(ATR) Overcomes Oxaliplatin Resistance and Promotes Antitumor Immunity in Colorectal Cancer Eve Combes� 1,2, Augusto F

Published OnlineFirst April 15, 2019; DOI: 10.1158/0008-5472.CAN-18-2807

Cancer Translational Science Research

Inhibition of Ataxia-Telangiectasia Mutated and RAD3-Related (ATR) Overcomes Oxaliplatin Resistance and Promotes Antitumor Immunity in Colorectal Cancer Eve Combes 1,2, Augusto F. Andrade1,2, Diego Tosi1,2, Henri-Alexandre Michaud1,2, Flavie Coquel3, Veronique Garambois1,2, Delphine Desigaud1,2, Marta Jarlier2, Arnaud Coquelle1,2, Philippe Pasero3, Nathalie Bonnefoy1,2, Jerome Moreaux3, Pierre Martineau1,2, Maguy Del Rio1,2, Roderick L. Beijersbergen4, Nadia Vezzio-Vie1,2, and Celine Gongora1,2

Abstract

Although many patients with colorectal cancer initially respond Oxaliplatin + ATRi to the chemotherapeutic agent oxa- ICD signals liplatin, acquired resistance to this Immune cell death CRT treatment remains a major chal- ATP lenge to the long-term management Antitumoral HMGB1 of this disease. To identify molecu- immunity lar targets of oxaliplatin resistance DNA damage in colorectal cancer, we performed Replication stress an shRNA-based loss-of-function genetic screen using a kinome library. We found that silencing of Cytoplasmic Proliferation inhibition IFN production ataxia-telangiectasia mutated and DNA Apoptosis induction RAD3-related (ATR), a serine/thre- Cell autonomous cell death onine protein kinase involved in the response to DNA stress, restored

oxaliplatin sensitivity in a cellular © 2019 American Association for Cancer Research model of oxaliplatin resistance. Combined application of the ATR inhibitor VE-822 and oxaliplatin resulted in strong synergistic effects in six different colorectal cancer cell lines and their oxaliplatin-resistant subclones, promoted DNA single- and double-strand break formation, growth arrest, and apoptosis. This treatment also increased replicative stress, cytoplasmic DNA, and signals related to immunogenic cell death such as calreticulin exposure and HMGB1 and ATP release. In a syngeneic colorectal cancer mouse model, combined administration of VE-822 and oxaliplatin signiﬁcantly increased survival by promoting antitumor T-cell responses. Finally, a DNA repair gene signature discriminated sensitive from drug-resistant patients with colorectal cancer. Overall, our results highlight the potential of ATR inhibition combined with oxaliplatin to sensitize cells to chemotherapy as a therapeutic option for patients with colorectal cancer.

Signiﬁcance: These ﬁndings demonstrate that resistance to oxaliplatin in colorectal cancer cells can be overcome with inhibitors of ATR and that combined treatment with both agents exerts synergistic antitumor effects. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2933/F1.large.jpg.

1Institut de Recherche en Cancerologie de Montpellier, INSERM U1194 Universite E. Combes and A.F. Andrade are the co-ﬁrst authors of this article. Montpellier, CNRS, France. 2Institut regional du Cancer–Montpellier–Val d'Aurelle, Corresponding Author: Celine Gongora, IRCM INSERM U1194, 208 rue des Montpellier, France. 3IGH UMR9002, CNRS-Universite de Montpellier, Montpellier, Apothicaires, Montpellier 34298, France. Phone: 334-6761-3745; Fax: 334- France. 4Division of Molecular Carcinogenesis and NKI Robotics and Screening 6761-3787; E-mail: [email protected] Center, The Netherlands Cancer Institute, Amsterdam, the Netherlands. Cancer Res 2019;79:2933–46 Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). doi: 10.1158/0008-5472.CAN-18-2807 N. Vezzio-Vie and C. Gongora contributed equally to this article. 2019 American Association for Cancer Research.

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Introduction SW48-R clones, respectively. The cell lines were tested and authen- ticated by short-tandem repeat profiling (LGC Standards and Colorectal cancer is the fourth most common cancer world- Eurofins Genomics). All experiments were performed at least wide. In 2015, the estimated colorectal cancer incidence was of three times. 1.65 million cases worldwide, and colorectal cancer caused more than 835, 000 deaths (1). Importantly, 30% of patients present synchronous metastases and 50%–60% will develop metastases Drugs and reagents that will require chemotherapy. The current management of VE-822 (ATR inhibitor) was kindly provided by A. Coquelle fi colorectal cancer is based on various drugs [5-fluorouracil (5- and dissolved to a nal concentration of 50 mmol/L in DMSO FU)/leucovorin (LV), capecitabine, irinotecan, oxaliplatin, bev- (Sigma Chemical Co.). Oxaliplatin (5 mg/mL) was from ICM acizumab, cetuximab, and panitumumab], either in combination (oxaliplatin ACCORD Cip: 3400957954642) and was diluted fi or as single agents (2). These treatments significantly improved in PBS to the nal concentration of 12 mmol/L. Both stock the patients' overall survival, though tumor resistance is still a solutions were aliquoted and stored at 20 C. Poly-L-ornithine frequent cause of therapy failure. was purchased from Sigma and formaldehyde (16%) from Among the drugs used in colorectal cancer treatment, oxali- Electron Microscopy Sciences. Antibodies against ATR platin is a third-generation platinum compound with a 1,2- #13934, ATM #2873, phosphorylated ATM (Ser1981) diaminocyclohexane carrier ligand, and is used in combination #13050, CDK2 #2546, phosphorylated CDK2 #2561, CHK1 with 5-fluorouracil and leucovorin (FOLFOX regimen). It causes 2360#, phosphorylated CHK1 (Ser345) #2341, CHK2 #2662, inter- and intrastrand DNA cross-links that stop DNA replication phosphorylated CHK2 (Thr68) #2661, GAPDH #5174, p21 and transcription, leading to apoptotic cell death (3). Oxaliplatin WAF1/CIP1 #2947, and phosphorylated p53 (Ser15) #9284 exerts its antitumor effect also by inducing immunogenic cell were from Cell Signaling Technology. The anti-phosphorylated – death (ICD; ref. 4), where the host immune system is primed to ATR (Thr1989) GTX128145 antibody was from Genetex, anti recognize and eliminate tumor cells upon drug-mediated stimu- b-tubulin T4026 from Sigma, and anti-p53 (DO-1) sc-126 from lation of the host antitumor immunity. Resistance to oxaliplatin is Santa Cruz Biotechnology. mediated by several factors, such as reduced cellular uptake, impaired DNA adduct formation, alterations in DNA repair genes Pooled shRNA "dropout" screen (e.g., ERCC1 and XRCC1), apoptosis defects, and modifications in The TRC kinome library included 1,798 short-hairpin RNA the expression levels of copper transporters (ATP7A and ATP7B; vectors that target 518 genes selected from TRC collection. The refs. 3–7). library was used to generate lentiviral supernatants as described Drug combinations are often used to overcome drug resis- previously (8). HCT-116-R1 cells were infected with the lentiviral tance and a major effort is made to identify novel drug combi- pools at a multiplicity of infection < 0.3 and with cell numbers nations to improve therapeutic efficiency. High-throughput sufficient to represent the kinome library with a 1,000 times functional genetic screens (using shRNA or CRISPR/Cas9 coverage for each shRNA present in the library. After puromycin approaches) provide a powerful tool to identify not only the selection, infected cells were pooled and plated in 15-cm dishes mechanisms of drug resistance, but also new targets for efficient (1 105 cells/dish) with sufficient cell numbers to maintain the drug combinations. In this study, we wanted to identify mole- 1,000-fold coverage. After incubation in the absence or presence cules that could modify molecular alterations that occur in of 3 mmol/L oxaliplatin for 10 days, cells were harvested, genomic oxaliplatin-resistant colorectal cancer cell lines. To this aim, we DNA was isolated, and used for recovery of the shRNA inserts by performed a kinome-specific shRNA genetic screen showing PCR. Indexes and adaptors for deep sequencing (Illumina) were that ataxia-telangiectasia mutated and RAD3-related (ATR) is incorporated in the PCR primers. PCR products were purified implicated in oxaliplatin resistance. Importantly, we could using the QIAGEN PCR Purification Kit, according to the man- demonstrate that the combinationofanATRinhibitorwith ufacturer's protocol. DNA was quantified using a BioAnalyzer, oxaliplatin is synergistic in in vitro and in vivo colorectal cancer and samples were combined at the same molar ratio. The shRNA models by inducing DNA double-strand breaks, leading to sequences were extracted from the sequencing reads and aligned apoptosis, and promoting antitumor immunity. to the kinome library. The matched reads were counted and the read counts were used for analysis. The statistical analysis was done with DESeq, version 1.8.3, using the default settings for Materials and Methods "pooled." The results of the DESeq analysis were used to calculate Cell culture the ratio between treated and nontreated cells for each individual The human HCT116, SW48, SW480, SW620, HT29 (from the shRNA. ATCC and the murine CT26, kindly provided by N. Bonnefoy, colorectal cancer cell lines were grown in RPMI1640 with 10% ATR silencing by shRNAs FCS and 2 mmol/L L-glutamine at 37 C in a humidified atmo- Four different shRNA vectors with the puromycin selection sphere with 5% CO2. The murine colorectal cancer cell line MC38, marker and specifictoATR were used. Lentiviral production was provided by N. Bonnefoy, was grown in DMEM with 10% FCS and performed by cotransfecting 293T cells (for lentiviral packaging) 1 mmol/L sodium pyruvate at 37C in a humidified atmosphere with 1 mg of anti-ATR shRNAs, 1 mg of the packaging vector gag- with 5% CO2. The oxaliplatin-resistant clones (HCT116-R1, pol, and 1 mg of envelope vector, using FuGene6 (Invitrogen), HCT116-R2, SW48-R) were obtained as described previously (5). according to the manufacturer's instructions. Lentiviral particles Briefly, the oxaliplatin-sensitive parental HCT116 and SW48 cell were harvested and then used to infect 1 106 HCT116 and lines were grown in the presence of 5–10 mmol/L oxaliplatin and HCT116-R1 cells for 48 hours. Following shRNA transduction, cloned to obtain the resistant HCT116-R1 and HCT116-R2 and cells were selected with puromycin.

