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ATR Pathway Inhibition Is Synthetically Lethal in Cancer Cells with ERCC1 Deficiency

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ATR Pathway Inhibition Is Synthetically Lethal in Cancer Cells with ERCC1 Deficiency Published OnlineFirst March 24, 2014; DOI: 10.1158/0008-5472.CAN-13-3229 Cancer Therapeutics, Targets, and Chemical Biology Research ATR Pathway Inhibition Is Synthetically Lethal in Cancer Cells with ERCC1 Deficiency Kareem N. Mohni, Gina M. Kavanaugh, and David Cortez Abstract The DNA damage response kinase ATR and its effector kinase CHEK1 are required for cancer cells to survive oncogene-induced replication stress. ATR inhibitors exhibit synthetic lethal interactions, with deficiencies in the DNA damage response enzymes ATM and XRCC1 and with overexpression of the cell cycle kinase cyclin E. Here, we report a systematic screen to identify synthetic lethal interactions with ATR pathway–targeted drugs, rationalized by their predicted therapeutic utility in the oncology clinic. We found that reduced function in the ATR pathway itself provided the strongest synthetic lethal interaction. In addition, we found that loss of the structure-specific endonuclease ERCC1-XPF (ERCC4) is synthetic lethal with ATR pathway inhibitors. ERCC1- deficient cells exhibited elevated levels of DNA damage, which was increased further by ATR inhibition. When treated with ATR or CHEK1 inhibitors, ERCC1-deficient cells were arrested in S-phase and failed to complete cell-cycle transit even after drug removal. Notably, triple-negative breast cancer cells and non–small cell lung cancer cells depleted of ERCC1 exhibited increased sensitivity to ATR pathway–targeted drugs. Overall, we concluded that ATR pathway–targeted drugs may offer particular utility in cancers with reduced ATR pathway function or reduced levels of ERCC4 activity. Cancer Res; 74(10); 1–11. Ó2014 AACR. Introduction repair pathway such as homologous recombination or post- – Replicating DNA is sensitive to a wide array of endogenous replicative repair to remove the PARP DNA complexes (7). and exogenous damaging agents, which can lead to replication The synthetic lethality between PARP inhibitors and muta- BRCA1/2 fork stalling. To accomplish error-free replication, stalled tions in provides a paradigm for combining DNA fi replication forks need to be stabilized to prevent their collapse repair inhibitors with speci c mutations or in combination into double strand breaks (DSB), which can lead to genomic with chemotherapy agents. rearrangements and/or apoptosis (1, 2). The DNA damage Oncogene activation often initiates an ATR-dependent rep- response kinase ATR coordinates many of the activities at lication stress response that is needed for continued cell – stalled forks, including fork stabilization and restart (3). ATR growth (8 10). Thus, ATR pathway inhibitors are being devel- activation also slows the cell cycle to allow for DNA repair oped as cancer therapeutics. For example, CHK1 inhibitors through the phosphorylation of CHK1 (3). have shown promise in preclinical models, and there are The loss of one or more DNA damage response or repair several ongoing and completed phase I and II clinical trials – fi pathways drives tumorigenesis but also forces cancer cells to (11 13). Recently, speci c ATR inhibitors have been described be more reliant on other pathways, providing an opportunity by AstraZeneca (14), Vertex Pharmaceuticals (15, 16), and the for the development of targeted therapeutics (4). For exam- Fernandez-Capetillo laboratories (17). These inhibitors func- in vitro ple, cancers with mutations in BRCA1/2 are sensitive to tion to inhibit the growth of cancer cell lines and PARP inhibitors as the replication-associated DSBs caused synergize with DNA-damaging agents such as cisplatin (15). fi by PARP inhibition cannot be repaired when the BRCA- Furthermore, ATR inhibition demonstrated ef cacy in a xeno- dependent homologous recombination system is nonfunc- graft mouse model of pancreatic cancer in combination with tional(5,6).PARPinhibitorscanalsotrapPARPonDNA gemcitabine (18). creating a toxic intermediate that requires a second DNA ATR inhibitors also exhibit synthetic lethal interactions with ATM and XRCC1 deficiency as well as with cyclin E over- expression (15, 17, 19). To date, no systematic approach to – Authors' Affiliation: Department of Biochemistry, Vanderbilt University identify synthetic lethal interactions with ATR pathway tar- School of Medicine, Nashville, Tennessee geted drugs has been reported. This information could be used – Note: Supplementary data for this article are available at Cancer Research in the clinic to design better clinical trials with ATR pathway Online (http://cancerres.aacrjournals.org/). targeted drugs and improve patient outcomes. Corresponding Author: David Cortez, Department of Biochemistry, Van- The current study was initiated to identify genes that, when derbilt University School of Medicine, Nashville, TN 37232. Phone: 615- lost, exhibit synthetic lethal relationships with ATR pathway 322-8547; Fax: 615-343-0704; E-mail: [email