MR, Long JM, Charrier JD, Maccormick S, Charlton PA, et al. Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR. Nat Chem Biol. 2011; 7:428–30.

19. Nam AR, Kim JW, Cha Y, Ha H, Park JE, Bang JH, et al. Therapeutic implication of HER2 in advanced biliary tract cancer. Oncotarget. 2016; 7:58007–21.

20. Kim HJ, Min A, Im SA, Jang H, Lee KH, Lau A, et al. Antitumor activity of the ATR inhibitor AZD6738 in HER2 positive breast cancer cells. Int J Cancer. 2017; 140:109–19.

21. Min A, Im SA, Jang H, Kim S, Lee M, Kim DK, et al. AZD6738, a novel oral inhibitor of ATR, induces synthetic lethality with ATM deficiency in gastric cancer cells. Mol Cancer Ther. 2017; 16:566–77.

22. George E, Kim H, Krepler C, Wenz B, Makvandi M, Tanyi JL, et al. A patient-derived-xenograft platform to study BRCA-deficient ovarian cancers. JCI Insight. 2017; 2:e89760.

23. Ruiz S, Mayor-Ruiz C, Lafarga V, Murga M, Vega-Sendino M, Ortega S, et al. A genome-wide CRISPR screen identifies CDC25A as a determinant of sensitivity to ATR inhibitors. Mol Cell. 2016; 62:307–13.

24. Williamson CT, Miller R, Pemberton HN, Jones SE, Campbell J, Konde A, et al. ATR inhibitors as a synthetic lethal therapy for tumours deficient in ARID1A. Nat Commun. 2016; 7:13837.

25. Mohni KN, Kavanaugh GM, Cortez D. ATR pathway inhibition is synthetically lethal in cancer cells with ERCC1 deficiency. Cancer Res. 2014; 74:2835–45.

26. Gunaydin H, Weiss MM, Sun Y. De novo prediction of p-glycoprotein-mediated efflux liability for druglike compounds. ACS Med Chem Lett. 2013; 4:108–12.

27. Li CC, Yang JC, Lu MC, Lee CL, Peng CY, Hsu WY, et al. ATRChk1 signaling inhibition as a therapeutic strategy to enhance cisplatin chemosensitivity in urothelial bladder cancer. Oncotarget. 2016; 7:1947–59.

28. Buisson R, Niraj J, Rodrigue A, Ho CK, Kreuzer J, Foo TK, et al. Coupling of homologous recombination and the checkpoint by ATR. Mol Cell. 2017; 65:336–46.

29. Sangster-Guity N, Conrad BH, Papadopoulos N, Bunz F. ATR mediate cisplatin resistance in a p53 genotype-specific manner. Oncogene. 2011; 30:2526–33.

30. Vendetti FP, Lau A, Schamus S, Conrads TP, O'Connor MJ, Bakkenist CJ. The orally active and bioavailable ATR kinase inhibitor AZD6738 potentiates the anti-tumor effects of cisplatin to resolve ATM-deficient non-small cell lung cancer in vivo. Oncotarget. 2015; 6:44289–305.