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Cell growth inhibition assay (2D assay) 24 hours later they were incubated with oxaliplatin (5 mmol/L) Cell growth was evaluated using the sulforhodamine B (SRB) and/or VE-822 (1 mmol/L). After 48 hours, cells were washed in assay, as described by Skehan and colleagues (9). Briefly, 300– ice-cold PBS, fixed in 75% ethanol, and labeled with 40 mg/mL 1,000 cells/well were seeded in 96-well plates. After 24 hours, propidium iodide (Sigma) containing 100 mg/mL RNase A drugs were added in serial dilution. Cells were incubated for 96 (Sigma). Cell-cycle distribution was then determined with a hours, after which, they were fixed in trichloroacetic acid solution Gallios Cytometer (Beckman Coulter) and quantified using the (final concentration of 10%), and stained with 0.4% SRB solution Kaluza Software (Beckman Coulter). in 1% acetic acid (Sigma Chemical Co.). Fixed SRB was dissolved in 10 mmol/L Tris-HCl solution and absorbance at 560 nm was Apoptosis assay read using a Thermo Fisher Scientific Multiskan EX plate reader. A total of 2.5 105 HCT116 and 4 105 HCT-116-R1 cells The IC50 was determined graphically from the cell growth curves. were plated, and after 24 hours, they were incubated with the indicated drugs (alone or in combination) for 48 hours. 3D spheroid assay Cells were stained with FITC-labeled Annexin V and 7-amino- For spheroid generation, 100 mL/well of cell suspensions at actinomycin D (7-AAD; Annexin V/7-AAD-Beckman Coulter). optimized densities (50–300 cells/well) were dispensed in ultra- For apoptosis determination, Annexin V- and 7-ADD–positive low attachment 96-well round-bottomed plates (Corning B.V. cells were quantified using a Gallios Cytometer and the Kaluza Life Sciences) and cultured at 37 C, 5% CO2, 95% humidity, for 4 Software (Beckman Coulter). days. Depending on the cell sensitivity, spheroids were incubated with VE-822 (0 to 2.5 mmol/L) and/or oxaliplatin (0–30 mmol/L) Western blot analysis at day 4 and at day 10 after plating. After treatment, cells (1 104 cells/mL) were washed (1 PBS) At day 14, images were captured with the Celigo Imaging and directly lysed in Laemmli buffer (4% SDS, 20% glycerol, 1% Cytometer (Nexcelom Bioscience) using the "Tumorosphere" 2-b mercaptoethanol, 0.004% bromophenol blue, 0.125 mol/L application. Cell viability was measured using a CellTiter-Glo Tris HCL) þ benzonase. After denaturation at 95 C for 5 minutes, Luminescent Cell Viability Assay (Promega), according to the protein extracts were deposited and separated on SDS-PAGE manufacturer's instructions. Luminescence was measured on a polyacrylamide (10%, 12%, or 4%–20% gradient) gels. They 1450 MicroBeta TriLux Luminescence Microplate Reader (Perkin were then transferred (transfer buffer containing 20% ethanol) Elmer). The IC50 was determined graphically from the cytotoxicity to nitrocellulose membranes (10% and 12% gels: semi-dry trans- curves. fer for 1 hour; 4%–20% gradient gels: liquid transfer for 2 hours). Membranes were then blocked in PBS/0.1% Tween/5% milk at Quantification of the interaction effect room temperature for 1 hour under agitation. They were then The interaction between the drugs tested in vitro was investi- incubated with the primary antibodies at 4 C by gentle agitation gated with a concentration matrix test, in which increasing con- overnight. After three washes in PBS/0.1% Tween, membranes centration of each single drug were assessed with all possible were incubated with the relevant antispecies secondary antibody combinations of the other drugs. For each combination, the coupled to horseradish peroxidase diluted to 1/5,000 in PBS/ percentage of expected growing cells in the case of effect inde- 0.1% Tween/5% milk with gentle agitation for 1 hour. After pendence was calculated according to the Bliss equation (10): washing, immunoreactions were revealed by chemiluminescence (ECL RevelBlot Plus, and ECL RevelBlot Intense Kits) and visu- fuc ¼ fuAfuB alized using the G-Box Imaging System (Syngene). Western blot analysis was performed using ImageJ software, and quantification fu where c is the expected fraction of cells unaffected by the drug were calculated relatively to the loading control associated. The fu fu combination in the case of effect independence, and A and B untreated condition was arbitrary fixed at 1 to easily observe the A B, are the fractions of cells unaffected by treatment and respec- changes induced by transfections or treatments. Each Western blot fu tively. The difference between the c value and the fraction of was performed and quantified between two and four times in each living cells in the cytotoxicity test was considered as an estimation cell line except for ATM total in HCT116 cells, for which quan- of the interaction effect, with positive values indicating synergism tification could only be done once. and negative values antagonism. RPA32 and gH2AX analysis EdU assay HCT-116 (4,000 to 9,000 cells/well) or HCT116-R1 (8,000 to Cell proliferation was measured with the Click-iT EdU Assay 16,000 cells/well) cells were plated in black-sided 96-well, flat- (Invitrogen) according to the manufacturer's protocol. Briefly, bottomed plates (Greiner) precoated with poly-L-ornithine. After 2.5 105 HCT116 and 4 105 HCT-116-R1 cells were plated in 24 hours, cells were incubated with VE-822 (1 mmol/L) and/or 25 cm2 flasks, and 24 hours later they were incubated with oxaliplatin (12.5 or 50 mmol/L) for 3, 6, 16, and 24 hours. Then, oxaliplatin and/or VE-822 for 48 hours. Twenty hours before RPA not bound to chromatin was extracted with PBS/0.2% Triton harvesting, EdU was also added. Cells were then washed in ice- X-100 on ice for 90 seconds. All steps were carried out carefully to cold PBS, fixed in 75% ethanol, and then incubated with the keep cells attached to the plate. Between each step, cells were Click-iT reaction cocktail for 30 minutes. Cells were washed with washed with PBS containing 1 mg/mL BSA. First, cells were fixed PBS and then analyzed by flow cytometry. in PBS/4% paraformaldehyde (PFA) for 30 minutes and permeabilized with PBS/0.2% Triton X-100 at room temperature for Cell-cycle analysis 10 minutes. After blocking with PBS/1% BSA for 30 minutes, To determine the cell-cycle distribution, 2.5 105 HCT116 and primary antibodies against RPA32 (Abcam, ab2175) and gH2AX 4 105 HCT-116-R1 cells were plated in 25 cm2 flasks, and (Cell Signaling Technology, #9718) were diluted in PBS with

