ATRX-Mutant Cancers to WEE1 Inhibition Junbo Liang1, Hong Zhao2,3, Bill H

Published OnlineFirst September 24, 2019; DOI: 10.1158/0008-5472.CAN-18-3374

CANCER RESEARCH | TRANSLATIONAL SCIENCE

Genome-Wide CRISPR-Cas9 Screen Reveals Selective Vulnerability of ATRX-Mutant Cancers to WEE1 Inhibition Junbo Liang1, Hong Zhao2,3, Bill H. Diplas4, Song Liu5, Jianmei Liu2,3, Dingding Wang1, Yan Lu1, Qing Zhu6, Jiayu Wu1, Wenjia Wang1, Hai Yan4, Yi-Xin Zeng6, Xiaoyue Wang1, and Yuchen Jiao2,7

ABSTRACT ◥ The tumor suppressor gene ATRX is frequently mutated in a induced apoptosis. AZD1775 also selectively inhibited the prolif- variety of tumors including gliomas and liver cancers, which are eration of patient-derived primary cell lines from gliomas with highly unresponsive to current therapies. Here, we performed a naturally occurring ATRX mutations, indicating that the synthetic genome-wide synthetic lethal screen, using CRISPR-Cas9 genome lethal relationship between WEE1 and ATRX could be exploited in a editing, to identify potential therapeutic targets specific for ATRX- broader spectrum of human tumors. As WEE1 inhibitors have been mutated cancers. In isogenic hepatocellular carcinoma (HCC) cell investigated in several phase II clinical trials, our discovery provides lines engineered for ATRX loss, we identified 58 genes, including the the basis for an easily clinically testable therapeutic strategy specific checkpoint kinase WEE1, uniquely required for the cell growth of for cancers deficient in ATRX. ATRX null cells. Treatment with the WEE1 inhibitor AZD1775 robustly inhibited the growth of several ATRX-deficient HCC cell Significance: ATRX-mutant cancer cells depend on WEE1, lines in vitro, as well as xenografts in vivo. The increased sensitivity which provides a basis for therapeutically targeting WEE1 in to the WEE1 inhibitor was caused by accumulated DNA damage– ATRX-deficient cancers.

Introduction variety of human cancers, including pancreatic neuroendocrine tumors (PanNET), glioma, liver cancer, and neuroblastoma. Although Over 20 years ago, germline variations in ATRX, a member of the ATR-X syndrome-related germline variations are mainly missense in SWI/SNF chromatin remodeling superfamily, were found to underlie a the histone methylation recognition domain and the ATPase rare developmental disorder, X-linked mental retardation with alpha- domain (6), the cancer-associated somatic ATRX mutations are thalassemia (ATR-X syndrome; ref. 1). More recently, genomic predominantly truncating mutations, resulting in loss of ATRX studies (2–5) have revealed that ATRX is frequently mutated in a expression (7), indicating that ATRX functions as a tumor suppressor gene in these cancers. Although genomic sequencing has enabled the rapid discovery of 1 State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical the mutations driving the development of human cancer, significant Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, challenges remain regarding the effective translation of these data into Beijing, China. 2State Key Lab of Molecular Oncology, National Cancer Center/ National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy viable therapies. Most driver mutations are damaging the functions of of Medical Sciences and Peking Union Medical College, Beijing, China. 3Depart- suppressor genes and cannot serve as a direct therapeutic target. One ment of Hepatobiliary Surgery, National Cancer Center/National Clinical strategy for developing targeted therapies against such loss-of-function Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical mutations is “synthetic lethality.” Synthetic lethality refers to the 4 Sciences and Peking Union Medical College, Beijing, China. The Preston Robert relationship between 2 genes for which loss of both genes results in Tisch Brain Tumor Center at Duke, Pediatric Brain Tumor Foundation Institute at cell death but loss of either one alone does not. For example, poly Duke, and Department of Pathology, Duke University Medical Center, Durham, PARP1 BRCA1/2 North Carolina. 5Department of Central Laboratory, Peking Union Medical (ADP)-ribose polymerase 1 ( ) and are synthetic lethal College Hospital, Peking Union Medical College & Chinese Academy of Medical because loss of both genes abolishes 2 parallel DNA damage repair Sciences, Beijing, China. 6Department of Experimental Research, Sun Yat-sen pathways and causes cell apoptosis (8). University Cancer Center, State Key Laboratory of Oncology in Southern China, Motivated by the clinical success of PARP inhibitors in treatment of Collaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong BRCA1/2-deficient tumors, scientists have performed genome-wide 7 Province, China. Department of Clinical Laboratory, National Cancer Center/ screens in human cancer cells to identify synthetic lethal partners National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. relevant to other frequently mutated tumor suppressor genes. In such screens, genes essential for each cell line are first identified by high- Note: Supplementary data for this article are available at Cancer Research throughput perturbation of genes using RNA interference (RNAi) or Online (http://cancerres.aacrjournals.org/). CRISPR-Cas9 genome editing. Synthetic lethal interactions are then J. Liang, H. Zhao, B.H. Diplas, and S. Liu contributed equally to this article. inferred by comparing the gene dependency profiles across a panel of Corresponding Authors: Xiaoyue Wang, Institute of Basic Medical Sciences, either independently derived cell lines with or without mutations at a Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing specific locus, or paired isogenic lines with engineered mutations. Such 100005, China. E-mail: [email protected]; and Yuchen Jiao, National screens have been used to identify potential pharmacologic targets that Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and enable selective killing of cancer cells with deficiencies in tumor Peking Union Medical College, Beijing 100021, China. Phone: 86-010- TP53 ARID1A 87788045; E-mail: [email protected] suppressors or (9, 10), or even with activation of “undruggable” oncogenes including RAS or MYC (11, 12). Cancer Res 2020;XX:XX–XX Among ATRX-mutant tumor types, hepatocellular carcinoma doi: 10.1158/0008-5472.CAN-18-3374 (HCC) is the 5th most common cancer in the world (13) and glioma 2019 American Association for Cancer Research. is the most common primary malignant brain tumor in adults. There

