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Translational Science

Differential Activity of ATR andWEE1 Inhibitors inaHighly Sensitive Subpopulation ofDLBCLLinkedto Replication StressLucy A.Young1, Lenka Oplustil O'Connor1, Christelle de Renty1,Margaret H. Veldman-Jones2, Thierry Dorval3, Zena Wilson2, David R. Jones2,Deborah Lawson4, Rajesh Odedra2, Apolinar Maya-Mendoza5, Corinne Reimer4,Jiri Bartek5,6, Alan Lau1, and Mark J. O'Connor1

Abstract

DNA damage checkpoint kinases ATR and WEE1 are amongkey regulators of DNA damage response pathways protectingcells from replication stress, a hallmark of cancer that haspotential to be exploited for therapeutic use. ATR and WEE1inhibitors are inearly clinical trials andsuccesswill requiregreaterunderstanding of both their mechanism of action and biomar-kers for patient selection. Here, we report selective antitumoractivity of ATR andWEE1 inhibitors in a subset of non-germinalcenter B-cell (GCB) diffuse large B-cell lymphoma (DLBCL) celllines, characterized by high MYC protein expression andCDKN2A/B deletion. Activity correlated with the induction ofreplication stress, indicated by increased origin firing and retar-dation of replication fork progression. However, ATR andWEE1inhibitors caused different amounts of DNA damage and celldeath in distinct phases of the cell cycle, underlying the increasedpotency observed with WEE1 inhibition. ATR inhibition causedDNA damage to manifest as 53BP1 nuclear bodies in daughter

G1 cells leading to G1 arrest, whereas WEE1 inhibition causedDNA damage and arrest in S phase, leading to earlier onsetapoptosis. In vivo xenograftDLBCLmodels confirmeddifferencesin single-agent antitumor activity, but also showed potential foreffective ATR inhibitor combinations. Importantly, insights intothe different inhibitor mechanisms may guide differentiatedclinical development strategies aimed at exploiting specific vul-nerabilities of tumor cells while maximizing therapeutic index.Our data therefore highlight clinical development opportunitiesfor both ATR andWEE1 inhibitors in non-GCBDLBCL subtypesthat represent an area of unmet clinical need.

Significance: ATR and WEE1 inhibitors demonstrate effec-tive antitumor activity in preclinical models of DLBCL asso-ciatedwith replication stress, but newmechanistic insights andbiomarkers of response support a differentiated clinical devel-opment strategy.

IntroductionReplication stress (RS), broadly defined as the slowing or

stalling of replication fork (RF) progression, poses a significantproblem for genome stability and cell survival (1). Generation ofaberrant RF structures containing single-strand DNA activates areplication stress response (RSR)mediatedby ataxia telangiectasiaand Rad3-related (ATR) and checkpoint kinase 1 (CHK1; ref. 2),leading to stabilization of RF structures, and delayed cell-cycle

progression for DNA repair and completion of DNA synthesisbefore mitosis (2, 3). Regulation of replication origin firing helpsprevent unscheduled DNA synthesis and rescue stalled RFs (2, 4).ATR and CHK1 negatively regulate cyclin-dependent kinase(CDK) activity through inhibition of the CDC25 family of CDKphosphatases (5). The WEE1 kinase that regulates mitotic entrythrough CDK1 phosphorylation, also plays an important role inregulating replication initiation through phosphorylation ofCDK2 (6, 7).

Compelling evidence links oncogene-induced RS to tumorprogression and its prevalence in human cancers (8). Thus,cancer-specific dependency on the RSR for survival could beexploited by pharmacologic inhibition of ATR, CHK1, orWEE1, ultimately causing cancer cell death (9–12). According-ly, it has been demonstrated that CHK1 and WEE1 inhibitorsinduce strong antitumor effects in MYC-driven lymphomamouse models and neuroblastoma (13, 14). Furthermore,hypomorphic Atr alleles in mouse models prevent the devel-opment of MYC-driven B-cell lymphomas (15), suggesting thatMYC-driven tumors are dependent on ATR for survival, andthat MYC-driven patient tumors could be preferentially tar-geted by ATR inhibitors. Diffuse large B-cell lymphoma(DLBCL), the most common form of non-Hodgkin lympho-ma (16), is associated with MYC overexpression. Although asubset of patients with DLBCL are cured with chemotherapeutic

1Bioscience, Oncology R&D, AstraZeneca, Cambridge, United Kingdom.2Bioscience, Oncology R&D, AstraZeneca, Alderley Park, United Kingdom.3Discovery Sciences, R&D, AstraZeneca, Cambridge, United Kingdom.4Bioscience, Oncology R&D, AstraZeneca, Boston, MA. 5Danish Cancer SocietyResearch Centre, Copenhagen, Denmark. 6Division of Genome Biology,Department of Medical Biochemistry and Biophysics, Science for Life Labora-tory, Karolinska Institute, Stockholm, Sweden.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Mark J. O'Connor, AstraZeneca UK Ltd., 1 Francis CrickAvenue, Cambridge CB2 0AA, UK. Phone: 44-7919596445; E-mail:[email protected]

Cancer Res 2019;79:3762–75

doi: 10.1158/0008-5472.CAN-18-2480

�2019 American Association for Cancer Research.

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regimens, approximately 40% have refractory disease or willrelapse after an initial response (17).

ATR, CHK1, and WEE1 inhibitors potentiate the genotoxicproperties of cytotoxic drugs and radiotherapy, and clinical trialsassessing tolerability and efficacy of this therapeutic strategy areongoing. However, broad clinical utility, especially as singleagents, will likely depend on identifying and targeting patienttumors with high RS. A lack of robust biomarkers for detecting RSin human tumors has limited clinical testing of this hypothesis.Furthermore, understanding how these agents kill cancer cells willbe essential for optimizing patient treatments. In this study, weidentified that the non-GCB subtype of DLBCL, as well as MYCoverexpression and CDKN2A/B deletion, are associated withincreased RS and in vitro and in vivo sensitivity to ATR and WEE1inhibition. Our data also demonstrate different modes of actionfor ATR and WEE1 inhibition and highlights the potential for aneffective combination.