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1 mg/mL BSA and added to each well at room temperature for 822 (1 mmol/L) for 24 hours and 48 hours. Supernatants were 1 hour. Then, the secondary antibodies (anti-mouse A448, collected and centrifuged to remove dying cells. ATP release Invitrogen, A11029; and anti-rabbit A568, Thermo Fisher Scien- was measured with the ATPlite Luminescence Assay System tific, A11011), diluted in PBS/1 mg/mL BSA, were added at (PerkinElmer), according to the manufacturer's instructions. room temperature for 45 minutes. After this step, cells were kept HMGB1 release was quantified with the HMGB1 ELISA Kit (IBL in the dark and incubated with 1 mg/mL DAPI at 37C for International), according to the manufacturer's instructions. 30 minutes. Cells were then washed and left in PBS for analysis with the Celigo cytometer imaging system. Cytosolic DNA Cells grown on glass coverslips were fixed in 80% ice-cold fi DNA ber spreading methanol for 20 minutes. Control samples were preincubated at 6 HCT116-R1 cells were seeded in 6-well plates (0.5 10 / 37C with 200 mg/mL RNase A (Sigma) and/or with DNase for 20 well). After 1 day, cells were incubated with VE-822 (1 mmol/L) minutes. Samples were blocked with 1% BSA/PBS/Triton X-100 and/or oxaliplatin (12.5 mmol/L) for 24 hours. Then, cells were for 1 hour, and then incubated with the primary antibodies labeled by two consecutive pulses of 15 minutes/each of the overnight [anti-ssDNA (Developmental Studies Hybridoma fi thymidine analogues iodo-deoxyuridine (IdU; nal concentra- Bank, The University of Iowa, Iowa City, IA) or anti-dsDNA fi tion: 20 mmol/L) and chloro-deoxyuridine (CldU; nal concen- (MAB1293, Millipore) in PBS/0.1% Tween]. After removal of tration: 200 mmol/L). Then, the medium was removed and unbound primary antibodies by washes with PBS/0.1% Tween, replaced by fresh medium containing 400 mmol/L of thymidine secondary antibodies were added at room temperature for 1 hour. for 2 hours (chase period). After this step, cells were trypsinized, Coverslips were then washed in PBS/0.1% Tween before mount- 6 washed, and diluted in cold PBS to 1 10 cells. ing with DAPI-containing Prolong Gold. Images were taken using In a humid chamber, the DNA of approximately 2,000 cells a63 objective and a Zeiss microscope. from the cell suspension was spread along a microscopy slide using lysing buffer (200 mmol/L Tris-HCL, 50 mmol/L EDTA, 0.5% SDS in milliQ H O), and fixed in acetic acid:methanol (1:3) for In vivo studies 2 6 10 minutes, after which, slides were dried. Xenografts. HCT116/R1 and MC38 tumor cells (2 10 and 5 5 fl For immunostaining, slides were washed with H O(2 5 10 , respectively) were injected subcutaneously in the left ank of 2 nu/nu minutes) and denatured in 2.5 mol/L HCL for 1 hour. After 8-week-old female athymic and C57BL/6 mice (Charles n ¼ – washing in PBS, slides were saturated in PBS/5% BSA for 1 hour, River Laboratories; 6 8). Tumors were detected by palpation and then incubated with the anti-Cldu antibody (Bio-Rad and measured with a calliper weekly. Mice were euthanized when 3 OBT0030) in PBS/0.1% Triton X-100 (PBST) at 37C for 1 hour. the tumor volume reached 1,500 mm . Ethical approvals were After washes in PBST (2 5 minutes) and in PBS (1 5 minutes), obtained by the local ethics committee [Ethics Committee slides were incubated with the secondary antibody (Thermo approved by the French Ministry, animal facility approval C34- Fisher Scientific A11006) in PBST at 37C for 45 minutes (in the 172-27, personal approval (Celine Gongora) 34.142 and protocol dark). After washing, slides were incubated with anti-IdU (BD approval CEEA-LR- 1193 and CEEA-LR-1194)]. Biosciences 347580) and anti-ssDNA (DHSB supernatant) anti- 3 bodies in PBST at 37C for 1 hour, washed, and incubated with the Tumor treatment. When tumors reached the volume of 100 mm , relevant secondary antibody (Thermo Fisher Scientific A21123 mice were treated for 4 weeks with: (i) 0.2 mL of 0.9% sodium þ and A21241) in PBST at 37C for 45 minutes. Then, slides were chloride solution by intraperitoneal injection D-a-tocopherol washed, air-dried, and mounted with Prolong Gold (Thermo polyethylene glycol succinate (TPGS; gavage; control group), Fisher Scientific P10144). Micrographs of labeled fibers were twice per week; (ii) 5 mg/kg oxaliplatin by intraperitoneal injec- taken with a Fluorescence Microscope (Zeiss or Leica DMRM) tion once per week; (iii) 30 mg/kg VE-822 (dissolved in TPGS) by þ and for each condition, at least 100 track lengths were measured. gavage twice per week; or (iv) 5 mg/kg oxaliplatin once per week Each experiment was repeated at least two or three times. 30 mg/kg VE-822 twice per week.

Calreticulin exposure Statistical analysis. A linear mixed-regression model, containing fi A total of 1.5–2 105 cells were plated in 6-well plates, and both xed and random effects, was used to determine the rela- after 24 hours, the medium was changed and cells were incubated tionship between tumor growth and days after grafting. Data were fi fi with oxaliplatin (25 mmol/L) and/or VE-822 (1 mmol/L) for 48 rst transformed using the natural log scale to better t the fi hours. Cells were collected, washed in cold PBS/10% FBS, and assumptions of the linear mixed model. The xed part of the stained with 7-AAD (Beckman Coulter) for 30 minutes. After model included variables corresponding to the number of post- washing in PBS/10% FBS, cells were fixed with 0.25% PFA for 5 graft days and the different treatments. Interaction terms were minutes. After washing, cells were incubated with the anti–CRT- built into the model; random intercepts and random slopes were fi Alexa647 antibody (ab196159, Abcam) for 1 hour, followed by included to take time into account. The model coef cients were fi washing, and fixation in 1% PFA for 5 minutes. Calreticulin (CRT) estimated by maximum likelihood and considered signi cant at exposure was assessed using a Gallios Cytometer (Beckman the 0.05 level. Statistical analyses were performed using the STATA Coulter). The fluorescent intensity of CRT-positive cells was gated 10.0 Software (StataCorp). relative to 7-AAD–negative cells. Detection of CD8-positive T cells ATP and HMGB1 release Treated and control mice harboring mouse MC38 cell xeno- ATP and HMGB1 release in the cell supernatant were analyzed grafts were sacrificed 3 weeks after graft/treatment. Spleens were after incubation with oxaliplatin (12.5–100 mmol/L) and/or VE- recovered in ice-cold PBS containing 0.5% BSA and 2 mmol/L