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are currently no effective targeted therapies available for HCC or with DNAMAN multiple sequence alignment tools. The primers used glioma, making them among the most lethal tumors in the world. Here, for amplifying the flanked region of the target site were as follows: we performed a genome-wide pooled CRISPR-Cas9–based screen in 0 0 paired isogenic HCC cells with or without engineered mutations in ATRX_forward: 5 -CCGTGACTCAGATGGAATGGA-3 ATRX – WEE1 ATRX 0 0 . The G2 M checkpoint control kinase, , was one of the _reverse: 5 -GGTTACAGAGCCAGAACAGG-3 several candidate genes identified as potentially lethal in the context of ATRX deficiency. The lethality of WEE1 loss was further investigated Lentivirus production 7 2 with small molecule inhibitors of WEE1 through the treatment of Low passage HEK293T cells (4 10 ) were seeded onto 500 cm engineered ATRX-mutant HCC cell lines and xenografts, as well as culture plates 1 day before transfection. When cells reached 80% to primary cell lines from naturally occurring gliomas. The results 90% confluence, a mixture containing the following library and support inhibition of WEE1 as the basis for the development of new packaging plasmids was transfected into HEK293T cells with Neofect: therapeutic strategies to treat multiple tumor types harboring ATRX GeCKO v2 library (63 mg; Addgene plasmid 1000000048), psPAX2 (45 mutations. mg; Addgene plasmid #12260), and pMD2.G (18 mg; Addgene plasmid #12259). Culture medium was changed 12 hours after transfection. The supernatant containing lentivirus was collected 60 hours post- Materials and Methods transfection, filtered through a PVDF filter membrane (0.45 mm; Ethics statement Millipore SteriCup 250 mL; Millipore) to remove cells, aliquoted, and All animal procedures were approved by and performed according stored at 80 C. to the animal ethical committee at the Cancer Hospital, Chinese Synthetic lethal screens Academy of Medical Sciences (Beijing, China). PLC/PRF/5 parental cells or PLC/PRF/5 ATRX KO cells (200 106) Cell culture were transduced with lentivirus from the human GeCKO v2 library at PLC/PRF/5, HuH-7, and HeLa cell lines were obtained from the an MOI of 0.3 and an average of 500-fold coverage of the library. As the human GeCKO v2 library contains 123,411 sgRNAs, at least 6 National Infrastructure of Cell Line Resource (Beijing, China) and are 7 routinely authenticated via their short tandem repeat profile (latest 10 cells need to be infected to achieve 500-fold coverage. At 48 hours verification for PLC/PRF/5 and HeLa: April 2019; latest verification for postinfection, cells were selected in puromycin (2 mg/mL; Thermo fi 7 HuH-7: November 2018). PLC/PRF/5, HuH-7, HeLa, and HEK293T Fisher Scienti c) for 2 days. At this time point, 6 10 transduced cells cells were cultured in DMEM supplemented with 10% FBS and were harvested as reference samples (T0), and the remaining cells were fi penicillin (100 U/mL)/streptomycin (0.1 mg/mL; Thermo Fisher passaged every 3 days. A nal pool of cell populations was collected at Scientific). After thawing, cells were used for up to 15 passages. Glioma day 21 (T21), after approximately 14 population doublings. Cell pellets cell line U251MG was a kind gift from Alan Meeker (JHMI) and harvested from T0 and T21 were stored at 80 C for isolation of matches the ECACC STR profiling reference. Primary glioma cell lines genomic DNA (gDNA), which was performed with the Blood & Cell derived from IMA, 12-0160, 13-0302, and 08-0537, were obtained Culture DNA Maxi Kit (Qiagen). fi from the Preston Robert Tisch Brain Tumor Center at Duke Univer- Two rounds of PCR were performed for identi cation of sgRNA fi sity, Durham, NC (latest verification were done in April 2019). The 3 inserts. In the rst round PCR, 200 mg of gDNA was used as template to achieve an average of 250-fold coverage of the sgRNA library assuming primary glioma cell lines were maintained under adherent conditions 6 on laminin-coated plates in human neural stem cell medium, which 10 cells contain 6.6 mg of gDNA. We performed 67 separate PCR consisted of NeuroCult NS-A NSC proliferation medium supplemen- reactions (3 mg gDNA/50 mL reaction volume) for each sample using ted with EGF (20 ng/mL), FGF (10 ng/mL), and heparin (0.0002%; NEBNext High-Fidelity 2 PCR Master Mix (New England Biolabs) Stem Cell Technologies). IMA was maintained in 50% neural stem cell and combined resulting amplicons. medium and 50% DMEM with 10% FBS and 1% penicillin/strepto- The second-round PCR was performed to attach Illumina adaptors fi mycin. U251MG was cultured in RPMI1640 with 10% FBS and 1% and index samples. A volume of 2 mL of PCR products from the rst m penicillin/streptomycin. All cell lines were tested negative for round PCR were used as templates for each 50 L reaction volume. The Mycoplasma. following primers were used: PCR1_forward: Small molecule inhibitors 50-AATGGACTATCATATGCTTACCGTAACTTGAAAG- AZD1775 (S1525), roscovitine (S1153), hydroxyurea (S1896), and 0 TATTTCG-3 ; gemcitabine (S1714) were purchased from Selleckchem. PCR1_reverse: 50-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACT- Generation of ATRX knockout cells GACGGGCACCGGAGCCAATTCC-30. The lentiCRISPR v2 plasmid (Addgene Plasmid #52961) containing PCR2_forward: ATRX single-guide RNA (sgRNA; sequence: TGGA- 0 5 -AATGATACGGCGACCACCGAGATCTACACTCTTTCCC- CAACTCCTTTCGACCA) was transfected into PLC/PRF/5 and TACACGACGCTCTTCCGATCTTCTTGTGGAAAGGAC- HuH-7 cells using Neofect (Neo Biotech). Single-cell colonies were 0 GAAACACCG-3 ; selected, and knockout (KO) status was validated by Western blot PCR2_reverse: analysis and Sanger sequencing. For Sanger sequencing, genomic DNA 0 5 -CAAGCAGAAGACGGCATACGAGATXXXXXXGT- was extracted from PLC/PRF/5 parental and ATRX KO cells. PCR was 0 GACTGGAGTTCAGACGTG-3 (XXXXXX represents a 6-bp performed to amplify the region flanked by the target site, and the index). 736 bp PCR products were subcloned into pCloneEZ vector (CloneS- marter), transformed into competent cells, and at least 20 clones were Amplifications were carried out for 20 cycles for the first-round PCR picked separately for Sanger sequencing. The sequences were aligned and 10 cycles for the second-round PCR. The resulting amplicons were

