Modeling Therapy Resistance in BRCA1/2-Mutant CancersWT BRCA2 BRC Repeats (1-8) DNA-Binding Domain TR2/NLS 0 500 1,000 1,500 2,000 2,500 3,000 3,418 2,002 CAPAN1 6174delT BRCA2 0 CAPAN1.B2.S*

Models and Technologies

Modeling Therapy Resistance in BRCA1/2-MutantCancersAmy Dr�ean1, Chris T.Williamson1, Rachel Brough1, Inger Brandsma1,Malini Menon1, Asha Konde1, Isaac Garcia-Murillas2, Helen N. Pemberton1,Jessica Frankum1, Rumana Rafiq1, Nicholas Badham1, James Campbell1, Aditi Gulati1,Nicholas C. Turner2, Stephen J. Pettitt1, Alan Ashworth1, and Christopher J. Lord1

Abstract

Although PARP inhibitors target BRCA1- or BRCA2-mutanttumor cells, drug resistance is a problem. PARP inhibitorresistance is sometimes associated with the presence of sec-ondary or "revertant" mutations in BRCA1 or BRCA2. Whethersecondary mutant tumor cells are selected for in a Darwinianfashion by treatment is unclear. Furthermore, how PARPinhibitor resistance might be therapeutically targeted is alsopoorly understood. Using CRISPR mutagenesis, we generatedisogenic tumor cell models with secondary BRCA1 or BRCA2mutations. Using these in heterogeneous in vitro cultureor in vivo xenograft experiments in which the clonal compo-sition of tumor cell populations in response to therapy wasmonitored, we established that PARP inhibitor or platinumsalt exposure selects for secondary mutant clones in a Darwin-

ian fashion, with the periodicity of PARP inhibitor adminis-tration and the pretreatment frequency of secondary mutanttumor cells influencing the eventual clonal composition of thetumor cell population. In xenograft studies, the presence ofsecondary mutant cells in tumors impaired the therapeuticeffect of a clinical PARP inhibitor. However, we found thatboth PARP inhibitor–sensitive and PARP inhibitor–resistantBRCA2 mutant tumor cells were sensitive to AZD-1775, aWEE1 kinase inhibitor. In mice carrying heterogeneoustumors, AZD-1775 delivered a greater therapeutic benefit thanolaparib treatment. This suggests that despite the restoration ofsome BRCA1 or BRCA2 gene function in "revertant" tumorcells, vulnerabilities still exist that could be therapeuticallyexploited. Mol Cancer Ther; 16(9); 2022–34. �2017 AACR.

IntroductionHeterozygous germline mutations in the BRCA1 or BRCA2

tumor suppressor genes strongly predispose to cancers of thebreast, ovary, pancreas, and prostate (1, 2). BRCA1 and BRCA2are involved in homologous recombination (HR), a process usedto repair DNA double-strand breaks (DSB), and other DNAlesions that impair replication forks (3–6). Extensive preclinicaland clinical data has established that the loss of BRCA1 or BRCA2function is associated with sensitivity to small-molecule PARPinhibitors (7). Recently, the PARP inhibitor (PARPi) Lynparza(olaparib/AZD2281 –KuDOS/AstraZeneca)was approved for the

treatment of platinum-responsive, BRCA1- or BRCA2-mutanthigh-grade serous ovarian carcinomas (HGSOC; ref. 8).