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EDTA, and were mechanically dissociated. Red blood cells were characteristics in terms of MSI/MSS status, origin, and KRAS, then removed by adding 2 mL ammonium-chloride-potassium BRAF, PIK3CA, and TP53 gene mutation status (Supplementary lysing buffer. White blood (effector cells) cells were recovered by Table S1). Using 2D (SRB) and 3D (spheroid growth) cell culture centrifugation, washed with PBS, and resuspended in RPMI1640 assays, we confirmed that the three oxaliplatin-resistant cell lines plus 10% FBS. MC38 target cells were cultured as described displayed the highest oxaliplatin IC50 values (Fig. 1A). previously, and then trypsinized, washed, and resuspended in To identify genes the inhibition of which confers sensitivity to RPMI1640 plus 10% FBS. A total of 10.5 cells were plated in oxaliplatin in HCT116-R1 cells (synthetic lethal interactions with 100 mL of RPMI1640 in 96-well U-shaped plates, and then oxaliplatin; ref. 8), we performed a loss-of-function genetic screen irradiated (50 Gy). Splenocytes were added at the 1:5 (target: using the TRC lentiviral kinome shRNA library that targets 518 effector) ratio and cultured overnight. GolgiPlug and GolgiStop human kinases (Fig. 1B). After infection of HCT116-R1 cells with were added to each well to block cytokine secretion (according to the library and culture in the absence/presence of 3 mmol/L the manufacturer's protocol; BD Biosciences) for 6 hours. To oxaliplatin (the oxaliplatin concentration required to kill all identify IFNg-secreting CD8-positive T cells, cells were washed sensitive HCT116 cells, but inefficient in HCT116-R1 cells, once with PBS and incubated with antibodies against surface see Fig. 1B) for 10 days. shRNAs were amplified by PCR and their markers CD45-BV786 (clone 30F11), CD8-BV605 (clone 53-6.7), relative abundance was determined by next-generation sequenc- and the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen) ing using the barcode identifiers present in each shRNA vector. We at 4C for 1 hour. After extracellular staining, cells were fixed and considered only shRNA vectors that were significantly depleted permeabilized, according to the eBioscience fixation and permea- (by at least 50%) upon incubation with oxaliplatin, and selected bilization procedures, and intracellular staining [(anti-IFNg anti- genes represented by multiple shRNAs matching this criterion. body V421 (clone XMG1.2)] was performed at 4C overnight. Only few of the 1,798 shRNAs in the library met these criteria Then, samples were washed, fixed in 1% PFA, and processed for (Fig. 1C), among which, three (out of four) were independent data acquisition and analysis using a Cytoflex Flow Cytometer shRNAs targeting ATR. To validate this finding, we infected (Beckman Coulter) and the Flowjo 10 software. HCT116-R1 cells with shRNAs against ATR or luciferase (control; Fig. 1D) and confirmed that ATR silencing sensitized Gene expression profiling and DNA repair score HCT116-R1 cells to oxaliplatin (Fig. 1E; Supplementary Fig. S1A ATR expression was assessed using the dataset GSE62322 and S1B). Moreover, comparison of the oxaliplatin IC50 values of [(n ¼ 17 normal mucosa, n ¼ 20 primary tumor, and n ¼ 19 HCT116-R1-shATR and HCT116 cells indicated that upon ATR hepatic metastasis tissue samples from patients with metastatic knockdown, resistant HCT116-R1 cells became as sensitive to colorectal cancer (mCRC)] from the prospective single-center oxaliplatin as HCT116 cells (Fig. 1F). These findings demonstrate study REGP (9, 10). that inhibition can be sufficient to overcome oxaliplatin resis- To build the DNA repair score, the gene expression datasets tance, at least in HCT116-R1 cells. from two cohorts were used. The Tsuji dataset (GSE28702) We then tested whether ATR or its target protein checkpoint included 80 patients with stage IV colorectal cancer treated with kinase 1 (CHK1) were constitutively activated (i.e., phosphory- FOLFOX. Shingo Tsuji (Genome Science Division, Research lated) in HCT116-R1 cells compared with sensitive HCT116 cells. Center for Advanced Science and Technology, The University of Western blot analysis showed that neither ATR nor CHK1 were Tokyo, Tokyo, Japan) kindly provided the OS data. The Del Rio activated in HCT116-R1 cells (Fig. 1G). In addition, analysis of a dataset (GSE72970) included 36 patients with stage IV colorectal transcriptome dataset from patients with mCRC (18) showed that cancer treated with FOLFOX and also the clinical data (11). Gene ATR expression was significantly higher in primary tumors (n ¼ expression data were normalized with the MAS5 algorithm and 20) and hepatic metastasis (n ¼ 19) than in normal tissue (n ¼ 17) analyzed with GenomicScape (http://www.genomicscape.com; samples (Fig. 1H). This indicates that ATR is aberrantly expressed ref. 12) and R.2.10.1 and Bioconductor, version 2.5. A consensus in colorectal cancer tumor samples and might be a potential list of genes encoding proteins involved in the DNA repair therapeutic target. pathways was obtained using the REPAIRtoire database (http:// repairtoire.genesilico.pl; ref. 13) and by reviewing Medline, as The ATR inhibitor VE-822 and oxaliplatin combination is described previously (14). The DNA repair score was built using synergistic in colorectal cancer cells our previously published methodologies to develop prognostic Currently, two ATR inhibitors (ATRi) are clinically available scores using sets of prognostic genes (14–17). The DNA repair (VX-970 and AZD-6738). As VX-970 (called VE-822 thereafter) is score was the sum of the Cox beta coefficients of each of the more advanced in terms of clinical development, we used this 12 DNA repair genes with a prognostic value, weighted by 1if ATRi. We evaluated the interaction between oxaliplatin and VE- the patient tumor signal for a given gene was above or below 822 in HCT116-R1 cells and in seven additional colorectal cancer the probeset maxstat value for that gene (14–17). cell lines, using a full-range concentration matrix approach and the SRB cytotoxicity assay (Fig. 2A). Specifically, we quantified the percentage of cell growth (blue matrix) and the additive, syner- Results gistic and antagonistic effects (black, red, and green matrices, A functional genetic screen reveals that ATR is implicated in respectively). We observed a synergistic interaction between oxa- oxaliplatin resistance in colorectal cancer cells liplatin and VE-822 in all tested colorectal cancer cell lines, with We assessed oxaliplatin sensitivity in five colorectal cancer cell the highest values in HCT116-R1, HCT116-R2, and SW48-R cells lines (HCT116, SW48, SW480, SW620, and HT29) and three and the smallest for SW480. This indicates that VE-822 can oxaliplatin-resistant derivatives (HCT116-R1, HCT116-R2, and efficiently overcome oxaliplatin resistance. The distribution of SW48-R) that were established by long-term growth in the pres- the interaction effects was not always homogeneous over the ence of oxaliplatin (5). These cell lines display different molecular concentration matrices. For instance, focal areas with higher

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Figure 1. Identiﬁcation of the ATR gene using a synthetic lethality assay in oxaliplatin-resistant colon cancer cells. A, The colorectal cell lines HCT116 and its resistant clones (HCT116-R1 and HCT116-R2), SW48 and its resistant clone (SW48-R), SW480, SW620, and HT29 were incubated with increasing concentrations of

oxaliplatin (0–30 mmol/L). The IC50 values were obtained using the SRB and spheroid formation assays (2D and 3D cell culture assay, respectively). Data are the mean SD (error bars) of at least three independent experiments. B, Schematic workﬂow of the "dropout" shRNA screen to identify genes, the inhibition of which was selectively lethal in oxaliplatin-resistant colon cancer cells. Oxaliplatin-resistant HCT116-R1 cells were infected with viral particles expressing the 1,798 shRNA vectors that target 518 genes from the kinome library and screened for shRNAs that cause lethality after addition of oxaliplatin. C, Three shRNAs targeting ATR were signiﬁcantly depleted in HCT116-R1 cells incubated with oxaliplatin. D, ATR knockdown was evaluated by Western blot analysis. The shLuc vector was used as a control. E and F, HCT116-R1 cells in which ATR was knocked down or not (shATR and shLuc) were incubated with increasing concentrations of

oxaliplatin (x-axis) and viability was assessed with the SRB assay (y-axis; E), and the IC50 values calculated (F). G, Analysis of the ATR pathway activation in HCT116 and HCT116-R1 cells. ATR, phosphorylated ATR (p-ATR), CHK1, and p-CHK1 were evaluated by Western blot analysis. Tubulin levels were used as loading control. H, Differential ATR gene expression in tissue samples from patients with mCRC. HM, hepatic metastasis; NM, normal mucosa; PT, primary tumor.

synergy were often located in concentration regions that corre- extending survival (Kaplan–Meier survival analysis, P ¼ 0.0216; sponded to high VE-822 concentration. Fig. 2C). This result confirms our previous in vitro findings, and Next, we assessed whether ATR inhibition influenced oxaliplatin provides evidence that ATR inhibition also enhances oxaliplatin cytotoxic activity in mice xenografted subcutaneously with HCT116- therapeutic effect in resistant colorectal cancer cells in vivo. R1 cells. To this aim, we treated mice with 0.9% sodium chloride solution (control group), oxaliplatin, VE-822, or the oxaliplatin þ The VE-822 þ oxaliplatin combination induces apoptosis, VE-822 combination. Oxaliplatin and VE-822 alone had no effect reduces cell proliferation, and activates the ATM–CHK2 on tumor growth compared with control. Conversely, the VE-822 þ pathway oxaliplatin combination was effective in limiting tumor growth, We then investigated whether the ATRi VE-822 þ oxaliplatin even not really marked but significant (P ¼ 0.018; Fig. 2B) and combination had a synergistic effect also on cell death and/or cell

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Figure 2. ATR inhibition combined with oxaliplatin alters colorectal cancer cell viability in vitro and in vivo. A, The indicated colorectal cancer cell lines were incubated with increasing concentrations of oxaliplatin and the ATR inhibitor VE-822, and cell viability was assessed with the SRB assay (2D) and by measuring spheroid growth (3D) to obtain the viability matrix. Drug concentrations were as follows: VE-822 (from 0.0075 to 2.56 mmol/L for 2D and from 0.01 to 2.52 mmol/L for 3D), oxaliplatin (from 0.184 and 30 mmol/L for 2D and from 0.09 and 30 mmol/L for 3D). The synergy matrix was calculated as described in Materials and Methods. B, Effect of the VE-822 þ oxaliplatin combination on the tumor volume in nude mice xenografted with HCT116-R1 cells. Mice received oxaliplatin, VE-822, or both or vehicle (NT, normal tissue). C, Kaplan–Meier survival analysis of the mice described in B.