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pooled and gel purified using the QIAquick Gel Extraction Kit qRT-PCR (Qiagen). Combined elutions were purified on AMPure XP beads Total RNA was isolated using TRizol reagent (Ambion), and 2 mgof (Beckman Coulter), quantified on a Bioanalyzer 2100 (Agilent), and total RNA was used to prepare cDNA using the RevertAid First Strand sequenced on a HiSeq X10 platform (Illumina). cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. qRT-PCR was performed in triplicate Analysis of the screen data for each target sequence using LightCycler 480 SYBR Green I Master At a basic level of interpretation, the abundance of the sgRNAs reaction mix (Roche) on a LightCycler 480 Real-Time system (Roche). targeting individual candidate genes will differ significantly between The following primers were used. ATRX wild-type (WT) and KO cells because of lethality. Therefore, 0 0 the fundamental goal is to generate a count of reads from the WEE1_forward: 5 -CAATTACTGAAAGCAATATGAAG-3 0 0 sequencing data for each sgRNA in the human GeCKO v2 library. WEE1_reverse: 5 -ACATGAGAATGCTGTCCAAG-3 0 0 To generate these counts, the sgRNA sequence containing reads VCP_forward: 5 -CTCATCTACATCCCACTTCCT-3 0 0 were first selected from the paired end sequence files using the VCP_reverse: 5 -CGTTCTCGCCTAATCTCAC-3 0 0 FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html) PKMYT1_ forward: 5 -CTACTTCCGCCACGCAGAA-3 0 0 with the fastx_barcode_splitter.pl command. The sgRNA sequences PKMYT1_reverse: 5 -CGCACCTTGAAGACCTCTCC-3 were clipped out with the fastx_trimmer command, leaving only the sgRNA sequence for further analysis. The sgRNA reads were Western blot analysis mapped to the GeCKOv2 sgRNA library with Bowtie (version Cells were lysed in RIPA buffer (Beyotime Biotechnology) 1.1.2; ref. 14), which aligns short sequencing reads, with tolerance containing protease inhibitor and PhosStop cocktails (Roche). Cell ofasinglenucleotidemismatch.ForeachsgRNAinthelibrary,the lysates were separated on an SDS-polyacrylamide gel and trans- count of mapped reads was calculated for each time point, T0 or ferred to a PVDF membrane (Millipore). The following anti- T21. The raw sgRNA count file was uploaded to the Model-based bodies were used for immunoblotting: anti-ATRX (HPA001906, Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) Sigma-Aldrich); anti-gH2A.X (05-636; Millipore); anti-phospho- algorithm to generate a maximum-likelihood estimation (mle) of RPA32 (S4/S8; A300-245A-M, Bethyl); anti-GAPDH (TA-100; gene essentiality score (b score) for each gene using the MAGeCK Zhongshanjinqiao Biotechnology); anti-cleaved caspase 3 (9661); mlemodule(15).TheATRX synthetic lethal genes were filtered as anti-b-actin (4970);anti-phospho-ATM (Ser1981; 5883); anti-ATM those with b score < 0andaFDR< 0.3 in the ATRX KO group, but (2873); anti-phospho-CHK2 (Thr68; 2197); anti-CHK2 (6334); with a P value of > 0.05 in the ATRX WT group. anti-phoso-CDK1 (Tyr15; 4539); anti-phoso-CDK1 (Thr14; 2543); Using this approach, we identified 58 potential synthetic lethal anti-CDK1 (9116); anti-Wee1 (13084); anti-PKMYT1 (4282; Cell genes in the context of ATRX mutation from the 2 replications. We Signaling Technology). used PANTHER, an online tool to calculate overrepresented gene clusters. A single gene, RGPD6, was not recognized by the PANTHER Cell viability assay database. Therefore, we used the remaining genes (n ¼ 57) to calculate For inhibitor studies, PLC/PRF/5 parental cells (2500/well) and the enrichment using Fisher exact test, and the P values were adjusted ATRX KO single colonies were seeded in 96-well plates for overnight using the Bonferroni correction. incubation and treated with different doses of AZD1775 for 3 days. Cell viability was assessed by measuring the absorbance at 450 nm siRNA transfections with the Cell Counting Kit-8 (CCK-8; DOJINDO). Glioma cells PLC/PRF/5 parental and PLC/PRF/5 ATRX KO cells were trans- (2,500 cells/well or 1,000 cells/well for U251) were incubated fected with WEE1 or VCP or PKMYT1 siRNAs (50 nmol/L final overnight in 96-well plates and subsequently exposed to increasing concentration; RiboBio) using Lipofectamine RNAiMAX (Thermo doses of AZD1775 in DMSO or DMSO as vehicle control. Viable Fisher Scientific) according to the manufacturer's instructions. Knock- cells were measured after 5 days using the CellTiter-Glo assay down efficiency was tested by qRT-PCR 24 hours after transfection. (Promega) according to the manufacturer's instructions. Each AZD1775 siRNA sequences used for WEE1, VCP, and PKMYT1 were the dose was tested in triplicate, and the average luminescence is presented following: as a percent of the DMSO control. The IC50 value was derived from the curve fitting of the dose–response data using GraphPad Prism siWEE1-1: 50-UUCUCAUGUAGUUCGAUAUUU-30; v6.0. The normalized growth rate inhibition (GR) metric was calculated siWEE1-2: 50-UAAUAGAACAUCUCGACUUAU; according to the protocol in Hafner and colleagues (16). siVCP-1: 50-CCUGAUGUGAAGUACGGCAAA; siVCP-2: 50-GAUGGAUGAAUUGCAGUUGUU; Clonogenic survival assay siPKMYT1-1: 50-GGACAGCAGCGGAUGUGUU-30; The clonogenic survival assay was performed according to the siPKMYT1-2: 50-GGAACCUCCUCAGCCUGUU-30; protocol as described previously (17). PLC/PRF/5 cells (400 cells/well) siNC (negative control): 50-UUCUCCGAACGUGUCACGU-30 or HuH-7 cells (5,000 cells/well for the ATRX WT group; 10,000 cells/ well for the ATRX KO group for its very low plating efficiency) were For cell viability assay, PLC/PRF/5 and HeLa cells were transfected plated in triplicate on a 6-well plate, incubated for 24 hours, and treated with either negative control siRNA (siNC) or ATRX siRNAs (siATRX-1 with AZD1775 at the indicated concentrations for 12 days. Surviving and siATRX-2; 50 nmol/L final concentration; RiboBio). Forty-eight colonies were fixed with formalin and stained with 0.1% crystal violet. hours after transfection, knockdown efficiency was tested by Western Colonies containing more than 50 cells were counted. For the sites blot analysis. siRNA sequences used for ATRX were the following: with colony overlapping, the number of overlapping colonies was estimated based on the shape and size of the colony cluster. The 0 0 siATRX-1: 5 -GCAGAUUGAUAUGAGAGGAAU-3 ; relative colony-forming efficiency was determined by normalizing the 0 0 siATRX-2: 5 -CGACAGAAACUAACCCUGUAA-3 . colony numbers to the untreated control.