Materials and MethodsCell lines and compounds

DLBCL cell lines were purchased from DSMZ unless otherwisespecified. Pfeiffer and Toledo were purchased from ATCC; HBL-1was from Professor Masafumi Abe, under license from TokyoMedical and Dental University Tokyo, Japan; TMD8 was fromDr. Daniel Krappmann, German Research Center for Environ-mental Health, Munich, Germany; OCI-LY10 was from Dr. LouisStaudt, Center for Cancer Research, NCI, Bethesda, MD.GM14680 and GM03567 were purchased from the CoriellInstitute forMedical Research, Camden,NJ. All cells were culturedin RPMI supplemented with 10% to 15% FBS and 2 mmol/LL-glutamine, except OCI-LY-10, which was cultured in IMDMsupplemented with 20% FBS, 2 mmol/L L-glutamine, and50 mmol/L b-mercaptoethanol. Cells with �90% viability,determined using trypan blue dye exclusion, were used in experi-ments. Cell line identification was validated using the CellCheckassay (IDEXX Bioanalytics). All cell lines were validated free ofvirus by IMPACT tests (IDEXX Bioanalytics) and validated free ofMycoplasma contamination using the MycoSEQ assay (ThermoFisher Scientific) or STAT-Myco assay (IDEXX Bioanalytics). Thegenomics of the cell lines was acquired from the AZ Cell LineExtraction Tool, a database containing genetic characterization ofcell lines from AstraZeneca, CCLE, and COSMIC. AZD6738 (18)and AZD1775 (19) were made by AstraZeneca and solubilizedin DMSO.

In vitro growth inhibition and cell viability assaysCells in 96-well plates were compound dosed using an Echo

555 (LabCyte) and viability determined by alamarBlue assay (LifeTechnologies). Fluorescence intensity wasmeasured using a Spec-traMax i3 (Molecular Devices). Percentage growth was deter-mined using fluorescence values in the equation (T � T0)/(C �T0) � 100, where T is the drug-treated cells, T0 the cells at timezero, andC is the control cells. Dose–response curveswere plottedin GraphPad prism V.6 using the nonlinear regression model,sigmoidal dose response. Fifty percent growth inhibition (GI50)and 100% total growth inhibition (TGI) values were determinedusing data from �3 independent experiments. Cell viability andapoptosis were assessed using the Guava Viacount and Nexin Kitsand analyzed on the Guava easyCyte HT Flow Cytometer (Merck-Millipore).

Cell-of-Origin subtype classificationTotal RNA extracted from cells was applied to a NanoString

code set for determination of Cell-of-Origin (COO) gene expres-sion subtyping, as described previously (20).

Western analysisEqual amounts of whole cell lysates, prepared in RIPA buffer

(Sigma-Aldrich) supplemented with protease inhibitors (Roche)and phosphatase inhibitors (Sigma-Aldrich), were analyzed bystandard SDS-PAGE/immunoblotting, using antibodies listed inSupplementary Methods Table S1.

DNA fiber analysisCells were compound treated for 1 hour before labeling with

25 mmol/L CldU for 20 minutes, then collected by centrifugationand incubated for 20 minutes in media containing compoundand 250 mmol/L IdU. Labeled cells were harvested and DNA fiberspreads prepared as described previously (21). Antibodies listedin SupplementaryMethods Table S1. Fibers were imaged using anLSM710 (Carl Zeiss Microscopy) confocal microscope and 63�objective with numerical aperture of 1.2. The lengths of CldUtracks (�300 per condition and experiment)weremeasured usingZenLite software (Carl Zeiss Microscopy).

DNA molecular combing assayDNA combing was performed using the Molecular Combing

Platform (Genomic Vision). Briefly, cells were compound treatedfor 1 hour before adding 25 mmol/L IdU and incubating the cellsfor 20 minutes. Fifty mmol/L CldU was then added to the mediaand the cells were incubated for another 20 minutes. Cells wereharvested andDNAwas extracted in agarose plugs using FiberPrepDNA Extraction Kit (Genomic Vision). DNAmolecules were thenstretched onto silanized coverslips (CombiCoverslips) usingFiberComb Molecular Combing System (Genomic Vision).Immunodetection of IdU, CldU, and ssDNA was performed withantibodies listed in SupplementaryMethods Table S1. Slides wereentirely scanned with automated FiberVision scanner and ana-lyzed with dedicated FiberStudio software (Genomic Vision). Todetermine the median replication fork velocity >700 tracks percondition were measured, and >100 tracks per condition for themedian inter-origin distance.

53BP1 nuclear bodies assayCells grown on 96-well poly-D-lysine coated black-walled

plates were dosed with compounds (Echo 555; LabCyte) for40 hours, then fixed and permeabilized with 4% paraformalde-hyde and 0.5% Triton X100, blocked in 3% BSA in TBS-Tween(0.05%) and incubated with primary antibodies at 4�C, followedby Alexa Fluor secondary antibodies and Hoechst 33342 (Sup-plementaryMethods Table S1). A series of 48 frames (8 per well, 6wells per condition) were acquired using a 40� objective on theOperetta (Perkin Elmer). Harmony software was used to definenuclei, cyclin A intensity, and quantify 53BP1 foci in G1 phase.

Cell-cycle analysisTo measure the S-phase population, cells were treated with

compound for the indicated times, then 1 hour prior to fixationlabeled with 10 mmol/L EdU (pulse-harvest). To measure cell-cycle progression, cells were first labeled with 10 mmol/L EdU for1 hour, washed, then resuspended in media containing com-pounds (pulse-chase). Cellswerefixed andEdUdetectedusing the

Differential Activity of ATR and WEE1 Inhibitors in DLBCL

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Click-iT Plus Kits (Life Technologies). Cells were subsequentlyincubatedwith fluorescently-conjugated antibodies (Supplemen-taryMethods Table S1) andDAPI.Datawere acquired using aflowcytometer (FACS Aria II) and analyzed in FlowJo (TreeStar).