proliferation. First, cell-cycle analysis showed that each com- p21 expression levels were increased by the drug combination, pound had only a minor effect on the cell-cycle distribution of whereas phosphorylation of the ATR target CDK2 was decreased HCT116-R1 cells. Conversely, the VE-822 þ oxaliplatin combi- compared with cells without treatment or incubated with each nation reduced significantly the number of S-phase cells and single drug (Fig. 3D; Supplementary Fig. S2B and S2C). Altogeth- increased that of cells in the sub-G1 phase (Fig. 3A). We confirmed er, these results demonstrate that the synergy between the ATRi this finding using the EdU assay. Oxaliplatin and VE-822 alone and oxaliplatin impairs cell-cycle progression and proliferation, had no effect on cell proliferation, whereas in combination they and promotes cell death. inhibited cell proliferation, as indicated by the reduction of EdU- To assess the effect of these drugs on the ATR signaling pathway, positive HCT116-R1 cells (from 75% in untreated cells to 18% in we performed immunoblotting analysis using HCT116-R1 oxaliplatin þ VE-822–treated cells; P ¼ 0.0094; Fig. 3B). (Fig. 3E) and HCT116 cells (Supplementary Fig. S2A–S2C) after þ Next, we assessed apoptosis by quantifying Annexin-V incubation with oxaliplatin and/or VE-822. In both cell HCT116-R1 cells (Fig. 3C). Only the VE-822 þ oxaliplatin com- lines, oxaliplatin treatment promoted ATR and CHK1 phosphor- bination significantly and strongly induced apoptosis compared ylation. As expected, this effect was inhibited when cells were with untreated cells and the drugs alone. Moreover, p53 and incubated with oxaliplatin and VE-822. As the ATR and ATM

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Figure 3. ATR inhibition combined with oxaliplatin alters the cell-cycle distribution profile and induces cell death. HCT116-R1 cells were incubated or not with oxaliplatin (5 mmol/L) and/or VE-822 (1 mmol/L) as indicated for 48 hours, and then the cell-cycle distribution was analyzed using the propidium iodide staining method (A), and the number of EdU-positive cells (B) and Annexin-V–positive cells (C) was quantified by flow cytometry. Data are the mean SD of at least three independent experiments. , P < 0.05, compared with control; #, compared with the drug combination. D and E, Western blot analysis of p53, p21, CDK2 (D), the ATR-CHK1 and ATM-CHK2 signaling pathways (E) in HCT116-R1 cells incubated or not with oxaliplatin (2.5 mmol/L) and/or VE-822 (1 mmol/L) as indicated for 24 hours. Tubulin and GAPDH were used as loading controls.

pathways cross-talk with each other, we also evaluated ATM and persisting ssDNA and gH2AX, marker of DNA damage, in CHK2 expression (Fig. 3E, right). VE-822 induced CHK2 phos- HCT116-R1 cells incubated with VE-822 and/or oxaliplatin. For phorylation, whereas oxaliplatin did not have detectable effect. this, we used a specific fluorimetric assay that allows to correlate Conversely, the oxaliplatin and VE-822 combination significantly the respective protein levels in cell lysates (Fig. 4A). Compared increased ATM and CHK2 phosphorylation, indicating that in the with untreated cells, RPA levels were significantly increased in cells presence of oxaliplatin, the VE-822–induced inhibition of the ATR incubated with oxaliplatin, but not with VE-822 (Fig. 4B). This signaling pathway leads to activation of the ATM pathway. Alto- effect was even more pronounced when oxaliplatin administra- gether, these results demonstrate that the combination of oxali- tion was combined with VE-822. Similar results were obtained for platin þ VE-822 has a cytotoxic and cytostatic effect in colorectal gH2AX, indicating that the VE-822 þ oxaliplatin combination cancer cell lines, and that it can effectively inhibit the ATR pathway acts synergistically to induce DNA damage (Fig. 4B). and activate ATM signaling. Temporal analysis of RPA (ssDNA) and gH2AX (DNA damage) labeling showed that oxaliplatin alone induced only a slight The VE-822 þ oxaliplatin combination induces replication increase in RPA staining, but not in gH2AX or RPAþgH2AX stress, leading to DNA break formation labeling (Fig. 4C, left). Notably, combined VE-822 þ oxaliplatin ATR plays an essential role in the cell response to replication treatment increased the number of double gH2AX- and RPA- stress (RS; ref. 19). As accumulation of ssDNA and DNA damage is positive cells over time (Fig. 4C, right), indicating replication an RS feature, we quantified levels of RPA, a protein covering catastrophe driven by excessive ssDNA accumulation (20).

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Figure 4. ATR inhibition combined with oxaliplatin induces replication stress, DNA break formation, and cytosolic DNA. A, Illustration of the method used to determine the RPA, gH2AX, and RPA þ gH2AX signal intensity. B, Analysis of total RPA and total gH2AX signal intensity after 16 hours of incubation with VE-822 and/or oxaliplatin (Ox) as indicated. Data are the mean SD of at least three independent experiments. , P < 0.01; , P < 0.001, compared with control; #, compared with oxaliplatin. C, Separated analysis of RPA, gH2AX, and RPA þ gH2AX labeling as a function of time after addition of oxaliplatin (50 mmol/L; left) or of VE-822 (1 mmol/L) þ oxaliplatin (50 mmol/L; right). D, Replication fork speed measurements (CldU track length in mmol/L) in HCT116-R1 cells incubated with oxaliplatin (12.5 mmol/L) and/or VE-822 (1 mmol/L) or not (untreated); the bottom right graph summarizes the results of all experiments relative to untreated cells set to 1. Bold line, mean. AU, arbitrary unit. , P < 0.01; , P < 0.001. E, HCT116-R1 cells were incubated or not (CTR) with 1 mmol/L VE-822 and/or 12.5 mmol/L oxaliplatin (as indicated) for 48 hours and stained for cytosolic dsDNA or ssDNA (green) and with DAPI (blue). Images were acquired by microscopy and analyzed usingthe ZEN software. Data are representative of three independent experiments.

To explore the impact of both drugs on RS, we analyzed own, and in some cases even led to replication fork arrest. These replication fork speed in HCT116-R1 cells (Fig. 4D). VE-822 results show that the VE-822 þ oxaliplatin combination (1 mmol/L for 24 hours) strongly affected replication in induces a dramatic decrease in replication fork speed, leading HCT116-R1 cells, decreasing fork speed by about 40%, as to RS and replication catastrophe. previously shown with the ATRi VE-821 (21). Oxaliplatin As exposure to genotoxic agents and replication fork stalling adducts were also reported to affect polymerase progres- increase the levels of cytosolic DNA (23), we assessed whether the sion (22), and we describe here for the ﬁrst time that oxaliplatin VE-822 þ oxaliplatin combination affected the cytosolic DNA (12.5 mmol/L for 24 hours) decreased signiﬁcantly replication levels. In fact, we observed that VE-822 or oxaliplatin alone fork speed by 10%. The VE-822 þ oxaliplatin combination slightly increased the cytosolic DNA level, whereas the drug inhibited replication fork speed stronger than each drug on its combination had a much stronger effect (Fig. 4E). This concurs