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Cell-cycle and apoptosis analysis mula: (length width2)/2. When the mean tumor volumes reached For cell-cycle analysis, cells were harvested by trypsinization, 150 to 200 mm3, mice bearing tumors derived from PLC/PRF/5 ATRX washed with PBS, fixed in cold 70% ethanol, incubated with 50 mg/ WT cells or ATRX KO cells were divided into 2 subgroups and orally mL RNaseA at 37C for 30 minutes, and then stained with 50 mg/mL administered with either AZD1775 (50 mg/kg body weight) or vehicle propidium iodide. For HuH-7 cells, DAPI (1 mg/mL) was used to stain control once daily for 15 days. The in vivo doses used in our study are the DNA without RNase A incubation. Cells were analyzed with a BD following those used in previous studies (19–21). Using the body Flow Cytometer. ModFit software was used for processing the data. surface area (BSA) scaling method (22) recommended by the U.S. Apoptotic assays were performed using the FITC Annexin V Apo- FDA, we translate the animal dosage to the human clinical trials. This ptosis Detection Kit (556547; BD Biosciences) according to the calculation results in a human equivalent dose of 4 mg/kg, which manufacturer's instructions. The apoptotic cells were analyzed with translates into a 240 mg dose of AZD1775 for a person weighing 60 kg. a BD Flow Cytometer. Data were analyzed with the FlowJo software. This dose is within a clinically achievable range as this drug has been used at a dose of 225 mg twice per day over 2.5 days per week for Neutral comet assay 2 weeks per 21-day cycle in phase I clinical trials (23, 24). We also The neutral comet assay was performed using the CometAssay HT evaluated the effect of AZD1775 on the body weight of mice admin- Kit (4252-040-K; Trevigen) according to the manufacturer's instruc- istered orally with a dose of 50 mg/kg once daily. The body weight of tions. Briefly, cells (1 105/mL) were mixed with molten LMAgarose mice treated with AZD1775 was reduced less than 10% compared with (at 37C) at a ratio of 1:10 (v/v), and a volume of 30 mL of this mixture the vehicle control group. Nude mice were housed in a pathogen-free was immediately transferred onto the sample area of a CometSlide. environment and were given food and water ad libitum for the After the agarose/cells was evenly dispersed, slides were placed flat at duration of experiments. 4C in the dark for 30 minutes in a high humidity environments. The cells were then lysed overnight by immersing slides into lysis buffer. Statistical analysis After lysis, the slides were rinsed in distilled water and immersed in Data analyses were performed using GraphPad Prism v6.0 and neutral electrophoresis buffer for 30 minutes before application of an Microsoft Excel. Statistical significance was determined by the 2-tailed electric field. An electric field (typically 1 V/cm) were applied to the unpaired Student t test. P values are reported in the graphs. , P < 0.05; cells for 45 minutes at 4C, and cells were stained with SYBR gold , P < 0.01; and , P < 0.001. n.s. denotes not significant. (S11494; Thermo Fisher Scientific) for 30 minutes in the dark and photographed using a ZEISS microscope with an attached camera. The comets were analyzed using CASP software (18). Results CRISPR-Cas9 screen identifies synthetic lethal partners of ATRX Immunofluorescence To screen for synthetic lethal partner genes of ATRX, we first Cells (1 105) were seeded onto Fisherbrand microscope cover generated isogenic ATRX KO cell models using PLC/PRF/5 and glass (Thermo Fisher Scientific), which were placed in 12-well cell HuH-7, 2 HCC cell lines, which are WT for the gene (25). Both cell culture plates, incubated overnight, and subsequently exposed to 200 lines have TP53 mutations that are known to inactivate the TP53 nmol/L AZD1775 or DMSO as vehicle control. After 24 hours, cells (Supplementary Fig. S1A and S1B), making them good models for were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton studying ATRX function as ATRX and TP53 mutations often co-occur X-100, blocked with normal goat serum, and incubated with Alexa in tumors (7). We used CRISPR technology (26) to specifically target Fluor 488 conjugated anti-phospho Histone H2A.X (Ser139) antibody the ATRX gene locus, introducing inactivating mutations and enabling (05-636-AF488; Millipore) for 1 hour at room temperature. Nuclei the generation of isogenic clones with mutated ATRX (ATRX KO). We were counterstained with DAPI (1 mg/mL; Sigma-Aldrich). Images isolated 2 independent cell clones for PLC/PRF/5 and 1 clone for HuH- were obtained using laser confocal microscopy (Olympus FV1000). 7, with complete absence of ATRX protein expression because of frameshift mutations introduced into the coding region of the gene BrdUrd incorporation assay (Supplementary Fig. S1C–S1F). The parental PLC/PRF/5 line and Cells were incubated in medium with 10 mmol/L BrdUrd for 30 clone sc26, referred to as ATRX WT and ATRX KO, respectively, were minutes at 37C, harvested by trypsinization, and fixed in ice-cold 70% used for the screen. Before performing the screen, the sc26 clone was ethanol overnight. To enable access of the antibody to incorporated passed 15 passages and no ALT phenotype was observed. BrdUrd, cells were treated with 2 mol/L HCl containing 0.1 mg/mL We performed a genome-wide CRISPR-Cas9 screen (27) for essen- pepsin for 20 minutes at RT. Cells were rinsed once with PBS, tial genes in our ATRX null model system, using the GeCKOv2 sgRNA incubated with APC-labeled anti-BrdUrd antibody (BD Biosciences) library (28), which contains 123,411 sgRNAs targeting 19,050 genes at room temperature for 30 minutes in the dark, rinsed again with PBS, and 1,864 miRNAs. We infected ATRX WT and ATRX KO cells with incubated with 50 mg/mL RNase A at 37C for 30 minutes, and then the library at a multiplicity of infection (MOI) of 0.3 and selected for stained with 50 mg/mL propidium iodide. For HuH-7 cells, DAPI (1 sgRNA and Cas9-expressing cells in the presence of puromycin for mg/mL) was used to stain the DNA without RNase A incubation. 2 days (Fig. 1A). The cells were collected at 2 time points, including Samples were analyzed using a FACS Scan (Becton Dickinson). The immediately after puromycin selection (T0) and after approximately results were analyzed with NovoExpress software from ACEA 14 population doublings (T21; Fig. 1A). Biosciences. We used next-generation sequencing to measure the abundance of all sgRNAs in these 2 cell populations at both time points and Xenografts in nude mice compared them to calculate the essentiality score for each gene using PLC/PRF/5 parental cells or PLC/PRF/5 ATRX CRISPR KO cells (2 the algorithm MAGeCK (Supplementary Fig. S2A and S2B; ref. 15). 106 cells per mouse) were subcutaneously inoculated into female We then searched for genes targeted by the sgRNAs that were BALB/C nude mice ages 6 to 8 weeks (Huafukang). Tumor volumes represented at nearly the same level at T0 as at T21 in ATRX WT were calculated with caliper measurements using the following for- cells, but depleted in ATRX KO cells at T21 compared with T0. Under

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Figure 1. Genome-wide CRISPR-Cas9 screens for identifying synthetic lethal partners of ATRX in liver cancer cell lines. A, Screen ﬂowchart. B, MAGeCK mle beta scores for each gene between ATRX WT and ATRX KO groups. Red points, ATRX synthetic lethal genes. C, Essentiality scores for two screen hits, SMC5 and SMC6 in ATRX WT and ATRX KO cells. The cartoon depicts the requirement of the SMC5/6 complex for telomere maintenance. D, Essentiality scores for two screen hits, ASF1 and CABIN1 in ATRX WT and ATRX KO cells. The cartoon represents the ASF1A and CABIN1 complex functioning in the H3.3 deposition pathway. E, Pathway enrichment results for the ATRX synthetic lethal genes using the PANTHER database. F, Distribution of the screen hits in different regulatory points of cell cycle.