In vivo studiesAll in vivo studies were performed at United States and United

Kingdom locations within AstraZeneca. Study protocols at the U.S. site were reviewed and approved by the Institutional AnimalCare andUseCommittees,whereas thoseperformed in theUnitedKingdom were approved by the Home Office. All studies wereperformed in accordance with the Animal Scientific ProceduresAct 1986 (ASPA), and AstraZeneca Global Bioethics policy. Datawere reported following the Animal Research: Reporting In Vivo(ARRIVE) experiments guidelines (22). Female NOD.CB17-SCIDmice were purchased from Charles River Laboratories and usedbetween the ages of 8 and 12 weeks in accordance with institu-tional guidelines. Animals were randomized into vehicle andtreatment groups based onmean tumor volume of approximately0.1 to 0.2 cm3. For the OCI-LY-19 study, animal treatment groupsreceived AZD6738 at either 25 or 50 mg/kg orally or AZD1775 ateither 120, 90, or 60 mg/kg orally once daily as monotherapy, orin combination where AZD1775 was dosed 1 hour following theAZD6738 dose. AZD6738 was prepared in 10% DMSO þ 40%propylene glycol þ 50% water and AZD1775 was prepared in0.5% methylcellulose. For the TMD8 study, 5 � 106 cells in 50%Matrigel were inoculated subcutaneously on the left flank offemale NOD.CB17-SCID mice. Treatment groups were dosed asfollows: AZD6738 dosed as monotherapy at 50 mg/kg orallydaily; the combination of rituximab and bendamustine(R-Benda) was dosed for only one cycle: rituximab at 10 mg/kgi.p. on day 1 and bendamustine at 12.5mg/kg i.v. on days 1 and 2.AZD6738 dosed in combination with R-Benda was dosed for 1cycle (daily for 5 days). Tumor volume was measured bilaterallyby caliper using the formula p/6,000 x length x width2; animalbody weight and tumor condition was recorded twice weekly forthe duration of the study. Growth inhibition from the start oftreatment was assessed by comparison of the geometric meanchange in tumor volume for the control and treated groups.Statistical significance was evaluated using a one-tailed, t test.

ImmunohistochemistryDLBCL xenograft tumors grown in CB17-SCID mice were

harvested between 0.2 and 0.5 cm3 and fixed in 10% neutralbuffered formalin then subjected to routine vacuum processingthrough graded ethanol, xylene, and paraffin. Tumors embeddedin paraffin blocks were sectioned at a thickness of 3 mm, air dried,and heated to 65�C for 20 minutes. IHC was run on the VentanaDiscovery XT using primary antibodies listed in SupplementaryMethods Table S1. Detection was performed with Vectastainbiotinylated secondary reagents (antirabbit PK-6101 and anti-mouse PK-6102), and the DABMap Kits (Ventana Medical 760-124, and UltraMap Kit Ventana 760-152 for anti-P16). Digitalslide images were acquired with the Aperio Scanscope XT using a20� objective.

Statistical analysisA Student unpaired Mann–Whitney t test was used to deter-

mine statistical differences between 2 groups of data, whereasone-way ANOVA with Kruskal–Wallis multiple comparisons testwas used for the DNA fiber analysis and DNA combing analysis

(GraphPad Prism V6). A value of P < 0.05 was consideredstatistically significant.

ResultsThe ATR inhibitor AZD6738 reduces proliferation in non-GCBDLBCL cell lines

Tumors with RS are hypothesized to be sensitive to ATRinhibition. To identify cell line models of RS, we evaluated theantiproliferative effects of ATR inhibitor AZD6738 (18) across 12cancer cell line panels. The concentration of AZD6738 resulting inGI50 was lowest in hematologic cancer cell lines (Fig. 1A). Eight of26 DLBCL cell lines were highly sensitive to AZD6738 (GI50 <0.5mmol/L), therefore we examined this cell panel for correlativebiomarkers of response and levels of RS.

DLBCL can be classified into germinal center B-cell like (GCB)and activated B-cell like (ABC) subtypes, the latter associated withworse outcomes following standard chemo-immunotherapytreatments (23). Using a NanoString scoring system (20), weidentified 6 cell lines of ABC subtype, 7 of GCB, and 6 unclassified(Fig. 1B and C). However, the unclassified signature closelyresembles the ABC subtype (Fig. 1B). Greater AZD6738 activitywas observed in the non-GCB group comprising ABC and unclas-sified subtypes (median GI50 ¼ 0.372 mmol/L), compared withGCB (median GI50¼ 1.554 mmol/L) or lymphoblastoid cell lines(Fig. 1C and D; Supplementary Table S1). AZD6738 activity wasonly weakly associated with doubling rates of cell lines (Supple-mentary Fig. S1A), and not enhanced by prolonged exposure(Supplementary Table S1; Supplementary Fig. S1B,i–S1B,ii).

AZD6738 sensitivity is associatedwithMYCoverexpression andCDKN2A/B deletion

We looked for genetic correlations of increased AZD6738activity in non-GCB DLBCL cell lines. Aberrant expressionand activation of oncogenes involved in proliferation, suchas RAS, CCNE1, and MYC, can induce RS by causing prematureS-phase entry, transcription–replication conflicts, and nucleotideshortage (15, 24–27). Translocation and/or amplification ofMYCwaspresent in 11of 18of theDLBCL cell lines (Fig. 2A), consistentwith higher prevalence of MYC (28) compared with RAS andCCNE1 aberrations in DLBCL (29). AZD6738 sensitivity did notcorrelate with MYC genomics (Supplementary Fig. S1C) but wasassociated with higher c-MYC protein levels (Fig. 2A and B).

Cell-cycle dysregulation contributes to RS,with reports suggest-ing selective toxicity of ATR and CHK1 inhibitors in TP53-defec-tive cells (30, 31). TP53 mutations were present in 12 of 18 ofDLBCL cell lines but did not enrich for AZD6738 sensitivity(Supplementary Fig. S1C). Homozygous deletion of CDKN2A/B(p16INK4a, p14ARF, and p15INK4b), a key regulator of G1 to S,occurs in 30% of ABC and 4% of GCB patients with DLBCL (32).Five of eight sensitive cell lines were homozygous CDKN2A/Bdeleted (Fig. 2A), which statistically enriched for AZD6738 activ-ity (meanGI50CDKN2Adel¼0.195mmol/L,WT¼1.561mmol/L),whereas p16 protein levels did not (Fig. 2B). Undetectable levelsof BCL-6 protein in non-GCB cell lines also enriched for AZD6738activity, consistent with BCL-6 expression characterizing GCBDLBCL (33). In vivo protein expression of MYC, BCL6, and p16was determined by IHC and corroborated in vitro data (Supple-mentary Fig. S2). Separately, each biomarker identified a cell linepopulation enriched for AZD6738 sensitivity (Fig. 2C) as did thecombination of lowp16 andhigh c-MYCprotein (Supplementary

Young et al.