protected] inhibitors. We conducted a synthetic lethal screen with DNA doi: 10.1158/0008-5472.CAN-13-3229 repair proteins and identified reduced ATR pathway function Ó2014 American Association for Cancer Research. and the ERCC1-XPF nuclease as synthetic lethal with ATR or www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst March 24, 2014; DOI: 10.1158/0008-5472.CAN-13-3229 Mohni et al. CHK1 inhibition. ERCC1-XPF functions in the repair of bulky and Luc Friboulet in December 2013 and maintained as DNA adducts, double strand breaks, interstrand crosslinks described (25). All cells lines were thawed from early-passage (ICL), and separation of sister chromatids at fragile sites stocks and were passaged less than 30 times before use. No (20, 21). Importantly, ERCC1 is being explored as a potential further cell line authentication was performed. The ATR biomarker in lung and other kinds of cancer (22–25). Low levels inhibitor VE-821 (15) was synthesized by the Vanderbilt Insti- of ERCC1 expression correlate with greater sensitivity to tute for Chemical Biology Chemical Synthesis Facility. The cisplatin and higher 5-year survival rates. Several phase II CHK1 inhibitor AZD7762 was previously described (28). clinical trials are also underway using ERCC1 protein levels to determine whether to treat patients with platinum-based Synthetic lethal siRNA screen chemotherapy (22, 23). Our data demonstrate that both triple- The custom siRNA library targets 240 known DNA repli- negative breast cancer and non–small cell lung cancer cell lines cation and DNA repair genes with 4 unique siRNAs per gene depleted of ERCC1 exhibit increased sensitivity to ATR path- in individual wells. The siRNA was plated into 96-well plates way inhibition. Thus, ERCC1 status may be a useful indicator of in replicates and frozen at —80C.Theplateswerethawed sensitivity to ATR pathway–targeted drugs. and siRNA was resuspended in OptiMEM with Dharmafect1. U2OS cells were then added to each plate. The final siRNA Materials and Methods concentration was 10 nmol/L. Cells were split into two 96- Cells and reagents well dishes 72 hours after transfection and incubated in U2OS and 293T cells were obtained from American Type media with or without ATR inhibitor at a final concentration Culture Collection (ATCC) and maintained in Dulbecco's of 1 mmol/L for an additional 72 hours. Cell viability was Modified Eagle's Media (DMEM) supplemented with 7.5% FBS. measured with alamar blue (Invitrogen). Nonfluorescent Triple-negative breast cancer cells lines BT549 and HCC1806 cell-permeable alamar blue dye is reduced to a molecule and non–small cell lung cancer cells lines A549 and H157 were that fluoresces red in metabolically active cells, which allows obtained from ATCC and maintained in RPMI supplemented for a quantitative measure of viability. The percent viability with 10% FBS. The HCT-116–derived ATR flox/þ and ATR of ATR inhibitor treated to untreated was determined for flox/À were previously described (26). XPF-deficient fibro- each siRNA to take into account siRNA-specificeffectson blasts XP2YO and XP2YO þ XPF were provided by Orlando cell growth. The z-scores were calculated using the mean Scharer in August 2013 and maintained as described (27). The and SD of the log10(percent viability) values. The values ERCC1-null (clone 216) and complemented A549 cells (clone presented in Fig. 1 are the mean z-scores from three inde- 216 þ 202) were provided by Jean Charles Soria, Ken Olaussen, pendent transfections. AB96-well dish C2 Non targeting XPB XPA Transfect cells 1 XPD XPC with siRNA 100 siNT 0 n = 3 siATR Mean Z-score 80 −1 XRCC1 XPF 60 −2 ATM Untreated +ATRi ERCC1 40 −3 72 h Chk1 −4 HUS1 20 Claspin Percent viability − Measure cell 0 5 RPA ATRi/untreated Z-score viability Untreated ATRi −6 ATRIP −7 ATR shGFP HCT116 D120 E shATR 120 ATR flox/+ 100 shCHK1 100 ATR flox/− 1 P 80 R AT 80 shGF sh shCHK 60 ATR 60 6 CHK1 40 40 R flox/+ Ku70 HCT11AT ATR flox/− 20 20 ATR Percent of control Percent of control CHK1 0 0 0.1 1 10 0.1 1 10 ATRi concentration (μmol/L) ATRi concentration (μmol/L) Figure 1. siRNA screen identifies synthetic lethal interactions with ATR inhibition. A, schematic of the siRNA screen. U2OS cells were transfected with siRNAs and then were left untreated or treated with 1 mmol/L ATRi. Cell viability was determined with alamar blue. B, percent viability of the nontargeting (NT) and ATR siRNA controls from the screen. Values represent the mean Æ SD of three independent replicates. C, the z-scores of treated compared with untreated cell viability for each gene is shown. Each data point represents the mean of three independent replicates and calculation of the z-scores is described in Materials and Methods. D, U2OS cells were infected with lentiviruses expressing the indicated shRNA and selected with puromycin and then treated with increasing doses of ATRi for 72 hours. E, HCT-116 wild-type, ATR flox/þ, and ATR flox/À cells were treated with increasing doses of ATRi for 72 hours. For D and E, cell viability was determined with alamar blue and reported as a percent of the untreated control cells.
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