Despite a number of profound and sustained antitumorresponses in patients treatedwith PARP inhibitors, drug resistancelimits theoverall effectiveness of these agents (9–12).Anumber ofmechanisms of PARP inhibitor resistance have been identified,including upregulation of PgP drug transporters, loss of 53BP1 orREV7 function, or secondary "revertant" mutations within theBRCA1 or BRCA2 genes themselves (13, 14). These secondaryBRCA gene mutations restore BRCA1 or -2 open reading framesand encode proteins that have partial function (13–16). In someBRCA2-mutant patients, initial clinical responses to PARPi areseen, followed by the emergence of profound PARPi-resistantlesions (13). The gradual emergence of PARPi resistance duringtreatment has led to the hypothesis that PARPi treatment mightprovide a Darwinian-selective pressure effect, where a secondarymutant clonehas a selective advantageover nonsecondarymutanttumor clones, once PARPi treatment is applied (7, 17). Althoughthis hypothesis has not as yet been tested, if such a Darwinianprocess did exist, a secondary mutant clone might be expected togradually dominate a tumor cell population over the course ofPARPi therapy. Todate, only one approach for targeting tumor cellclones with secondary BRCA1/2 mutations has been proposed,namely the use of thiopurines (18). The wide application ofthiopurines in the treatment of cancer has been limited by safetyconcerns (18), suggesting that additional therapeutic approachesfor targeting secondary BRCA1/2-mutant tumor cells might alsobe required.

We set out to assess, both in vitro and in vivo, whether tumor cellswith secondary BRCA1 or -2 gene mutations are selected for by

1The CRUK Gene Function Laboratory and The Breast Cancer Now Toby RobinsBreast Cancer Research Centre, The Institute of Cancer Research, London,United Kingdom. 2Molecular Oncology Laboratory, The Breast Cancer NowToby Robins Breast Cancer Research Centre, The Institute of Cancer Research,London, United Kingdom.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Current address for A. Ashworth: UCSF Helen Diller Family ComprehensiveCancer Centre, San Francisco, California.

Corresponding Authors: Christopher J. Lord, The Institute of Cancer Research,237 FulhamRoad, LondonSW36JB,UnitedKingdom.E-mail: [email protected];and Alan Ashworth, UCSF Helen Diller Family Comprehensive Cancer Centre,1450 Third Street, San Francisco, CA 94158. E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-0098

�2017 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 16(9) September 20172022

on June 3, 2021. © 2017 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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PARPi treatment in a Darwinian fashion. To do this, we usedCRISPR-Cas9–mediated gene targeting in BRCA1- or BRCA2-mutant tumor cells to generate daughter clones with secondarymutations. By mixing these secondary mutant clones with paren-tal tumor cells in in vitro cocultures or in vivo tumor xenografts, weestablished that PARPi treatment can select for secondary mutantclones in a Darwinian fashion. Using these same systems, we alsofound that exposure to a clinical WEE1 kinase inhibitor (ref. 19;AZD-1775) minimized the selection of secondary mutant tumorcells, targeting parental and secondary mutant cells to a similarextent, while having minimal effects on nontumor cells.

Materials and MethodsCell lines

CAPAN1 and SUM149 cells were obtained from ATCC. DLD1-BRCA2WT/WT and DLD1-BRCA2–/– cells were purchased fromHorizon Discovery Inc. All cells were cultured according to themanufacturer's instructions. All cells were STR typed to confirmidentity and verified to be mycoplasma-free (Lonza MycoAlert)prior to the study.

Small-molecule inhibitorsOlaparib, talazoparib, and AZD-1775 were obtained from

Selleck Chemicals. Inhibitor stock solutions were prepared inDMSO and stored in aliquots at �20�C. Inhibitors were addedto cell cultures so thatfinalDMSOconcentrationswere constant at1% (v/v).

CRISPR-generated PARPi-resistant secondary mutant cell linesCAPAN1.B2.S� and SUM149.B1.S� were generated from

CAPAN1 and SUM149 parental tumor cell lines. Parental lineswere transiently transfected with 5 mg of gRNA (described below)and5mgof aCas9 pMA-T expression vector (GEHealthcare) usingLipofectamine 2000 (Invitrogen) according to themanufacturer'sinstructions. Twenty-four hours later, cells were replated into15-cm dishes at 2,000 cells/dish and exposed to 100 nmol/Ltalazoparib for two weeks after which clones were cultured intalazoparib-free media until visible. Clones were manually iso-lated, and replated into 96-well plates for expansion. DNA fromclones was isolated using DNeasy Blood and Tissue Kit (Qiagen)and PCR amplified using BRCA1 or BRCA2 primers describedbelow. PCRproducts were subcloned using TOPOTACloningKit,with pCR2.1-TOPO (Invitrogen). Sanger sequencing confirmedsecondary BRCA1 or BRCA2 mutations from 20 subcloned E.colicolony sequences per cell line.

gRNA (in pMA-T vector): BRCA2, 50-GAGCAAGGGAAAA-TCTGTCC-30 and BRCA1, 50-CCAAAGATCTCATGTTAAG-30. TheBRCA2 gRNA contains the c.6174delT mutation specific to theCAPAN1 cell line.