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with the observation that the VE-822 þ oxaliplatin combination Identification of DNA repair genes associated with overall increases ssDNA and dsDNA levels what could then lead to survival and response to oxaliplatin-based treatment in patients activation of the immune system. with colorectal cancer Our results indicated that the ATRi VE-822 strongly synergizes The VE-822 þ oxaliplatin combination promotes the antitumor with oxaliplatin in colorectal cancer cells. Therefore, we asked T-cell response whether some DNA repair genes that are deregulated in colorectal Oxaliplatin has been described to induce ICD in vitro and in vivo cancer are associated with overall survival (OS) and/or response in different preclinical cancer models (4, 24). ICD is a specific cell to oxaliplatin-based treatment. To this aim, we used microarray death characterized by the release or expression of damage-asso- gene expression data from primary tumors before treatment ciated molecular patterns (DAMP), such as adenosine-50-triphos- (FOLFOX regimen) from two independent cohorts of patients phate (ATP) and HMGB-1 release and CRT translocation. We with colorectal cancer: (i) Tsuji cohort [n ¼ 80 patients (26)], and therefore decided to test the effect of the combined VE-822 þ (ii) Del Rio cohort (n ¼ 36 patients; ref. 27). We defined a list of oxaliplatin administration in vivo. 176 genes involved in the six major DNA repair pathways [base For this, we first asked whether the ATRi VE-822 synergized with excision repair (BER), nucleotide excision repair (NER), mismatch oxaliplatin also in mouse CT26 and MC38 colorectal cancer cells. repair (MMR), homologous recombination repair (HRR), non- To this aim, we used the concentration matrix approach and SRB homologous end joining (NHEJ), and FANC] using the REPAIR- cytotoxicity tests with four oxaliplatin and five VE-822 concen- toire database (http://repairtoire.genesilico.pl) and literature tration in 2D (SRB) and 3D (spheroid growth) cell assays (Sup- data (14). Using the Maxstat R function and Benjamini–Hochberg plementary Fig. S3A). Like in human colorectal cancer cell lines, multiple testing correction, we found that 12 of these 176 genes we observed a strong synergistic interaction between oxaliplatin had a prognostic value (5 genes with poor and 7 with good and VE-822 in both mouse cell lines, with higher values in the 3D prognostic values; Fig. 6A, left). We used this DNA repair gene than in the 2D assay. signature to create the DNA repair score. In the Tsuji cohort, We then monitored ICD in vitro by assessing CRT exposure, patients with a high DNA repair score were characterized by ATP secretion, and HMGB1 release (25) in mouse MC38 cells stronger expression of the five bad prognostic genes and weaker and in oxaliplatin-resistant human HCT116-R1 cells (Fig. 5A– expression of the seven good prognostic genes (Fig. 6A, right). C; Supplementary Fig. S3B and S3C for HCT116 cells). Quan- Using the Maxstat R function, we found the maximum difference tification of CRT translocation to the outer leaflet of the plasma in OS with a DNA repair score ¼4.2464 that split patients in the membrane by flow cytometry of live cells (Fig. 5A) showed that Tsuji cohort into a low survival group (67.5% of patients, median oxaliplatin and the drug combination, but not VE-822 alone, OS ¼ 12 months), and a high survival group (32.5% of patients, strongly increased CRT translocation, in both cell lines. Also, median survival not reached; P ¼ 6.58 10 5; Fig. 6B). The DNA ATP secretion was promoted by the drug combination in both repair score, computed using the Tsuji cohort parameters, was also cell lines (Fig. 5B). HMGB1 release was significantly increased prognostic in the Del Rio cohort (Fig. 6C, left). The median OS of only in MC38 cells incubated with the VE-822 þ oxaliplatin patients with high DNA repair score (i.e., higher than 4.2464) combination (Fig. 5C). These results reveal that the ICD induc- was 17 months, whereas it was not reached for patients with tion by oxaliplatin is significantly increased in combination low DNA repair score (P ¼ 0.00181). The Del Rio cohort also with the ATRi VE-822. included data on the response after the first-line FOLFOX che- To test whether the VE-822 þ oxaliplatin combination motherapy, according to the WHO criteria. Using the same increased ICD also in vivo, we grafted the syngeneic MC38 cells DNA repair score threshold (4.2464), we found a significant in immunocompetent C57/Bl6 mice (Fig. 5D). When tumors difference in the median progression-free survival (PFS) between reached a volume of 100 mm3,wetreatedthemornotwith groups (8 months for patients with high DNA repair score and oxaliplatin, VE-822, or the oxaliplatin þ VE-822 combination. 21 months for patients with low DNA repair score; P ¼ 0.0189; Like in nude mice (Fig. 2B), VE-822 had no effect on tumor Fig. 6C, right). These findings confirm that the ATR and DNA growth. Conversely, oxaliplatin and particularly the oxaliplatin damage response pathways have an important prognostic and þ VE-822 combination decreased tumor growth (Fig. 5D). therapeutic value in patients with colorectal cancer. Importantly, Kaplan–Meier survival analysis showed that oxaliplatin þ VE-822 was more efficient than control (P ¼ 0.00471), and also oxaliplatin (P ¼ 0.0237) and VE-822 alone Discussion (P ¼ 0.0218; Fig. 5E). The higher efficiency of the drug com- Despite the advances in mCRC management, the 5-year sur- bination in immunocompetent mice suggests the establish- vival rate is still 12% (28). One of the causes of treatment failure is ment of a specific antitumor immune response. To test whether the resistance to therapy that occurs in 90% of patients with VE-822 amplifies oxaliplatin immunogenic effect, we sacrificed mCRC (29). Hence, the aim of this study was to identify new mice at week 3 after treatment and quantified IFNg-producing therapeutic targets to overcome oxaliplatin resistance in colorectal CD8-positive T cells in the spleen in untreated animals and cancer. Using a "chemical synthetic lethality screening," also mice treated with oxaliplatin alone or combined with VE-822 called "dropout screen," we found that ATR inhibition synergizes (Fig. 5F). After stimulation with irradiated MC38 cells (target with oxaliplatin to induce cancer cell death. The combination of cells), IFNg-producing CD8-positive T cells (effector cells) were oxaliplatin with the ATRi VE-822 was cytotoxic and cytostatic, significantly increased (P ¼ 0.0121) in mice that received both in vitro and in vivo. oxaliplatin þ VE-822 compared with those treated only with The ATR protein kinase is a member of the PI3K-related kinase oxaliplatin. Altogether, these results provide evidence that ATR (PIKK) family and activates CHK1 (30). ATR is activated by RPA- inhibition can reverse resistance to oxaliplatin and amplify coated ssDNA during S-phase to promote genomic stability in oxaliplatin-induced ICD in vitro and in vivo. response to DNA damage, cell-cycle arrest, or even stabilization

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Figure 5. Oxaliplatin combined with the ATR inhibitor VE-822 induces immune death signaling. A, CRT exposure at the cell surface of mouse MC38 and human HCT116-R1 colorectal cancer cells was assessed after 48-hour incubation with oxaliplatin (25 mmol/L) and/or VE-822 (1 mmol/L), or not (control) by ﬂow cytometry analysis. , P < 0.05, compared with control; #, compared with the drug combination. B and C, ATP and HMGB1 release in the supernatant was assessed using luminescence and ELISA assays, respectively, in MC38 and HCT116-R1 cells incubated with oxaliplatin (12.5 or 100 mmol/L) and/or VE-822 (1 mmol/L) for 24 hours or 48 hours. Data are the mean SD of at least three independent experiments. , P < 0.05, compared with control; #, compared with the drug combination. D and E, Effect of treatment with VE-822 (30 mg/kg) and/or oxaliplatin (5 mg/kg) on tumor growth in C57BL/6J mice xenografted with MC38 colorectal cancer cells (n ¼ 5–7 mice/group; D) and Kaplan–Meier survival curves (E). F, Autologous mixed lymphocyte-MC38 tumor cell culture. Peripheral antitumor CD8- positive T cells were detected in the spleen of treated and control mice on the basis of their capacity to secrete IFNg after coculture with target (irradiated MC38) cells. Living IFNg-secreting T cells were detected by ﬂow cytometry. Left, dot plots, representative results for splenocytes isolated from treated or control (na€ve) mice. Right, histogram, percentage of IFNg-secreting CD8-positive T cells from three independent experiments in na€ve (n ¼ 5), oxaliplatin (Ox) þ VE-822– treated (n ¼ 8), and oxaliplatin-treated mice (n ¼ 3). , P < 0.05 (nonparametric Mann and Whitney t test). NT, normal tissue.

and repair of replication forks (31–33). Here, we found that the age increases, promoting the activation of the ATM–CHK2 path- ATRi þ oxaliplatin combination promoted cell death and inhib- way and the presence of cytosolic DNA. Finally, these molecular ited cell proliferation, as observed in the presence of high repli- events lead to the inhibition of replication and induction of cell cation stress, DNA damage, and activation of the ATM pathway. death. Moreover, as cancer cells may be defective in G1–S check- These data are in agreement with the model presented by Toledo points, they might require the ATR–CHK1 pathway to repair DNA and colleagues (20), suggesting that VE-822 might increase rep- damage (34), thus making them more sensitive/suitable to ATR licative stress generated by oxaliplatin and causing RPA shortage. inhibition than normal cells. If ssDNA is not stabilized by RPA, its phosphorylation by ATR To the best of our knowledge, this is the ﬁrst report that clariﬁes becomes unstable. Thus, in unstable ssDNA regions, DNA dam- the molecular mechanisms underlying the synergism between

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Figure 6. Identiﬁcation of prognostic genes that are regulated by oxaliplatin treatment in two independent cohorts of patients. A, Left, The DNA repair gene signature obtained after Maxstat R function and Benjamini–Hochberg multiple testing correction. Five genes have poor and seven genes good prognostic value. Right, clustergram of the DNA repair score genes ordered from best to worst prognosis. The level of the probe set signal is displayed from low (deep blue) to high (deep red) expression. B, Patients with colorectal cancer (N ¼ 80; Tsuji cohort) were ordered according to the increasing DNA repair score. C and D, High DNA repair score in the primary colorectal cancer could predict shorter OS and PFS. Patients from the Tsuji cohort (N ¼ 80) and the Del Rio cohort (N ¼ 36) were divided into two groups based on the DNA repair score threshold of 4.2464 (group 0, low DNA repair score, score lower than 4.2464; group 1, high DNA repair score, score higher than 4.2464).