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these stringent criteria, we identified 58 genes, which were essential for WEE1 small molecule inhibition in ATRX mutated cells for possible viability in ATRX KO cells but nonessential for ATRX WT cells, as translation into the clinic. We treated ATRX WT and ATRX KO clonal potential synthetic lethal partners for ATRX (Fig. 1B; Supplementary cells with different doses of the WEE1 inhibitor AZD1775 for 3 days, Table S1). and determined IC50 values from cell viability curves (Fig. 2A). Both From the top 15 candidates that having the most deleterious effects ATRX KO cell clones exhibited an increased sensitivity to AZD1775 to ATRX KO cells, we chose 3 (WEE1, VCP,orPKMYT1) to validate based on the IC50 value of 0.38 and 0.31 mmol/L, which were about ATRX the screen results. We used RNAi to knockdown the expression of the 4-fold lower than the IC50 value of 1.4 mmol/L for WT cells. 3 genes in PLC/PRF/5 ATRX KO and WT cells. Cell growth was Because ATRX KO cells had a slightly slower growth rate than ATRX evaluated in colony formation assays. siRNAs for VCP, WEE1, and WT cells (Supplementary Fig. S4A), we used a normalized growth rate PKMYT1 efficiently reduced corresponding mRNAs to <50% of inhibition (GR) metrics to correct for the confounding effects of – ATRX controls in both cell backgrounds (Supplementary Fig. S3A S3C). growth rate on IC50 (16). The KO cells had a lower GR50 Cell growth was inhibited more significantly by these siRNAs in ATRX compared with ATRX WT cells (1.79 mmol/L vs. 0.47 mmol/L and 0.59 KO cells than in the parental cell lines (Supplementary Fig. S3D–S3I). mmol/L, ATRX WT vs. ATRX KO sc26 and sc5; Supplementary Thus, suppression of these genes selectively inhibited colony forma- Fig. S4B), suggesting that they are more sensitive to AZD1775 than tion of ATRX KO cells, indicating that the hits in our screen were true the WT cells in 2D cell culture. Similarly, enhanced sensitivity to synthetic lethal partners of ATRX. AZD1775 was observed in ATRX KO cells compared with ATRX WT HuH-7 cells (Fig. 2B), indicating that this effect of ATRX KO was Synthetic lethal partners of ATRX are enriched in cell-cycle reproducible in a different cell line. Consistently, ATRX depletion by regulators RNAi also increased sensitivity to AZD1775 in PLC/PRF/5 and HeLa Among the 58 candidates identified in the screen, several groups of cells (Fig. 2C and D; Supplementary Fig. S5A and S5B). genes encode proteins that are physically interacting in a complex. For AZD1775 treatment also resulted in significantly reduced clono- example, both proteins of the SMC5/6 complex were identified in the genic survival of ATRX KO relative to ATRX WT cells (Fig. 2E and F). screen (Fig. 1C). The SMC5/6 complex functions in homologous After treatment with 50 to 200 nmol/L AZD1775, the number of recombination during replication and has been implicated in the colonies generated by ATRX KO cells was significantly reduced relative maintenance of heterochromatin (29, 30). The synthetic lethality to ATRX WT cells (Fig. 2E and F). Similar results were observed in between ATRX and the SMC5/6 complex is consistent with ATRX's HuH-7 cells; HuH-7 isogenic ATRX KO clones were also more known function in heterochromatin formation and gene silencing (31). sensitive to AZD1775 treatment relative to WT cells (Fig. 2G and Similarly, 2 components of the HIRA: ASF1A: UBN1: CABIN1 H). Taken together, this observed increase in sensitivity of ATRX complex were identified with similar lethality scores in the screen KO cells to AZD1775 was consistent with the synthetic lethal inter- (Fig. 1D). The HIRA complex is a histone 3.3 chaperone required for action between ATRX and WEE1 identified in our genetic screen. heterochromatinization of senescent human cells (29). ATRX is involved in deposition of histone H3.3 to telomeres in a HIRA- WEE1 inhibition causes S-phase arrest and DNA damage in independent way (32). The synthetic lethal relationship between ATRX KO cells ATRX and HIRA complex members raised questions on the com- We next investigated the mechanism by which loss of ATRX pensation of the 2 H3.3 deposition pathways in certain contexts. sensitized cells to WEE1 inhibition. WEE1 regulates cell-cycle pro- Because our screens have successfully identified genes associated gression by inactivating CDK1 through phosphorylation at tyrosine with well-known ATRX functions, we annotated the functions of all 15. To confirm that AZD1775 inhibits the kinase activity of WEE1 in the hits for new mechanistic insights into the activity of ATRX. We PLC/PRF/5, we examined the protein levels and phosphorylation found that these 58 genes were enriched in pathways involved in a few levels of CDK1 by Western blot at different time points. As expected, cellular processes, including mRNA processing, mitosis, chromatin the Y15 phosphorylation significantly decreased after 1-hour treat- organization, and cell-cycle regulation (Fig. 1E). Strikingly, 19 of the ment with AZD1775 in both ATRX WT and ATRX KO cells, whereas 58 hits are involved in cell cycle and checkpoint pathways (Fig. 1F). the CDK1 protein levels did not change (Supplementary Fig. S6A– These genes include those recruited to the replication fork complex at S6C). As a control, phosphorylation of CDK1 threonine 14, which is intra-S phase checkpoint (TIPIN, TOPBP1, MCM4, SMC5, SMC6, and mainly controlled by another kinase PKMYT1, did not change after RRM2 ; ref. 33), redundant G2–M checkpoint regulators WEE1 and AZD1775 treatment (Supplementary Fig. S6D). Meanwhile, WEE1 or PKMYT1 (34), proteins critical for centromere and spindle assembly in PKMYT1 protein levels did not significantly change during 24-hour M phase (SMC1A, CENPN, CDCA8, SKA3, TUBGCP6, TUBB, treatment of AZD1775 in either ATRX WT or KO cells (Supplemen- TUBA1B, and DYNLRB1; refs. 35, 36), and proteins involved in tary Fig. S6E and S6F). These results suggested that AZD1775 inhibit heterochromatin formation (ASF1A, CABIN1, HAT1; refs. 29, 37, 38). WEE1 activity on cell-cycle regulation in both ATRX WT and KO cells. These results suggest a central role for ATRX in cell-cycle progression Some ATRX-deficient cells have been shown to exhibit defects in cell and the possibility that different regulatory points in the cell cycle can cycle with an increase in DNA replication stress (39, 40). Therefore, we be targeted to treat ATRX mutant cancers. investigated whether the enhanced sensitivity of ATRX KO cells to WEE1 inhibition was a result of combined deficiencies in cell-cycle ATRX KO cells are selectively killed by the WEE1 inhibitor regulation. AZD1775 To test this hypothesis, we first examined the cell-cycle distribu- Among the 19 cell-cycle regulators identified in the screen, WEE1 is tion of ATRX WT and KO cells under AZD1775 treatment as a the only one with small molecule inhibitors designed and tested in measure of DNA content with propidium iodide using FACS clinical trials. One of the inhibitors, AZD1775 (MK1775), has passed (Fig. 3A). Consistent with previous reports (39, 40), the ATRX KO regulations for patient safety and is currently in several phase II clinical cells had a slightly increased proportion of S-phase cells (Fig. 3A). trials in combination with chemotherapies for advanced solid tumors After AZD1775 treatment, a robust increase of cells in S-phase (http://www.clinicaltrials.gov). We therefore investigated the effects of occurred in ATRX KO cells (70.34 1.90% vs. 31.34 2.25%, ATRX