Primers for PCR amplification were BRCA1, 50-TGCT-TTCAAAACGAAAGCTG-30, 50-ACCCAGAGTGGGCAGAGAA-30;BRCA2, 50-CTGTCAGTTCATCATCTTCC-30, 50-ATGCAGCCAT-TAAATTGTCC-30.

Confocal microscopyCells on glass coverslips were exposed to 5 Gy IR using an X-ray

machine and then fixed and stained 5 hours later as describedpreviously (20). Nuclei were stained using DAPI diluted in PBS(1:10,000 v/v), RAD51 using the Abcam (ab137323) antibodyand gH2Ax using the Millipore (05-636) antibody. At least 100

cells were assessed per coverslip. Cells scoring positive had >5 fociper nucleus.

Immunoprecipitation and Western blottingImmunoprecipitation (IP) and Western blotting were per-

formed as described previously (15, 21). Antibodies targetingphospho-CDC (Y15; #4539), PARP1 (#9542), gH2AX (#9718; allCell Signaling Technology), and tubulin (#T6074, Sigma) wereemployed in Western blots.

Cell-based assaysShort-term drug exposure and clonogenic assays were per-

formed as described previously (22). In brief, cells were seededeither into 384-well or 6-well plates at a concentration of 500 to2,000 cells per well. After 24 hours, cells were exposed to olaparib,talazoparib, or AZD-1775. For short-term drug exposure, cellviability was assessed after five days of drug exposure usingCellTiter-Glo Luminescent Cell Viability Assay (Promega) as perthe manufacturer's instructions. For clonogenic assays, drug wasreplenished every three days for up to 14 days, at which pointcolonies were fixed with TCA and stained with sulforhodamine B.Colonies were counted and surviving fractions calculated bynormalizing colony counts to colony numbers in vehicle-treatedwells. Survival curves were plotted using a four-parameter logisticregression curve fit as described in ref. 23.

In vitro coculture drug exposure assaysCells were plated in a fixed starting ratio of secondary mutant:

parental cells in either 24 well or 6 well plates, or T75 flasks andexposed to either olaparib, talazoparib, or AZD-1775 for 14 or 21days. In "constant drug exposure" experiments, media containingdrug were replenished every three days. In "intermittent drugexposure" experiments, cells were exposed to media containingdrug for 24 hours after which, media were removed, cells werewashed using PBS, and then cells were recultured in mediawithout drug for 48 hours, at which point cells were "refed" withmedia containing drugs as before.

DLD1 drug exposure assayDLD1-BRCA2WT-GFP andDLD1-BRCA2–/–-RFP cells were plat-

ed at one of the following ratios: 1:1, 1:10, 1:100, or 1:1,000.Twenty-four hours later, cells were exposed to DMSO, olaparib,or talazoparib. Aliquots of cell populations were analyzed byflow cytometry, every 3–4 days, (LSR II, Beckman-Coulter)for GFP and RFP cell populations. Drug was replenished everythree days.

Droplet digital PCR assaysCells were pelleted and genomic DNA was extracted using the

DNeasy Blood and Tissue Kit (Qiagen) following the manufac-turer's instructions. DNA concentration was measured usingQubit broad range detection kit (Invitrogen). Restriction diges-tion was performed with EcoRI (BD Biosciences) and final work-ing dilutions were made at 5 mg/uL per sample. DNA reactionmixtures were performed as described previously (24).