oxaliplatin and the ATRi VE-822 in human cells, a long time after etoposide, and SN38; ref. 37). However, none of these studies the studies on fission yeast by Perego and colleagues showing the reported that the combination of ATRi with chemotherapeutic relevance of ATR homologue (rad3) in the sensitivity to oxali- drugs or radiotherapy can overcome drug resistance, or its molec- platin (35). It also shows that oxaliplatin can induce replicative ular effects. Moreover, the role of ATR in oxaliplatin resistance is stress, the activation of ATR–CHK1 pathway and ssDNA accumu- somewhat controversial because at least two studies have shown lation (and RPA levels) on its own, and, as well as increased DNA opposite effects. First, Lewis and colleagues (2009) found that damages and ICD when combined with VE-822. ATR contributes to the survival of fibroblast and osteosarcoma Acquired resistance to oxaliplatin has limited the management cell lines after cisplatin, but not oxaliplatin treatment (38). of colorectal cancer and other tumor types. Our loss-of-function Conversely, Hall and colleagues, demonstrated in 35 lung genetic screen allowed showing that the kinase ATR is, at least in cancer cell lines that ATR inhibition sensitizes cells to five part, responsible of this resistance, and suggests that the ATRi and DNA-damaging drugs (cisplatin, oxaliplatin, gemcitabine, eto- oxaliplatin combination therapy could be useful in the clinic. poside, and SN38; ref. 37). Moreover, they found that while the Similar screenings have already demonstrated that ATR, CHK1, combination of the ATRi VX-970 with cisplatin or etoposide is and WEE1 are involved in the sensitivity to cisplatin in triple- always synergistic, the combination with oxaliplatin is syner- negative breast cancer (36). Moreover, ATR inhibition sensitized gistic in only 60% of the tested cell lines and antagonistic in the cancer cells to chemotherapy, for instance in ovarian cancer others (37). These findings suggest that the response to the (in combination with cisplatin and gemcitabine), in pancreatic oxaliplatin and ATRi combination is cell type–specific. Our cancer (with gemcitabine), in breast cancer (with camptothecin) results showed synergistic effects in all tested colorectal cancer and in lung cancer (with cisplatin, oxaliplatin, gemcitabine, cell lines, even in oxaliplatin-resistant colorectal cancer cell

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lines, suggesting that colorectalcancerisapotentialtargetfor resistance to this drug. This confirms the impact of the NER the synergistic interaction between oxaliplatin and ATRi. pathway in the repair of oxaliplatin-induced lesions. Moreover, The synergistic effects of oxaliplatin and VE-822 also promoted 50% of the genes in the DNA repair signature belong to the HRR ICD signals. It has been recently shown that oxaliplatin antitumor pathway (SCARP, XRCC2, DMC1, BARD1, C19orf40, and RPA2). activity is due, at least in part, to ICD activation (4). Tumor cells This could be surprising, but is consistent with our results showing incubated with the accurate concentration of oxaliplatin are able that ATR, the major protein of the HRR pathway, is implicated in to produce ATP (laborious to be measured), release the DNA- resistance to oxaliplatin. Thus, the DNA repair score could binding protein HMGB1 in the tumor microenvironment, and become a biomarker of oxaliplatin response in patients with expose the chaperone protein CRT to the outer part of the plasma colorectal cancer and might allow identifying the patients who membrane. These three signals favor the priming of the host will most benefit from the oxaliplatin þ ATRi combination. antitumor immune response, and thus an efficient elimination Altogether, our data indicate that the ATRi þ oxaliplatin com- of tumor cells, which could result in a long-term antitumor bination is efficient in colorectal cancer cells, and also in oxali- immunity. Here, we found that in the tested colorectal cancer platin-resistant colorectal cancer cells. Our results provide a cell lines, oxaliplatin could induce these three signals, as expected, rational mechanistic basis for clinical trials with the oxaliplatin and for the first time that ATRi VE-822 potentiates this effect. þ ATRi combination. Cytosolic DNA can be recognized by DNA sensors, leading to the expression of type I IFNs (39) that has tumor-suppressive Disclosure of Potential Conflicts of Interest effects (40). Moreover, replication fork stalling may induce the No potential conflicts of interest were disclosed. formation of extended ssDNA regions, dsDNA breaks, and the activation of the DNA damage response. Shen and colleagues (23) Authors' Contributions reported that DNA damage caused by replication fork stalling Conception and design: H.-A. Michaud, A. Coquelle, P. Pasero, N. Bonnefoy, leads to the presence of genome-derived DNA in the cytosol of R.L. Beijersbergen, N. Vezzio-Vie, C. Gongora tumor cells and to activation of immune responses. Here, we Development of methodology: E. Combes, A.F. Andrade, D. Tosi, found that incubation of colorectal cancer cells with VE-822 and R.L. Beijersbergen, N. Vezzio-Vie, C. Gongora Acquisition of data (provided animals, acquired and managed patients, oxaliplatin increases the cytosolic DNA levels. This could be the provided facilities, etc.): E. Combes, A.F. Andrade, H.-A. Michaud, consequence of replicative stress, and could promote the antitu- F. Coquel, D. Desigaud, M.D. Rio, N. Vezzio-Vie mor immunosurveillance. These in vitro results were confirmed by Analysis and interpretation of data (e.g., statistical analysis, biostatistics, the results in immunocompetent mice, where treatment with the computational analysis): E. Combes, A.F. Andrade, D. Tosi, H.-A. Michaud, oxaliplatin þ VE-822 combination promoted tumor growth M. Jarlier, J. Moreaux, R.L. Beijersbergen, N. Vezzio-Vie, C. Gongora inhibition and tumor specific CD8-positive T-cell activation. Writing, review, and/or revision of the manuscript: E. Combes, A.F. Andrade, þ D. Tosi, A. Coquelle, J. Moreaux, P. Martineau, C. Gongora Although the combination of oxalipatin ATR inhibition results Administrative, technical, or material support (i.e., reporting or organizing in immunogenic cancer cell death in vivo, it is not clear how much data, constructing databases): V. Garambois, C. Gongora of the observed tumor size reduction is actually due to a cancer cell Study supervision: N. Vezzio-Vie, C. Gongora autonomous effect of the combined therapy versus immunogenic cancer cell death. Acknowledgments The clinical efficacy of oxaliplatin has been largely demonstrat- We acknowledge the imaging facility MRI, member of the national infra- ed, and its mechanism of action is well known. As a platinum structure France-BioImaging supported by the French National Research Agency fl derivative, its primary target is DNA, where it can form intra- and (ANR-10-INBS-04, Investments for the Future) for ow cytometry, and Celigo experiments. We thank the IRCM animal facility. This work was supported by interstrand cross-links. The repair of these lesions mostly occurs INSERM and the Institut du cancer de Montpellier (ICM), SIRIC (SIRIC through the NER pathway that involves ERCC1, 2, 3, 4, 5. Indeed, Montpellier Cancer Grant, INCa-DGOS-Inserm 6045), and Canceropole GSO. the association between ERCC1 expression and response to E. Combes had a PhD studentship from the program Investissement D'avenir platinum derivatives has been widely reported, particularly in (grant agreement: Labex MabImprove, ANR-10-LABX-53-01). A.F. Andrade advanced non–small-cell lung, bladder, head-and-neck and was supported by S~ao Paulo Research Foundation (14/06947-2) and Fondation esophagus cancer (for cisplatin) and in colorectal cancer (for de France (postdoctoral fellowship). oxaliplatin). The costs of publication of this article were defrayed in part by the payment of In agreement, the gene expression–based predictive DNA repair page charges. This article must therefore be hereby marked advertisement in score we developed for OS and PFS of patients with colorectal accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cancer undergoing oxaliplatin treatment validates the importance of ERCC1 in oxaliplatin resistance, and identifies four other NER Received September 11, 2018; revised January 3, 2019; accepted April 5, 2019; genes (GTF2H3, HMGN1, RPA2, and GTF2H2) as involved in published first April 15, 2019.

