Cancer Res Treat. 2019;51(3):1167-1179.

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

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제1저자 남아롱, 김미화 · 교신저자 오도연

PMID 30514066 PMC6639230 DOI 10.4143/crt.2018.526 IF 4.2 혈액종양내과

소속 (2)

Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.
Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea.

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.

Keywords

ATMATR inhibitorBiliary tract neoplasmsDNA damage responsep53

Figures

**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.

그림 출처: PubMed Central 공개 논문 (PMC6639230)

Conflict of interest statement

D-Y Oh received AstraZeneca-KHIDI (Korea Health Industry Development Institute) Oncology Research Program Grant and Y-J Bang is a consultant/advisory board member of AstraZeneca. No conflicts of interest were disclosed by the other authors.

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