Primers and probes were as follows:CAPAN1:

ddPCR Probe: VIC-CTGGACAGATTTTC,Forward primer: 50- TCTCATCTGCAAATACTTGTGGGATT-30

Reverse primer: 50- TTGTCTTGCGTTTTGTAATGAAGCA-30

Modeling Secondary Mutations in BRCA1 and BRCA2

www.aacrjournals.org Mol Cancer Ther; 16(9) September 2017 2023



http://mct.aacrjournals.org/

CAPA

N-1

CAPA

N-1.B

2.S*

020406080

100

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ANOVAP < 0.0001

P < 0.0001

Olaparib (mmol/L)

Olaparib (mmol/L)

Nuclear RAD51 Foci

SUM1

49

SUM1

49.B

1.S*

020406080

100

% C

ells

with

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ControlIR - 5 Gy

P = 0.01

NS

Nuclear RAD51 Foci

CAPAN-1 CAPAN-1.B2.S*

DAPI g-H2AX RAD51

γ H2AX

RAD51

DAPI

Untreated +IR Untreated +IR

10 μμm

Merged

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CAPAN-1

5-bp Deletion

BRCA2 32,340,298

BRCA2 32,340,298

A

gRNA CRISPR/Cas9 targeting either BRCA1 or BRCA2

Parental tumor cell line:either SUM149 (BRCA1 mutant) or CAPAN-1 (BRCA2 mutant)

PARPi resistant daughter clone with secondary mutation in BRCA1 or BRCA2

Parental clone mixed with secondary mutantdaughter clone

Drug exposure

Assessfrequency of parental and secondary mutant mutant daughter clone

B C

ED

G

H WT BRCA1 RING

NLS

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BRCT Repeats

SUM149 BRCA1 RING

NLS

0 762

T

SUM149.B1.S* BRCA1 RING

NLS

0 1,836

T

762 789

80-bp Deletion

SUM149.B1.S*

SUM149 BRCA1 43,096,339

BRCA1 43,096,339

WT BRCA2 BRC Repeats (1-8) DNA-Binding Domain TR2/NLS

3,418 3,000 2,500 2,000 1,500 1,000 500 0

2,002

CAPAN1 6174delT BRCA2

0

2,002 CAPAN1.B2.S* 6174delT6182del5

0 3,412

2,004

Cas9

F

JI

Figure 1.

Characterization of BRCA1 and BRCA2 secondary mutant, PARPi-resistant clones. A, Schematic showing experimental design. SUM149 and CAPAN1 cells weretransfectedwith Cas9 and CRISPR gRNA expression constructs targeting BRCA1 or BRCA2, respectively, to induce DSB and subsequently create a secondary BRCA1or BRCA2 mutation reinstating the open reading frame. B, DNA sequence for CAPAN1.B2.S� showing 5-bp (Continued on the following page.)

Dr�ean et al.

Mol Cancer Ther; 16(9) September 2017 Molecular Cancer Therapeutics2024




CAPAN1.B2.S�

ddPCR Probe: 6FAM- CTGATACCTGATTTTCForward primer: 50- TCTCATCTGCAAATACTTGTGGGATT -30

Reverse primer: 50- TTGTCTTGCGTTTTGTAATGAAGCA -30

SUM149ddPCR Probe: VIC- TTTGTCAACCTAGCCTTCCAForward primer: 50- TGACAGCGATACTTTCCCAGA -30

Reverse primer: 50- GAGATCTTTGGGGTCTTCAGC -30

SUM149.B1.S�

ddPCR Probe: 6FAM-ACCAGGTGCATTTGTTAACTTCAForward primer: 50- TGACAGCGATACTTTCCCAGA -30

Reverse primer: 50- GCAAAACCCTTTCTCCACTTACT -30

AZD-1775 sensitivity assessmentA total of 146 cancer cell lines were profiled as described

previously (22, 25). In brief, cells were plated at a density of250 or 500 cells per well. Twenty-four hours later, media contain-ing WEE1 inhibitor was added to adherent cells. After five days ofdrug exposure, cell viability was measured using CellTiter-Glo(Promega). Luminescence data was log2 transformed and cen-tered according to the plate median value. Surviving fractionswere calculated relative to the DMSO-exposed control wells togenerate AUC data.