Cancer Res; 79(14) July 15, 2019 Cancer Research3764




Fig. S3A). However, combination of the non-GCB subtype withhigh c-MYC protein and CDKN2A/B deletion was most accurate,identifying 7of 8highly sensitive and0of 7 less sensitive cell lines.

Constitutive activation of DNA damage response (DDR) pro-teins is a hallmark of RS and improved response to CHK1inhibitors in DLBCL cell lines (34). Surprisingly, we found nocorrelation between AZD6738 sensitivity and endogenous DDRactivation, ATR/CHK1 protein levels, or ATM functionality in theDLBCL panel (Supplementary Fig. S3B–S3D). Together, theseresults suggest MYC protein expression and CDKN2A/B deletioncould represent more reliable predictive biomarkers of ATRinhibitor response in DLBCL tumors than DDR pathway status.

AZD6738 induces RS in DLBCL cell linesDNA lesions formed inmitosis following RS can be detected as

53BP1 nuclear bodies (NB) in daughter G1 cells (35). We there-fore evaluated whether 53BP1 NB formation correlated with

AZD6738 sensitivity (Fig. 3A–C). AZD6738 sensitivity was notassociated with endogenous levels of 53BP1 NBs but was asso-ciated with a greater fold induction in 53BP1 NBs followingAZD6738 treatment (Fig. 3C). Moderate levels of RS are toleratedby cells, with slowedRF velocity causing chromatin recruitment ofATR pathway sensors andmediators without activation of CHK1,ATM, or cell-cycle checkpoints (36). We therefore speculated thatnon-GCB cell lines may have higher intrinsic levels of RS that,upon exacerbation by AZD6738 treatment, would result in more53BP1 NBs compared with GCB cell lines. To test this, weanalyzed RF progression by DNA fiber analysis (Fig. 3D andE). Basal RF velocities were significantly slower in non-GCB celllines OCI-LY-19, WILL-2, and TMD-8 compared with GCB celllines Karpas-422 and DB (Fig. 3D; Supplementary Table S2),except for TMD-8 versus Karpas-422 (Supplementary Table S3).AZD6738 treatment reduced RF velocity, with the greatest impactinnon-GCB cells (Supplementary Table S2). RF velocity inDBand

Figure 1.

AZD6738 activity in non-GCB subtype DLBCL cell lines. A, GI50 values of cancer cell lines and respective median of the panel 72 hours after AZD6738 treatment.B, DLBCL cell lines were classified and clustered into COO subtypes by gene expression profiling. C,GI50 values of COO-subtyped DLBCL cell lines. Mean andSEMwere calculated from n� 3 experiments.D,Median GI50 value of COO-subtyped DLBCL cell lines, where non-GCB includes ABC and unclassified subtypes.






Karpas-422 cells following AZD6738 treatment (0.81–0.86 kb/min) was comparable to DMSO-treated OCI-LY-19 and WILL-2cells (0.77–0.96 kb/min), thus fromour studywe propose that RFvelocities of approximately 0.8 kb/min represent a moderate, butmanageable level of RS requiring ATR for RF stability. AZD6738treatment slowed RF progression below this threshold in non-GCB cells, potentially leading to underreplicated DNA, whichtogether with abrogation of the G2–M checkpoint, increased

mitotic transmission of DNA lesions. Together, these data suggestthat underlying intrinsic levels of RS inDLBCL cell lines ultimatelydetermines response to ATR inhibition.

Pharmacologic inactivation of ATR and WEE1 increases RSlevels

ATR, WEE1, and CHK1 are all involved in the RSR (5, 12).Consistent with this, we found a similar trend in sensitivity for

Figure 2.

AZD6738 activity in DLBCL celllines is associated withCDKN2A/B deletion, high c-MYCprotein, and low BCL-6expression.A, Immunoblotanalyses of exponentially growingDLBCL cell lines. Genomic statusof indicated gene biomarkers,COO subtype, and sensitivity toAZD6738 for each cell line isshown. B, Correlation betweenGI50 AZD6738 values andgene/protein biomarkerexpression. � , P�0.05;�� , P�0.01; ns, not statisticallysignificant. C, Venn diagram ofthe COO subtype, gene, andprotein biomarkers in sensitiveand insensitive DLBCL cell lines.

Young et al.





AZD6738 and the WEE1 inhibitor AZD1775 across DLBCLcell lines (Fig. 4A; Supplementary Fig. S4A–S4B), withAZD1775 showing greater reduction in cell viability (Fig. 4B;Supplementary Fig. S4C) and shared biomarkers of response(Supplementary Fig. S4D). We next compared the effects ofboth inhibitors on DNA replication in OCI-LY-19 and DBcell lines (the most and least sensitive cells in the panel).Distribution of cells in S-phase was determined by measuringEdU incorporation (Fig. 4C). OCI-LY-19 cells were more abun-dant in early S-phase (Fig. 4D), indicating slower progression of

cells through S-phase, a sign of RS. ATR is critical in early S-phase to limit replication origin firing and suppress formationof ssDNA (37). Thus, OCI-LY-19 cells may be more vulnerablethan DB cells to ATR or WEE1 inhibition.

We performed DNA combing with DNA counterstaining todetermine the impact of AZD6738 and AZD1775 on RF progres-sion and origin firing. In agreement with previous DNA fiberanalyses, RFs inOCI-LY-19 cells progressed slower than inDBcells(Fig. 4E). RF velocity is closely correlated with origin firing (38),and both AZD6738 and AZD1775 treatments decreased RF

Figure 3.

AZD6738 treatment induces 53BP1 nuclear bodies and retardation of RF progression.A, Representative images of immunofluorescence staining for 53BP1 andcyclin A in cells treated with AZD6738 or DMSO for 40 hours acquired by wide-field microscopy (Operetta, Perkin Elmer). B,Quantification of 53BP1 nuclearbodies (NB) in G1 nuclei (Cyclin A

�) of untreated DLBCL cells using Harmony software (Perkin Elmer);�1,000 cells were counted per condition. Error bars arecalculated from�3 independent experiments. C, Induction of 53BP1 NBs by AZD6738 treatment. Data shown as the fold change in average number of 53BP1 NBsin AZD6738-treated cells relative to untreated cells. D, Cells were treated with 1 mmol/L AZD6738 or DMSO for 1 hour before sequentially labeling for 20minuteswith IdU, then CldU. Replication fork velocities were determined by DNA fiber assay. Dot plots showmedian velocity with interquartile range (IdU; n > 300 forksper cell line, n¼ 3 experiments; GM14680 and Karpas-422, n > 100 forks, n¼ 2 experiments). E, Representative fibers from each cell line (original magnification,�63).