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Figure 2. ATRX loss sensitizes cells to WEE1 inhibition by AZD1775 in multiple cell lines. A and B, CCK-8 assays showing cell viability of ATRX WT and ATRX KO PLC/PRF/5 cells (A) and HuH-7 cells (B) after treatment with indicated doses of AZD1775 for 3 days. C and D, CCK-8 assays showing cell viability of PLC/PRF/5 cells (C) and HeLa cells (D) transfected with either negative control siRNA (siNC) or ATRX siRNAs (siATRX-1 and siATRX-2) and exposed to AZD1775 for 3 days. E–H, Clonogenic assays of ATRX WT and ATRX KO cells exposed to different doses of AZD1775. Colonies containing more than 50 cells were counted in PLC/PRF/5 cells after 12 days of treatment (E and F) and in HuH-7 cells after 10 days of treatment (G and H). For all graphs, error bars represent SEMs from three independent experiments. , P < 0.01; , P < 0.001; n.s., not signiﬁcant. Unpaired and 2-tailed t tests were used for F and H.

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Figure 3. WEE1 inhibition causes S-phase arrest and DNA damage in ATRX KO cells. PLC/PRF/5 WT and ATRX KO cells treated with either DMSO or AZD1775 (200 nmol/L) for 24 hours for the analysis in A–G. A, Cell-cycle distribution of the cells as determined by flow cytometry. B, Representative flow cytometric analysis of DNA synthesis (BrdUrd) and DNA content (propidium iodide, PI). C, Percentages of nonreplicating S-phase cells, determined as cells with 2N < PI < 4N/BrdUrd in B. D, Representative flow cytometric analysis to show the cell-cycle distribution of gH2A.X-positive cells. E, Percentages of gH2A.X-positive cells in D are shown. F, þ Immunofluorescence staining of gH2A.X (green) and DAPI (blue) in the cells. G, Percentages of gH2A.X-positive (gH2A.X ) cells in F were quantified and are shown in the bar plots separately. H, Representative flow cytometric analysis to show the cell-cycle distribution of gH2A.X-positive cells in PLC/PRF/5 WT and ATRX KO cells treated with AZD1775 alone or together with 25 mmol/L CDK1/2 inhibitor roscovitine (Rosco) for 18 hours. Cells were stained with DAPI to assess DNA content. I, Percentages of gH2A.X-positive cells in H are shown. For all graphs, error bars represent SEMs from four independent experiments and unpaired and 2-tailed t tests were used to determine P values. , P < 0.01; , P < 0.001.

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Figure 4. Prolonged WEE1 inhibition causes replication forks collapse into DSBs and committed ATRX KO PLC/PRF/5 cells into apoptosis. A, Representative images of neutral comet assays performed in ATRX WT and ATRX KO cells treated with either DMSO or AZD1775 for 48 hours. For each of the three independent experiments, approximately 100 individual cells from 10 random ﬁelds were scored for the proportion of DNA in the COMET “tail.” (Continued on the following page.)