References 1. Global Burden of Disease Cancer Collaboration, Fitzmaurice C, Allen C, 3. Perego P, Robert J. Oxaliplatin in the era of personalized medicine: from Barber RM, Barregard L, Bhutta ZA, et al. Global, regional, and national mechanistic studies to clinical efﬁcacy. Cancer Chemother Pharmacol cancer incidence, mortality, years of life lost, years lived with disability, and 2016;77:5–18. disability-adjusted life-years for 32 cancer groups, 1990 to 2015: a sys- 4. Tesniere A, Schlemmer F, Boige V, Kepp O, Martins I, Ghiringhelli F, et al. tematic analysis for the Global Burden of Disease Study. JAMA Oncol 2017; Immunogenic death of colon cancer cells treated with oxaliplatin. Onco- 3:524–48. gene 2010;29:482–91. 2. Van Cutsem E, Nordlinger B, Cervantes A, ESMO Guidelines Working 5. Gourdier I, Del Rio M, Crabbe L, Candeil L, Copois V, Ychou M, et al. Drug Group. Advanced colorectal cancer: ESMO clinical practice guidelines for speciﬁc resistance to oxaliplatin is associated with apoptosis defect in a treatment. Ann Oncol 2010;21 Suppl 5:v93–7. cellular model of colon carcinoma. FEBS Lett 2002;529:232–6.

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6. Samimi G, Manorek G, Castel R, Breaux JK, Cheng TC, Berry CC, et al. 22. Chaney SG, Campbell SL, Temple B, Bassett E, Wu Y, Faldu M. Protein cDNA microarray-based identification of genes and pathways associated interactions with platinum-DNA adducts: from structure to function. with oxaliplatin resistance. Cancer Chemother Pharmacol 2005;55: J Inorg Biochem 2004;98:1551–9. 1–11. 23. Shen YJ, Le Bert N, Chitre AA, Koo CX, Nga XH, Ho SS, et al. Genome- 7. Virag P, Brie I, Fischer-Fodor E, Perde-Schrepler M, Tatomir C, Balacescu O, derived cytosolic DNA mediates type I interferon-dependent rejection of B et al. Assessment of cytotoxicity, apoptosis and DNA damages in Colo320 cell lymphoma cells. Cell Rep 2015;11:460–73. colorectal cancer cells selected for oxaliplatin resistance. Cell Biochem 24. Wang W, Wu L, Zhang J, Wu H, Han E, Guo Q. Chemoimmunotherapy by Funct 2011;29:351–5. combining oxaliplatin with immune checkpoint blockades reduced tumor 8. Prahallad A, Sun C, Huang S, Di Nicolantonio F, Salazar R, Zecchin D, et al. burden in colorectal cancer animal model. Biochem Biophys Res Commun Unresponsiveness of colon cancer to BRAF(V600E) inhibition through 2017;487:1–7. feedback activation of EGFR. Nature 2012;483:100–3. 25. Kepp O, Senovilla L, Vitale I, Vacchelli E, Adjemian S, Agostinis P, et al. 9. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, et al. New Consensus guidelines for the detection of immunogenic cell death. colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Oncoimmunology 2014;3:e955691. Inst 1990;82:1107–12. 26. Tsuji S, Midorikawa Y, Takahashi T, Yagi K, Takayama T, Yoshida K, et al. 10. Greco WR, Bravo G, Parsons JC. The search for synergy: a critical review Potential responders to FOLFOX therapy for colorectal cancer by Random from a response surface perspective. Pharmacol Rev 1995;47:331–85. Forests analysis. Br J Cancer 2012;106:126–32. 11. Del Rio M, Mollevi C, Bibeau F, Vie N, Selves J, Emile JF, et al. Molecular 27. Del Rio M, Molina F, Bascoul-Mollevi C, Copois V, Bibeau F, Chalbos P, subtypes of metastatic colorectal cancer are associated with patient et al. Gene expression signature in advanced colorectal cancer patients response to irinotecan-based therapies. Eur J Cancer 2017;76:68–75. select drugs and response for the use of leucovorin, fluorouracil, and 12. Kassambara A, RemeT,JourdanM,FestT,HoseD,TarteK,etal. irinotecan. J Clin Oncol 2007;25:773–80. GenomicScape: an easy-to-use web tool for gene expression data 28. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of analysis. Application to investigate the molecular events in the differ- worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010; entiation of B cells into plasma cells. PLoS Comput Biol 2015;11: 127:2893–917. e1004077. 29. Longley DB, Johnston PG. Molecular mechanisms of drug resistance. 13. Milanowska K, Krwawicz J, Papaj G, Kosinski J, Poleszak K, Lesiak J, et al. J Pathol 2005;205:275–92. REPAIRtoire–a database of DNA repair pathways. Nucleic Acids Res 2011; 30. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 39:D788–92. 3-kinases as regulators of growth and metabolism. Nat Rev Genet 2006;7: 14. Bret C, Klein B, Cartron G, Schved J-F, Constantinou A, Pasero P, et al. DNA 606–19. repair in diffuse large B-cell lymphoma: a molecular portrait. Br J Haematol 31. Kumagai A, Lee J, Yoo HY, Dunphy WG. TopBP1 activates the ATR-ATRIP 2015;169:296–9. complex. Cell 2006;124:943–55. 15. Kassambara A, Hose D, Moreaux J, Reme T, Torrent J, Rossi JF, et al. 32. Kumagai A, Dunphy WG. How cells activate ATR. Cell Cycle 2006;5: Identification of pluripotent and adult stem cell genes unrelated to cell 1265–8. cycle and associated with poor prognosis in multiple myeloma. PloS One 33. Cimprich KA, Cortez D. ATR: an essential regulator of genome integrity. 2012;7:e42161. Nat Rev Mol Cell Biol 2008;9:616–27. 16. Kassambara A, Hose D, Moreaux J, Walker BA, Protopopov A, Reme T, et al. 34. Fokas E, Prevo R, Hammond EM, Brunner TB, McKenna WG, Muschel RJ. Genes with a spike expression are clustered in chromosome (sub)bands Targeting ATR in DNA damage response and cancer therapeutics. and spike (sub)bands have a powerful prognostic value in patients with Cancer Treat Rev 2014;40:109–17. multiple myeloma. Haematologica 2012;97:622–30. 35. Perego P, Zunino F, Carenini N, Giuliani F, Spinelli S, Howell SB. Sensi- 17. Moreaux J, Reme T, Leonard W, Veyrune JL, Requirand G, Goldschmidt H, tivity to cisplatin and platinum-containing compounds of Schizosacchar- et al. Development of gene expression-based score to predict sensitivity omyces pombe rad mutants. Mol Pharmacol 1998;54:213–9. of multiple myeloma cells to DNA methylation inhibitors. Mol Cancer 36. Zheng H, Shao F, Martin S, Xu X, Deng CX. WEE1 inhibition targets cell Ther 2012;11:2685–92. cycle checkpoints for triple negative breast cancers to overcome cisplatin 18. Del Rio M, Mollevi C, Vezzio-Vie N, Bibeau F, Ychou M, Martineau P. resistance. Sci Rep 2017;7:43517. Specific extracellular matrix remodeling signature of colon hepatic metas- 37. Hall AB, Newsome D, Wang Y, Boucher DM, Eustace B, Gu Y, et al. tases. PLoS One 2013;8:e74599. Potentiation of tumor responses to DNA damaging therapy by the selective 19. Lopez-Contreras AJ, Fernandez-Capetillo O. The ATR barrier to replication- ATR inhibitor VX-970. Oncotarget 2014;5:5674–85. born DNA damage. DNA Repair 2010;9:1249–55. 38. Lewis KA, Lilly KK, Reynolds EA, Sullivan WP, Kaufmann SH, Cliby WA. 20. Toledo LI, Altmeyer M, Rask MB, Lukas C, Larsen DH, Povlsen LK, et al. Ataxia telangiectasia and rad3-related kinase contributes to cell cycle arrest ATR prohibits replication catastrophe by preventing global exhaustion of and survival after cisplatin but not oxaliplatin. Mol Cancer Ther 2009;8: RPA. Cell 2013;155:1088–103. 855–63. 21. Josse R, Martin SE, Guha R, Ormanoglu P, Pfister TD, Reaper PM, et al. ATR 39. Desmet CJ, Ishii KJ. Nucleic acid sensing at the interface between innate and inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase i adaptive immunity in vaccination. Nat Rev Immunol 2012;12:479–91. inhibitors by disabling DNA replication initiation and fork elongation 40. Dunn GP, Koebel CM, Schreiber RD. Interferons, immunity and cancer responses. Cancer Res 2014;74:6968–79. immunoediting. Nat Rev Immunol 2006;6:836–48.

2946 Cancer Res; 79(11) June 1, 2019 Cancer Research

Inhibition of Ataxia-Telangiectasia Mutated and RAD3-Related (ATR ) Overcomes Oxaliplatin Resistance and Promotes Antitumor Immunity in Colorectal Cancer

Eve Combès, Augusto F. Andrade, Diego Tosi, et al.

Cancer Res 2019;79:2933-2946. Published OnlineFirst April 15, 2019.

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