Figure 4.

AZD1775 is more potent than AZD6738 in DLBCL cell lines. A, Comparison of GI50 values in DLBCL cell lines treated with AZD6738 or AZD1775 for 72 hours. B,Cell viability measured by uptake of Viacount reagent in control and drug-treated DLBCL cells at the times indicated. Graphs show the mean percentage of viablecells (�SEM) relative to controls from three independent experiments. C and D, Cells were pretreated for 1 hour with 1 mmol/L AZD6738, AZD1775, or DMSO, thenEdU labeled (10 mmol/L, 40 minutes) before fixation. C, Representative cell-cycle distribution determined by flow cytometry analysis of EdU intensity and DNAcontent. D, EdUþ cells were gated into early-mid or mid-late S-phase populations based on DNA content. Graphs show the average� SEM from threeindependent experiments. E–F, Cells were treated with 1 mmol/L AZD6738, AZD1775, or DMSO for 1 hour before sequential IdU/CldU dual labeling, then analyzedby DNA combing. E, Distribution of replication fork velocity by treatment. All distributions are statistically different from each other (P < 0.0001), exceptDBþATRi vs. OCI-LY-19þDMSO. F, Distribution of inter-origin distance in indicated conditions. A summary of DNA replication parameters and track numbermeasurements with dot plots showing median and interquartile range from two independent experiments. �� , P¼ 0.0039; �� , P¼ 0.0003; �� , P < 0.0001. ns,not statistically significant.

Young et al.





velocities and increased origin firing, indicating RS (1, 39, 40).However, the overall impact was greatest in the OCI-LY-19 cellline (Fig. 4E and F), suggesting sensitivity to both inhibitors iscaused by a higher capacity for excessive origin firing. Median RFvelocities and inter-origin distances in AZD6738- or AZD1775-treatedDB cells were equal or greater than those in untreatedOCI-LY-19 cells (Fig. 4E and F). These data suggest that RS levelsinduced by AZD6738 and AZD1775 in OCI-LY-19 cells are likelygreater than the RS threshold, pushing cells towards replicationcatastrophe (41). In contrast, RS levels are likely below thethreshold in DB cells, providing better tolerance to AZD6738and AZD1775.

Differences in viability and replication dynamics betweenAZD6738- and AZD1775-treated cell lines was not caused bydifferential target inhibition, as measured by phosphorylatedCHK1 on serine 345 for AZD6738 (18) and phosphorylatedcdc2 (CDK1) on tyrosine 15 for AZD1775 treatment (Supple-mentary Fig. S5A; ref. 19). Because of cdc2 pY15 antibody cross-reactivity, we cannot exclude concurrent reduction in phos-phorylated CDK2 and CDK3. Re-phosphorylation of CHK1 byDNA-PK during sustained ATR inhibition (37) was notobserved in AZD6738-treated DLBCL cell lines (SupplementaryFig. S5), suggesting compensatory regulators of the RSR donot affect the activity of AZD6738 or AZD1775 in DLBCL.Whether this is a general feature of DLBCL or due to the use ofdifferent ATR inhibitors remains unclear. AZD1775 treatmentinduced phosphorylation of CHK1 in DLBCL cell lines (Sup-plementary Fig. S5A–S5B), indicating WEE1 inhibition causesactivation of the ATR–CHK1 pathway. Interestingly, AZD1775also decreased CHK1 and ATM protein expression between 24and 72 hours, with earlier protein reduction correlating withincreased drug sensitivity. We did not investigate the mecha-nism of DDR protein reduction, however proteasomal degra-dation of DDR proteins has been reported (42) and warrantsfurther investigation.

AZD6738 and AZD1775 differentially affect cell-cycleprogression and induce DNA damage and cell death in distinctcell-cycle phases

The differential effects of AZD6738 and AZD1775 on DNAreplication in OCI-LY-19 and DB cell lines prompted furtherevaluation of their mechanism of action. Cells were EdU pulse-labeled then released into EdU-free medium containing DMSO(control), AZD6738, or AZD1775 and assessed by flow cytometryto measure cell-cycle progression of S-phase (EdUþ labeled) cellsand DNA damage (gH2AX; Fig. 5A). Control EdUþ cells transi-tioned into the daughter cell cycle within 24 hours. However,OCI-LY-19 cells progressed through S-phase more slowly (com-pare DMSO at 8 hours, Fig. 5A), consistent with slower RFprogression. AZD1775 prevented transition of EdUþ OCI-LY-19 into daughter G1 phase, indicated by 3% G1-phase EdU

þ cellsafter 8 hours compared with 26% in control cells (Fig. 5B).Abrogation of S-phase progression correlated with gH2AX induc-tion at 8 to 24 hours (Fig. 5A) and an increased sub-G1 populationat 48 to 72 hours, indicative of cell death (Fig. 5B). Consistentwith this, 53BP1 NBs declined in OCI-LY-19 following AZD1775treatment (Supplementary Fig. S6), suggesting DNA-damagedcells do not complete mitosis. In contrast, AZD1775-treated DBcells displayed normal S-phase progression, and fewer weregH2AXþ (Fig. 5A and B). Replication catastrophe, characterizedby high levels of ssDNA and RPA exhaustion, causes DNA break-

age and S-phase pan-nuclear gH2AX (41). Increased origin firing,observed in AZD1775-treatedOCI-LY-19 cells, but not inDB cells(Fig. 4F), may cause excessive ssDNA and account for the largerproportion of gH2AXþ S-phase OCI-LY-19 cells. gH2AX wasmostly detected in EdU�DBcells entering S-phase at 8 to 12hourson AZD1775 treatment (Fig. 5A), suggesting delayed effects inDBcells.