Xenograft experimentsFemale BALB/c nude mice aged 4–6 weeks and 15–22 g in

weight (Charles River Laboratories) were inoculated subcutane-ously with 5 � 106 tumor cells into the right flank. When tumorsreached 100 mm3, 6 animals from each cohort were sacrificed assentinels to enable estimation of parental and secondary mutanttumor cell frequencies prior to treatment. Remaining animalswere randomized into different treatment arms (n ¼ 6) asdescribed in the main text. Mice were weighed once weekly;tumors were measured twice weekly. When tumors reached1,500 mm3, tumors were harvested and half of the tissue wasformalin fixed, the other half was snap frozen in liquid nitrogenfor DNA isolation.

Cell-cycle analysisAsynchronously growing CAPAN1 and CAPAN1.B2.S� cells

were plated in 10-cm dishes (5 � 105 cells/plate) and treatedwith 1 mmol/L AZD-1775 or DMSO for 72 hours. The cells werepulse labeled with 30 mmol/L bromodeoxyuridine (BrdUrd; Sig-ma, B5002) for 1 hour prior to collection. Cells were harvestedusing trypsin, washed with PBS, fixed in cold 70% ethanol andstored at �20�C overnight. Samples were washed with 2 mol/LNaCl/0.5% Triton X-100 and incubated for 30 minutes at roomtemperature. Cell pellets were resuspended in 0.1 mol/L sodiumtetraborate for 2 minutes and subsequent cell pellets were incu-bated at room temperature for 1 hour in anti-BrdUrd antibody

(BD Biosciences, 347580) diluted in 0.5% TWEEN-20/1%BSA/PBS (1 mg antibody per 1� 106 cells). Sample pellets were washedin PBS/1%BSA. Cells were then incubated for 30minutes at roomtemperature with goat anti-mouse IgG FITC antibody (Sigma,F0257) diluted in 0.5% TWEEN-20/1%BSA/PBS (1 mg antibodyper 1 � 106 cells). Cells were pelleted and resuspended in PBScontaining 10 mg/mL RNaseA (Sigma, R4642) and 20 mg/mLpropidium iodide (Sigma, P-4170) and incubated at roomtemperature for 30 minutes. Cell-cycle analysis was performedon a FACS LSR II and analyzed using FlowJo software (FlowJo).

ResultsGeneration of PARPi-resistant models harboring secondaryBRCA1 or BRCA2 mutations

We used CRISPR-Cas9–mediated gene targeting to generatenovel tumor cell models with secondary mutations in eitherBRCA1 or BRCA2 and then used these in in vitro and in vivococulture systems to assess the clonal evolution of tumor cellpopulations in response to therapy (Fig. 1A). To generate thesemodels, we used two PARPi-sensitive tumor cell lines, the pan-creatic ductal adenocarcinoma tumor cell line, CAPAN1 (BRCA2mutation c.6174delT, p.S1982fs�22; refs. 13, 15, 26), andthe breast tumor cell line SUM149 (BRCA1-mutant c.2288delT,p.N723fsX13; ref. 27). We designed specific CRISPR guide RNA(gRNA) expression constructs targeting PAM (protospacer adja-cent motifs) sequences close to either the BRCA2 c.6174delTmutation in CAPAN1 cells or the BRCA1 c.2288delT mutation inSUM149 cells, and transiently transfected these into cells, along-side aCas9 expression construct.We reasoned that the error-pronerepair of DSBs in these BRCA gene–defective tumor cell lineswould in some cells cause frameshift secondary mutations ineither BRCA1 or BRCA2 that restored the open reading frame. In aCAPAN1-derived daughter cell clone, CAPAN1.B2.S�, we identi-fied a five base pair (bp) BRCA2 secondary mutations,c.[6174delT;6182del5], in addition to the parental c.6174delTmutation (c.6174delT: c.[6174delT;6182del5] allele ratio of3:2; Fig. 1B; Table 1). TheBRCA2 c.[6174delT;6182del5] secondarymutation in CAPAN1.B2.S� was predicted to restore the openreading frame of the gene and to encode a 3612 amino acid (aa)BRCA2 protein (Fig. 1C), confirmed by Western blotting (Sup-plementary Fig. S1A). The secondary mutation in CAPAN1.B2.S�

was associated with olaparib and also talazoparib resistance (Fig.1D; Supplementary Fig. S1B, ANOVA P < 0.0001) and the abilityto form nuclear RAD51 foci in response to ionizing radiation(Student t test P < 0.001; Fig. 1E and F; Supplementary Fig. S1C), abiomarker of functional DNA repair by BRCA2. The CAPAN1.B2.S� clone also exhibited a similar doubling time to the parentalCAPAN1 clone, of 2.5 days (Supplementary Fig. S1D). Using thesame approach, we also identified other CAPAN1daughter cloneswith secondarymutations, including CAPAN1.B2.S�2, which had

(Continued.) deletion in BRCA2. PAM sequence is underlined in blue. C, Predicted BRCA2 protein structure for CAPAN1.B2.S�. The predicted aminoacid length is shown. D, Dose–response survival curves for CAPAN1.B2.S� (red) and CAPAN1 parental cell lines exposed to olaparib (P < 0.0001, ANOVA). Error barsrepresent SEM from triplicate experiments. E, Representative images for nuclear RAD51 foci formation in CAPAN1 and CAPAN1.B2.S� cells following IRexposure. Scale bar, 10 mm. F, Bar chart illustrating quantitation of nuclear RAD51 foci. Cells containingmore than five foci were counted as positive. Mean� SEM forthree independent experiments are shown. P values were calculated using Student t test. G, DNA sequence for SUM149.B1.S� showing 80-bp deletion inBRCA1. PAM sequence is underlined in blue. H, Predicted BRCA1 protein structure for SUM149.B1.S�. The predicted amino acid length is shown. I, Dose–responsesurvival curves for SUM149.B1.S� (green) and SUM149 parental cells exposed to olaparib (P < 0.0001, ANOVA). Error bars represent SEM from triplicateexperiments. J, Bar chart illustrating quantitation of nuclear RAD51 foci. Cells containing more than five foci were counted as positive. Mean � SEM for threeindependent experiments are shown. P values were calculated using Student t test.






three different BRCA2 alleles (BRCA2 c.[6174delT;6185del5],BRCA2 c.[6174delT;6183delG], and BRCA2 c.[6174delT;6184delTC]) and CAPAN1.B2.S�3, which also had three differentBRCA2 alleles (BRCA2 c.[6174delT;6183del6], BRCA2c.[6174delT;6183del5], and BRCA2 c.[6174delT;6185del3]; Sup-plementary Table S1).

Using a similar approach in SUM149 cells, we identifiedSUM149.B1.S�, a daughter clone which possessed a secondarymutation (an 80-bp BRCA1 deletion, c.[2288delT;2293del80]), aswell as the parental c.2288delT mutation (c.2288delT:c.[2288delT;2293del80] alleles in a 1:2 ratio; Fig. 1G; Table 2).This secondary mutation was predicted to restore the openreading frame in the parental BRCA1 c.2288delT allele and toencode an 1836 aa BRCA1 protein (Fig. 1H; SupplementaryFig. S2A). SUM149.B1.S� exhibited both olaparib and talazo-parib resistance (Fig. 1I; Supplementary Fig. S2B, ANOVA P <0.0001), and restoration of RAD51 nuclear localization inresponse to DNA damage (Fig. 1J; Supplementary Fig. S2C;Supplementary Fig. S2D, P < 0.01, Student t test). SUM149 andSUM149.B1.S� cells exhibited similar proliferation rates (Sup-plementary Fig. S2E).