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KO vs. WT; n ¼ 4; Fig. 3A) was observed. Labeling of cells with (Fig. 2E and F), we examined the possible mechanisms mediating cell BrdUrd supported this finding, as the percentage of nonreplicating lethality. Using FACS, we found that WEE1 inhibition caused a cells in S-phase (2N < PI < 4N/BrdUrd) was increased in ATRX KO significant increase in the portion of cells undergoing apoptosis cells under AZD1775 treatment (2.47 0.42% vs. 11.35 1.46%, (Annexin V staining: 4.06% vs. 15.05% and 19.12%, ATRX WT vs. ATRX WT vs. ATRX KO; n ¼ 4; Fig. 3B and C). Similarly, ATRX KO sc26 and sc5; Fig. 4E and F). Similar results were also proportions of S-phase cells and nonreplicating cells in S-phase observed in HuH-7 cells (Supplementary Fig. S10A and S10B). To were significantly increased in HuH-7 ATRX KO cells treated with determine whether caspase-3 was activated in the process of apoptosis, AZD1775 (Supplementary Fig. S7A–S7C). The above results indi- ATRX KO and WT cells were treated with 200 nmol/L AZD1775 over a cated that WEE1 inhibition caused S-phase arrest in ATRX KO cells. 72-hour time course. Western blot analysis revealed a greater increase To examine the biological conditions triggering S-phase arrest in in the level of activated (cleaved) caspase-3 in ATRX KO cells than in ATRX KO cells under WEE1 inhibition, we first investigated the levels ATRX WT cells, suggesting that cells were committed to apoptosis in a of DNA damage using phosphorylated H2A.X as a marker of stalled caspase-3–dependent manner (Fig. 4G). Similar changes in the level of replication forks and double-strand breaks (DSB). The percentage of gH2A.X were also observed, indicating that the induction of gH2A.X gH2A.X-positive ATRX KO cells increased immensely upon treatment upon AZD1775 preceded apoptosis (Fig. 4G). Taken together, these with AZD1775 compared with ATRX WT cells, as measured by FACS data demonstrate that DNA damage induced by replication stress analysis in both PLC/PRF/5 and HuH-7 cells (Fig. 3D and E; Sup- contributed to the increased sensitivity of ATRX deficient cells to plementary Fig. S7D and S7E). Indeed, these gH2A.X-positive cells WEE1 inhibition, which ultimately committed ATRX KO cells to were mainly distributed in S-phase (Fig. 3D; Supplementary Fig. S7D), apoptosis (Fig. 5A). indicating that the S-phase arrest in ATRX KO cells was partly because Based on the model, we proposed that other drugs that cause of the accumulated DNA damage. Immunofluorescence staining was increased replicative stress could also exacerbate ATRX loss. We tested consistent with these results, showing a sharp increase in the percent- several such drugs and found that ATRX deficiency cells exhibited an age of gH2A.X-positive cells compared with controls (16.98 0.57% enhanced sensitivity to hydroxyurea (IC50: 181.60 3.49 mmol/L vs. vs. 55.42 1.51%, n ¼ 4, AZD1775 treated WT vs. KO cells, P < 81.08 10.79 mmol/L, ATRX WT vs. ATRX KO, n ¼ 3, P ¼ 0.0009) 0.0001; Fig. 3F and G; Supplementary Fig. S8A and S8B). Addition of and gemcitabine (IC50: 0.0418 0.0029 mmol/L vs. 0.0258 0.0015 the CDK1/2 inhibitor roscovitine almost completely repressed the mmol/L, ATRX WT vs. ATRX KO, n ¼ 3, P ¼ 0.0081) than the WT cells increase of gH2A.X levels in both ATRX WT and KO cells after WEE1 (Fig. 5B and C). These results opened the possibilities for exploiting inhibition (Fig. 3H and I; Supplementary Fig. S9A and S9B), suggest- other replicative stress–inducing chemicals for future drug developing that the increased DNA damage was because of the elevated CDK1/ ment for ATRX mutant cancer. 2 activity induced by WEE1 inhibitors. AZD1775 inhibits growth of ATRX-deficient xenografts Prolonged WEE1 inhibition caused replication forks collapse To test the potential therapeutic efficacy of WEE1 inhibition in vivo, into DSBs and committed ATRX KO cells into apoptosis we generated a xenograft model in nude mice using PLC/PRF/5 ATRX To determine whether the increase in gH2A.X after WEE1 inhibi- WT and KO cell lines. Animals were inoculated subcutaneously with tion was because of an increase in DNA double-strand breaks, we used cells, and AZD1775 or vehicle control was administered orally for the neutral comet assay, which is a sensitive method for monitoring 15 days once tumors reached a volume of nearly 150 to 200 mm3. PLC/ DSBs. We observed that WEE1 inhibition caused a significant increase PRF/5 ATRX WT xenografts continued to grow, even under AZD1775 in comet tail moment in ATRX KO cells (Fig. 4A), confirming the treatment, reaching a several-fold increase in volume by the end of the increased DSB formation and replication fork collapse in these cells. experiment (Fig. 6A). In contrast, tumor growth was significantly Consistent with the presence of gH2A.X in ATRX KO cells, the inhibited in PLC/PRF/5 ATRX KO xenografts in response to AZD1775 activation of ATM-dependent DSB signaling events were detected at compared with the vehicle control treated group (230 mm3 vs. 800 ATRX KO cells without drug treatment, as revealed by the increased mm3, AD1775 vs. vehicle control; Fig. 6B). Furthermore, by day 5, levels of CHK2 phosphorylation (Fig. 4B). After AZD1775 treatment, volumes of ATRX KO xenografts had reached a plateau under treat- the DNA-PK–dependent phosphorylation of RPA32 on positions 4 ment (230 mm3; Fig. 6B), whereas ATRX WT and vehicle control and 8 increased more significantly in ATRX KO cells than in ATRX ATRX KO xenografts continued to increase in both size (Fig. 6A–C) WT cells (Fig. 4C and D), indicating that the ATRX loss augmented and weight (Fig. 6D). accumulation of DSBs. Growth inhibition in vivo as in vitro was also possibly mediated by Because long-term AZD1775 treatment resulted in significantly increases in DNA damage. Sections from ATRX KO compared with reduced clonogenic survival of ATRX KO relative to ATRX WT cells ATRX WT xenografts exhibited increased levels of gH2A.X staining,

(Continued.)Scalebar,100mm. B, Representative immunoblots for expression of DSB checkpoint proteins in cell lysates prepared from ATRX KO and ATRX WT cells treated with AZD1775 for indicated times. Normalized levels of ATM-pS1981/ATM and CHK2-pT68/CHK2 were quantifiedandareshowninthebar plots. Error bars represent SEMs from five independent experiments. C, Representative immunofluorescence staining for phosphorylated RPA levels in ATRX KO or ATRX KO cells treated with DMSO or AZD1775 for 24 hours. Cells were stained with phospho-RPA (S4/S8) antibody (red) and DAPI (blue). Scale bar, 50 mm. For each of the three independent experiments, approximately 800 individual cells from at least 10 random fields were scored for immunofluorescence density. D, Representative immunoblots analysis of phosphorylated RPA32 (pRPA32) in ATRX KO or ATRX WT cells treated with DMSO or AZD1775 for 24 hours. Nuclear extract (NE) and whole cell lysates (WCL) were extracted separately. E, Representative Annexin V and propidium iodide (PI) staining FACS plots for assessing apoptosis in ATRX WT and ATRX KO cells treated with either DMSO or AZD1775 for 72 hours. F, Quantification of Annexin V–positive cells as the percentage of cells in Q2 and Q3 of the FACS plots in E. G, Western blot analysis of gH2A.X and cleaved caspase-3 in ATRX WT and ATRX KO cells treated with either DMSO or AZD1775 for the indicated times. For all graphs, error bars represent SEMs (n ¼ 3)andunpairedand2-tailedt tests were used to determine P values unless noted. , P < 0.05; , P < 0.01; , P < 0.001.

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Figure 5. Loss of ATRX renders cells vulner- able to replications stress induced by drug treatment. A, Loss of the tumor suppressor gene ATRX leads to destabilization of the genome by causing replication stress and accumulated DNA damage. Upon AZD1775 treatment, CDK1/2 activities are elevated. The synergy of ATRX loss and increased CDK1/2 activities results in accumulation of replication stress and DNA damage, which triggers apoptosis. B and C, CCK-8 assays demonstrated increased vulnerability of ATRX KO cells to drugs, causing increased replicative stress. ATRX WT and KO cells were treated with indicated concentrations of hydroxyurea (B) or gemcitabine (C). Error bars represent SEMs from three independent experiments.