AZD6738-treated cells showed normal S-phase progression,and accelerated progression into daughter G1 compared withDMSO control (8 hours, Fig. 5B) consistent with the regulationof intrinsic S–G2 and G2–M checkpoints by ATR (3). AZD6738caused chronic accumulation of EdUþ OCI-LY19 cells in thedaughter G1–early-S phase, whereas DB cells continued throughsubsequent cell divisions (Fig. 5B). AZD6738 induced gH2AX in4% to 13% of OCI-LY-19 cells predominantly in early S (8–12hours) and daughter G1–S cells (24 hours), compared withdetection only in the second S-phase in DB cells (EdUþ cells,24 hours, Fig. 5A), consistent with increased 53BP1 NBs in OCI-LY-19, but not DB cells. Increasing AZD6738 to 3 mmol/L did notexacerbate the phenotype, suggesting differences betweenAZD6738 and AZD1775 aremechanistic and not dose dependent(Supplementary Fig. S7A).

Cell-cycle distribution and proliferation were also evaluated byEdU pulse-labeling cells before fixation (Fig. 5C,i). AZD1775 andAZD6738 decreased EdU intensity in S-phase OCI-LY-19, but notDB cells (Fig. 5C,i), consistent with slower DNA synthesis andhigher RS in OCI-LY-19 cells. AZD1775 increased the OCI-LY-19EdU� S-phase population, concurrent with decreased EdUþ cells(24 hours, Fig. 5C,ii), indicating S-phase arrest and inhibitedproliferation. S-phase arrest occurred at 48 hours in DB cells,consistent with transition into the successive S-phase (Fig. 5A).AZD6738 reduced the population of EdUþ OCI-LY-19 cellscompared with control cells at 48 to 72 hours, in agreement withG1–S arrest of daughter cells (Fig. 5A).

To assess where DNA damage occurred in the cell cycle,gH2AXþ cells were gated by DNA content (Fig. 6A). AZD1775induced gH2AX predominantly in G1–early-S phase in OCI-LY-19 cells, and in S and G2–M phases in DB cells, indicatingDNA damage was replication-associated. AZD1775 increasedthe population of pH3þ (mitotic) cells in both cell lines (2–8 hours, Fig. 6B,i), suggesting either unscheduled mitosis ormitotic arrest; conversely AZD6738 had no effect. The popu-lation of pH3þ AZD1775-treated OCI-LY-19 cells decreased by24 hours and supports the notion that WEE1 inhibitionimpedes replication, whereas the mitotic population of DBcells remained higher than control cells (Fig. 6B,i), correspond-ing with accumulation in G2–M. A fraction of mitotic DB cellswas gH2AXþ (Fig. 6B,ii), suggesting DNA damage sustainedduring S-phase may have been carried into mitosis, resulting installing. However, most AZD1775-induced DNA damageoccurred outside of mitosis (Supplementary Fig. S7B), and allpH3þ cells compromised 4N DNA (Supplementary Fig. S7C),suggesting premature mitosis is not the major source ofDNA damage. Cleaved caspase 3 (CC3) was predominantlydetected in gH2AXþ cells between 24 and 72 hours (Fig. 6C,iand ii), indicating DNA damage leads to apoptosis. Apoptosisand DNA damage occurred in the same cell-cycle phase;predominantly S–G2 in response to AZD1775 or the daughterG1–S phase in response to AZD6738 (Supplementary Fig. S7D).Some AZD6738-treated OCI-LY-19 cells undergoing apo-ptosis had DNA content consistent with early S-phase






(Supplementary Fig. S7D), suggesting that cells carrying DNAlesions into S-phase, in the absence of functional ATR, arepredisposed to higher levels of RS, replication catastrophe, andcell death. Interestingly, AZD1775-induced apoptosis occurredlater in the DB cell line, possibly due to differences in cell-cycledistribution of the DNA-damaged cells (Fig. 6A). However, wehave not tested whether differential apoptotic regulation con-tributes to the kinetics of cell death.

AZD1775 and AZD6738 demonstrate in vivo activity in DLBCLxenograft models

Consistentwith in vitrodata, once daily oral dosingofAZD6738or AZD1775 induced significant tumor growth inhibition (TGI)in the OCI-LY-19 xenograft model. The maximum-tolerated dose(MTD) of 120 mg/kg/day for AZD1775, or 50 mg/kg/day forAZD6738 caused complete TGI at day 8 (Fig. 7A; SupplementaryFig. S8A,i; Supplementary Table S4), without significant body

Figure 5.

S-phase–dependent cell-cycle effects of AZD6738and AZD1775.A and B, Cells pulse-labeled with EdU(10 mmol/L) for 1 hour were PBS washed then treatedwith AZD1775 (1 mmol/L), AZD6738 (1 mmol/L), orDMSO for durations indicated. Immunofluorescencestaining of EdU, gH2AX, and DNA intensity wasassessed by flow cytometry. A, Representative cell-cycle dot plots overlaid with gH2AX-positive cells(green). Percentage of gH2AXþ cells is indicated(green text). B, Histograms showing the percentageof EdUþ (S-phase cells actively replicating at time oftreatment) by cell-cycle phase. Cells were gated onEdU, then by DNA content (DAPI) to determine cell-cycle phase. Percentage of populations at 8 hourscorrespond to the G1 peak. C, Cells were treated withAZD1775 (1 mmol/L), AZD6738 (1 mmol/L), or DMSOfor durations indicated, then pulse-labeled with EdU(10 mmol/L; 1 hour) before fixation and flowcytometry analysis. The gating strategy is shown,where EdUþ cells represent active S-phase, andEdU� cells with DNA content between 2N and 4Nrepresent inactive S-phase. Data shown arepercentage cell population in active and inactive S-phase from two independent experiments (�SEM).

Young et al.





weight loss (Supplementary Fig. S8A,ii). However, body weightloss was observed by day 17 in some animals, leading to termi-nation of dosing and reduced numbers of animals used tocalculate end-of-study tumor volume (Supplementary TableS4). These data suggest that at high doses, either inhibitor wouldneed to be administered using shorter cycles to allow for recovery.At the end of study (21 days), the average tumor volume inmice treated with AZD6738 (50 mg/kg/day) or AZD1775(120 mg/kg/day), was 0.35 or 0.1 cm3, respectively. These in vivo

data suggest AZD1775 has greater single-agent antitumor activitythan AZD6738 at their respective MTDs.

Given the differential mechanisms of action observed inthe in vitro experiments, we tested whether combination ofAZD6738 and AZD1775 in vivo would improve efficacy. A lessdose-intensive 5 days on, 9 days off schedule, using AZD1775(60 mg/kg/day) and AZD6738 (25 mg/kg/day), resulted intumor regressions comparable to the AZD1775 single-agentMTD (120 mg/kg/day; Fig. 7B; Supplementary Fig. S8B,i;

Figure 6.

Cell-cycle–dependent DNA damage andcell death induced by AZD1775 andAZD6738. Cells treated with AZD1775(1 mmol/L), AZD6738 (1 mmol/L), or vehicle(DMSO) for the durations indicated werepulse-labeled with EdU (10 mmol/L; 1 hour)before fixation and staining. Intensity ofEdU, gH2AX, Histone 3 pS28 (pH3), CC3,and DNA content (DAPI) was assessed byflow cytometry. A, Cells were gated forgH2AX, then DNA content used todetermine cell-cycle phase. Shown is thepercentage gH2AXþ population by cell-cycle phase. B, Cells gated on intensity ofgH2AX and Histone 3 pS28 (pH3). Shown isthe percentage of cells in mitosis (pH3þ)(i), and those in mitosis with DNA damage(ii). C, Cells gated on intensity of gH2AXand CC3. Shown is the percentage of dual-positive gH2AXþ/CC3þ cells (i) and CC3only (ii). Data from two independentexperiments (�SEM).






Supplementary Table S5), but with better tolerability (Supple-mentary Fig. S8B,ii).

AZD6738 demonstrated lower potency compared withAZD1775, suggesting that chemotherapy combinationapproaches may be required in the clinic. R-Benda is a commonchemotherapy regimen for DLBCL, however the ABC-subtype isfrequently refractory, or patients soon relapse (43). We evalu-ated AZD6738 in combination with R-Benda in the TMD-8model (Fig. 7C,i). Single-agent AZD6738 (50 mg/kg/day con-tinuous) resulted in 82% TGI after 15 days and was superior to

R-Benda (53% TGI; Fig. 7C,ii; Supplementary Fig. S8C,i).Strikingly, the combination of R-Benda with 5 days ofAZD6738, resulted in tumor regression with completeresponses in 8 of 8 animals, no relapse within 100 days(Fig. 7C,ii; Supplementary Table S6), and good tolerability(Supplementary Fig. S8C,ii). AZD1775 in combination withR-Benda would also be expected to demonstrate efficacy. How-ever, only limited (2.5 days) exposure of AZD1775 has beentolerated with chemotherapy in the clinic (44), and thereforewe did not test this combination in vivo.

Figure 7.

In vivo efficacy of AZD6738 and AZD1775 in DLBCLxenograft models. A and B, TGI in the subcutaneousOCI-LY-19 xenograft model in CB17-SCID mice.A,AZD6738 or AZD1775 were given orally at theindicated once daily doses for 21 days. Dosing in the50mg/kg AZD6738 and 120 mg/kg AZD1775 groupswas terminated on day 17 due to body weight loss. B,Treatment groups received two cycles of 25 mg/kgAZD6738 and 60mg/kg AZD1775 once daily, or incombination, on a 5 days on, 9 days off schedule. C,TGI in the subcutaneous TMD8 xenograft model inCB17-SCID mice. i, Combination dosing schedule ofrituximab, bendamustine, and AZD6738. ii,Treatment groups received AZD6738 at 50 mg/kgdaily for 15 days, rituximab at 10 mg/kg i.p. on day 1only, and bendamustine at 12.5 mg/kg i.v. on days 1and 2 only. AZD6738 dosed in combination with R-Benda was given as 5 daily doses. Data representedas mean� SEM (n¼ 10 for OCI-LY-19 groups; n¼ 8for TMD8 groups).

Young et al.





DiscussionPreclinical activity of ATR, WEE1, and CHK1 inhibitors has

been reported in numerous cancer subtypes, including hemato-logic cancers (13, 30). However, the genetic and moleculardeterminants of sensitivity and how this could be exploited inthe clinic is still being defined. In this study, we identified a groupof non-GCB DLBCL cell lines sensitive to both ATR (AZD6738)and WEE1 (AZD1775) inhibitors and provided preclinical evi-dence that RS and specific genetic features of DLBCL represent keypredictive biomarkers of response. Furthermore, we have provid-ed some of the first head-to-head mechanistic comparisons forATR and WEE1 inhibitors in DLBCL.

Although constitutive activation of theDDR inDLBCL cell lineswas previously associated with CHKi response (34), we found nocorrelation with ATR inhibitor activity, consistent with otherstudies profiling CHK1i in hematologic cell lines (45). Theseresults suggest endogenous DDR activation is not a reliablepredictive biomarker of sensitivity to RSR inhibitors, at least innonsolid tumors. However, we showed the non-GCB subtype,together with high expression of c-MYC and/or deletion of theCDKN2A/B locus in DLBCL cell lines, enriches for AZD6738sensitivity, similar to that reported for CHK1 inhibition inMYC-driven lymphomas (13, 15) andCDKN2A/p16 deleted headand neck cancer cells (46). The DLBCL COO classification hasprofound prognostic implications, with the ABC subtype associ-ated with an inferior outcome compared with GCB (43). Poorprognosis is also associated with CDKN2A deletion, found in30%ofABCDLBCL (32, 47) andhighMYCexpression, reportedin30% of newly diagnosed patients with DLBCL (28). Further-more, a recent comprehensive genetic analysis of primary DLBCLrevealed new robust DLBCL subsets based on gene signatures,including an ABC/GCB-independent group with biallelic inacti-vation of TP53, CDKN2A loss, genomic instability, and increasedlevels of E2F targets (48). Together, our data suggest that ATR andWEE1 inhibitors could provide therapeutic benefit for patientswith DLBCL subgroups characterized by CDKN2A/B deletionand/or c-MYC overexpression.