which were further enhanced under treatment with AZD1775 (15.66% damage response. Furthermore, we demonstrated the efficacy of vs. 2.67%, AD1775 treated ATRX KO vs. ATRX WT, P < 0.01; Fig. 6E targeting WEE1 with the small molecule inhibitor AZD1775 in and F). in vitro and in vivo models deficient in ATRX. ATRX is mutated in the majority of progressive gliomas and WEE1 inhibition selectively impairs the growth of ATRX- secondary GBMs (7). In a recent study, AZD1775 exhibited good deficient cell lines derived from patients with glioma penetration across the human blood–brain barrier (BBB) and exten- As ATRX mutations are frequently found in human glioma, we sively accumulated in human GBM tumors in an acidic tumor investigated whether WEE1 dependency also exists in this tumor microenvironment to reach potentially therapeutic concentra- type in the context of naturally occurring mutations. Patient- tions (41, 42). This requirement of an acidic basolateral pH is based derived primary glioma cell lines with WT or mutant ATRX were on the inactivation of ABCB1/ABCG2-mediated efflux clearance and treated with increasing doses of AZD1775, and IC50 values were activation of the uptake transporter OATP1A2 (41) of AZD1775. determined from the cell viability curves (Fig. 7). Two independent Interestingly, almost all ATRX-mutant gliomas/GBMs harbored the cell lines, 08-0537 and IMA, harboring ATRX nonsense mutations, IDH1/2 hotspot mutations (7), which are associated with the produc- exhibited 2- to 15-fold increased sensitivity to AZD1775 relative tion of D-2-hydroxyglutarate (D2HG), the altered catalytic flux and to ATRX WT cell lines (Fig. 7). The differences in sensitivity the promotion of the acidic tumor microenvironment (43). In this case, to AZD1775 still held after adjusting for the differences in growth AZD1775 has the potential to be an effective therapeutic choice for a rate (Supplementary Fig. S11A and S11B). These results indicate significant number of patients with gliomas/GBM. These results have that the synthetic lethality between ATRX and WEE1 may be implications for the treatment of diverse tumor types with ATRX present in different cancer types harboring ATRX mutations. mutations and for the feasibility of functional screens for the identi- Because AZD1775 exhibits good penetration in brain tumors fication of new pharmacologic targets in cancer. (41, 42), it could be investigated as a potential therapeutic agents Based on the results of our study and others, ATRX loss confers in the treatment of ATRX-deficient malignant glioma. sensitivity to WEE1 inhibition by further destabilizing the genome, which ultimately leads to apoptosis. ATRX is required for the restart of stalled replication forks and recovery from replicative stress in S- Discussion phase (40). Our data and previous studies have shown that loss of In this study, we used an unbiased genome-wide CRISPR-Cas9 ATRX alone induces replication stress and delayed S-phase pro- KO screen to discover synthetic lethal interactions specifictoATRX gression (39, 40, 44). WEE1 inhibition also has a direct effect on deficiency, which affects a variety of tumor types (2–5). We iden- DNA replication in S-phase through activation of CDK1/2 (45). tified several candidate synthetic lethal partners specifictoATRX Previous work has shown that depletion of WEE1 activity results in deficiency with this genetic screen. The most promising candidate unscheduled DNA replication, nucleotide shortage, stalling of was WEE1, a kinase involved in cell-cycle regulation and DNA replication forks and accumulation of DNA damage (46–49).

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Figure 6. ATRX-deﬁcient tumors exhibit increased sensitivity to WEE1 inhibition in a xenograft model in nude mice. A and B, PLC/PRF/5 WT or PLC/PRF/5 ATRX KO cells were inoculated subcutaneously in 6 to 8 weeks old female nude mice. When xenografts reached 150 to 200 mm3, mice with PLC/PRF/5 WT- or PLC/PRF/5 ATRX KO–derived xenografts were divided into two subgroups (n ¼ 6 mice for each group) and orally administered with either AZD1775 or vehicle control for 15 days. PLC/PRF/5 WT (A) or PLC/PRF/5 ATRX KO (B) tumor volumes (mm3) for each treatment group calculated from caliper measurements made each day for 15 days and plotted as a function of time (days). C, Represen- tative gross images of xenograft tumors obtained in A and B. D, Tumor weights after dissection; n ¼ 6 per group. E and F, Representative images and quantitation of immunostaining for gH2A.X in xenograft tumors from each group. Scale bars, 50 mm. For all graphs, unpaired and 2-tailed t tests were used to calculate P values. , P < 0.01; n.s., not signiﬁcant.

Together our observations supported the model that the synergistic effects of ATRX loss and WEE1 inhibition on DNA replication leads to accumulation of DNA damage and S-phase arrest of cells, and eventually apoptosis (Fig. 5A). In summary, CRISPR technology coupled with next-generation sequencing made it possible to identify WEE1 and other candidate genes as critical to the survival of ATRX deficient HCC and glioma cells. This experimental strategy that combines genomic with functional data directly addresses the challenges in designing therapeutic agents with high specificity but low toxicity. Although our study was limited to 2 tumor types, our findings are of clinical interest to other cancers with frequent ATRX mutation. Finally, other synthetic lethal

Figure 7. WEE1 inhibitor AZD1775 selectively inhibits ATRX-mutant glioma primary cells derived from patients. Viability of glioma cell lines treated with increasing doses of AZD1775. Glioma cell lines were treated with increasing doses of AZD1775 for 5 days. Viability was assessed using luminescence values from the CellTiterGlo assay represented as a percentage of the DMSO control. Error bars indicate SEMs of four independent experiments.

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candidates identified in our screen may provide additional effective Acknowledgments targets or critical insight into the tumor suppressive roles of ATRX in We thank Drs. B. Vogelstein, K.W. Kinzler, N. Papadopoulos, and S. Zhou for cancer. helpful discussions. We thank Ms. Guangyu Li for data processing of the GR values for cell viability assays. We appreciate Ms. Ran Gao's help on all the clonogenic survival fl Disclosure of Potential Conflicts of Interest assays and ow cytometry experiments. This work was supported by the National Natural Science Foundation Fund (81472559 to Y. Jiao and 81502420 to J. Liang), the fl No potential con icts of interest were disclosed. National Key Basic Research Program of China (973 program no. 2015CB553902 to Y. Jiao), the CAMS Initiative for Innovative Medicine (2017-I2M-4-003 to X. Wang; Authors’ Contributions 2017-I2M-4-002 to H. Zhao), the State Key Project on Infection Diseases of China Conception and design: J. Liang, H. Zhao, S. Liu, H. Yan, Y.-X. Zeng, X. Wang, Y. Jiao (2017ZX10201021-007-003 to H. Zhao), and the National Laboratory Special Fund Development of methodology: J. Liang, X. Wang (2060204). Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Liang, H. Zhao, B.H. Diplas, J. Liu, D. Wang, Y. Lu, Q. Zhu, W. Wang The costs of publication of this article were defrayed in part by the Analysis and interpretation of data (e.g., statistical analysis, biostatistics, payment of page charges. This article must therefore be hereby marked computational analysis): H. Zhao, S. Liu, X. Wang advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Writing, review, and/or revision of the manuscript: J. Liang, H. Zhao, B.H. Diplas, this fact. S. Liu, X. Wang, Y. Jiao Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Liang, H. Zhao Study supervision: X. Wang, Y. Jiao Received November 1, 2018; revised April 28, 2019; accepted September 17, 2019; Other (perform the experiments): J. Wu published first September 24, 2019.

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OF14 Cancer Res; 2020 CANCER RESEARCH

Genome-Wide CRISPR-Cas9 Screen Reveals Selective Vulnerability of ATRX-Mutant Cancers to WEE1 Inhibition

Junbo Liang, Hong Zhao, Bill H. Diplas, et al.

Cancer Res Published OnlineFirst September 24, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-3374

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2019/09/24/0008-5472.CAN-18-3374.DC1

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