The roles of CDKN2A/B genes and MYC in regulating cell-cycleprogression and replication (26) prompted us to assess whethernon-GCB DLBCL cell lines exhibited higher levels of RS. Weobserved increased origin firing, slower RF velocity, and increased53BP1 NB formation following ATR inhibition, specifically in theAZD6738 sensitive non-GCB cell lines, which correlated withsubsequent G1 arrest and cell death. Based on these findings, wepropose that AZD6738 sensitivity inDLBCL cell lines is due to RS-associated ATR dependency. Consistent with this, CDKN2A/p16deleted HNSCC cell lines were hypersensitive to CHK1 inhibitionand reduced RF velocity was mitigated by reexpression ofp16 (46). MYC depletion is lethal across both ABC and GCB celllines (29), thus we were unable to confirm a causal link betweenMYC and RS levels and cannot exclude the possibility that othergenetic drivers may be involved. Our results are also consistentwith recent data showing sensitivity to ATR inhibition is pre-vented by inactivation of the CDC25A phosphatase, which delaysentry into mitosis, allowing increased time for completion ofDNA replication (49). Accordingly, uponAZD6738 treatment, weobserved a modest increase in CDK activity and cell-cycle tran-sition in sensitive, but not insensitive, cell lines.

Interestingly, although ATR inhibition caused increased 53BP1NBs, inhibition of WEE1 did not, suggesting a differential

mode of action of the two drugs. Cytotoxicity induced by WEE1inhibitors is attributed to aberrant CDK1 and CDK2 activationleading to abrogation of the DNA-damage G2–M checkpoint (5)and RS (7, 50), respectively. Therefore, it has been proposed thatWEE1 inhibitors would behave similarly to ATR inhibitors, pref-erentially targeting cancer cells with high RS and promotingmitotic catastrophe by forcing cells with under-replicated DNAto prematurely enter mitosis (51). Although we did observe anincrease in mitotic cells at early time points following WEE1inhibition, these cells had a full complement of DNA and onlya fractionwere positive for DNA damage. In contrast, themajorityof DNA damage occurred in S-phase cells, which, in the highlysensitive OCI-LY-19 model, underwent cell death before enteringmitosis, presumably due to replication catastrophe (41).

Through both in vitro and in vivo studies, we have shown thatAZD1775 and AZD6738 are effective at killing cancer cells withhigher levels of endogenous RS.However, AZD1775 appears to bea more potent option for a monotherapy approach, whereasAZD6738 may have utility in combination therapy. Our in vivodata demonstrated enhanced AZD6738 efficacy in combinationwith AZD1775 or chemotherapy. In the latter example, combi-nation with R-Benda induced complete tumor regression in anABC-DLBCL CDKN2A/B�/� xenograft model. This finding isimportant when considering the high-unmet need for this patientpopulation (47). Given the tolerability challenges of AZD1775 incombination with chemotherapy (51), our data suggest develop-ment of a WEE1 inhibitor as a monotherapy option in non-GCBDLBCL with CDKN2A/B deletion and/or MYC overexpression,whereas ATR inhibitor development in this same populationmaybe more likely to succeed using a combination approach.

Finding functional biomarkers or assays relevant for the manygenetic drivers of RS is amajor challenge for effective deploymentof RSR inhibitors in the clinic (12).Our results showdrug-induced53BP1NBs are an indication of intrinsic RS and predict AZD6738sensitivity inDLBCL cancer cells, whereas endogenous levels werenot predictive, potentially limiting the application of such anassay as a patient selection biomarker. In addition, we showedpotential to predict cancer cell sensitivity to ATR inhibitors usingRF velocity measurements, thus warranting the investigation ofthis approach inmore relevant in vivo settings. Of note, CDKN2A/B deletion and MYC overexpression are both prevalent in othertumor types such as T-ALL, TNBC, and squamous NSCLC. Addi-tional studies providing functional and prognostic links betweenthese biomarkers, as well as high levels of RS and ATR and WEE1inhibitor sensitivity, has the potential to expand the use of theseDDR targeted agents to additional tumor indications.

Disclosure of Potential Conflicts of InterestR. Odedra has ownership interest (including stock, patents, etc.) in

AstraZeneca. C. Reimer has ownership interest (including stock, patents,etc.) in AstraZeneca. M.J. O'Connor has ownership interest (including stock,patents, etc.) in AstraZeneca. No potential conflicts of interest were disclosedby the other authors.

Authors' ContributionsConception and design: L.A. Young, C. Reimer, J. Bartek, A. Lau,M.J. O'ConnorDevelopment of methodology: L.A. Young, A. Maya-MendozaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L.A. Young, L.O. O'Connor, C. de Renty,M.H. Veldman-Jones, T. Dorval, D.R. Jones, R. Odedra, C. ReimerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L.A. Young, L.O. O'Connor, C. de Renty,






M.H. Veldman-Jones, T. Dorval, Z. Wilson, D.R. Jones, R. Odedra, A. Maya-Mendoza, C. Reimer, J. Bartek, A. Lau, M.J. O'ConnorWriting, review, and/or revision of the manuscript: L.A. Young,L.O. O'Connor, C. de Renty, C. Reimer, J. Bartek, A. Lau, M.J. O'ConnorAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L.A. Young, Z. Wilson, D.R. Jones, D. LawsonStudy supervision: J. Bartek, A. Lau, M.J. O'Connor

AcknowledgmentsWe thank staff in Laboratory Animal Sciences at AstraZeneca for tech-

nical support and Mark Wappett for bioinformatic support. A. Maya-Mendoza and J. Bartek are funded by the Danish Cancer Society(R1123-A7785-15-S2) and the Swedish Research Council (VR-MH 2014-46602-117891-30).

Data and materials availability: Researchers may obtain AZD6738,AZD1775, and AZD7762 with a material transfer agreement from AstraZeneca.All reasonable requests for collaboration involving materials used in theresearch will be fulfilled provided that a written agreement is executed inadvance between AstraZeneca and the requester (and his or her affiliatedinstitution). Such inquiries or requests should be directed to the correspondingauthor.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 11, 2018; revised December 23, 2018; accepted May 20,2019; published first May 23, 2019.

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2019;79:3762-3775. Published OnlineFirst May 23, 2019.Cancer Res Lucy A. Young, Lenka Oplustil O'Connor, Christelle de Renty, et al. Sensitive Subpopulation of DLBCL Linked to Replication StressDifferential Activity of ATR and WEE1 Inhibitors in a Highly

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http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-18-2480

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differentialactivityofatrandwee1inhibitorsin ... · regimens, approximately 40% have refractory...

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