Darwinian selection of BRCA1- or BRCA2-proficient clones byPARPi treatment in vitro

To assess whether a Darwinian-selective process might operatein the case of PARPi resistance, we mixed parental and secondarymutant tumor cells in in vitro cocultures (i.e., CAPAN1 parentalwith CAPAN1.B2.S� secondary mutant cells, or SUM149 withSUM149.B1.S� cells) and then exposed the cocultures to twodifferent BRCA gene–selective drugs, olaparib or the platinumsalt cisplatin (Fig. 2A). We then monitored the relative frequencyof each clone in response to drug exposure using droplet digitalPCR (ddPCR; ref. 28). To do this, we used duplex PCR reactionsthat included fluorophore-labeled digital PCR probes that werecomplementary to either parental or secondary mutant alleles(Supplementary Table S2, see Materials and Methods). In pilotexperiments, wherewemixedCAPAN1andCAPAN1B2.S� cells in1:1, 1:10, 1:100 ratios, we were able to accurately detect thesedifferent ratios using the ddPCR assay (Supplementary Fig. S3).Using this ddPCR approach, we assessed whether olaparib orcisplatin exposure would preferentially select for the secondarymutant clones in either CAPAN1plusCAPAN1B2.S� cocultures or

SUM149plus SUM149.B1.S� cocultures. In these experiments, weexposed cocultures to either: (i) DMSO (the drug vehicle), (ii)constant exposure to olaparib (drug replenished every three days),(iii) intermittent exposure to olaparib (where drug was appliedfor 24 hours then washed out and replenished with media notcontaining drug for 48 hours), (iv) constant exposure to cisplatin(drug replenished every three days), or (v) intermittent 24-hourpulses of cisplatin (where drug was applied for 24 hours thenwashed out and replenished with media not containing drug for48 hours; Fig. 2A). As expected, in both CAPAN1 and SUM149cocultures, constant exposure to either olaparib or cisplatincaused a greater reduction in the total tumor cell population sizethan intermittent drug exposure (Fig. 2B). For example, in theCAPAN1 coculture, 37% of the cell population survived after 14days when exposed to constant olaparib, compared with 55%when intermittent drug exposurewas used (Fig. 2B).Despite thesereductions in population size, both olaparib and cisplatin expo-sure caused an increase in the relative frequency of the secondarymutant clones compared with the parental clone (Fig. 2C), effectsreplicated when mixed cultures were exposed to a chemicallydistinct PARP inhibitor, talazoparib (Supplementary Fig. S4A).We found that the enrichment in the secondary mutant clonescompared with the parental clones was most profound whencultures were constantly exposed to either olaparib or cisplatin,compared with intermittent drug exposures (Fig. 2C). In culturesexposed to the drug vehicle, the proportion of CAPAN1.B2.S� andSUM149.B1.S� in DMSO-exposed cultures remained the samethroughout the experiment (1:20 secondary mutant:parentalclone, Supplementary Fig. S4B). We therefore concluded thatwhile constant drug exposure elicited a more profound reductionin the size of the tumor cell population, it did enrich for secondarymutant clones.

We then assessed whether the initial frequency of a secondarymutant clone in a tumor cell populationmight influence the timetaken for this clone to dominate the population when it wasexposed to the selective pressure of PARPi therapy. To do this, wegenerated CAPAN1.B2.S�: CAPAN1 mixed in vitro cultures with1:1, 1:20, and 1:40 clone ratios and then exposed these toolaparib.We then estimated the temporal evolution of the culturein response to drug treatment by using ddPCR to measureCAPAN1.B2.S�: CAPAN1 ratios over time (Fig. 2D). We foundthat in each culture, olaparib exposure caused an increase in the

Table 2. Sequencing of BRCA1 in SUM149.B1.S�

Cell line

Reference sequence for BRCA1 CAATCCTAGCCTTCCAAGAG-AAGAAAAAGAAGAGAAACTAGAAACAGTTAAAGTGTCT-AATAATGCTGAAGACCCCAAAGATCTCATGTTAAG

CRISPR-Inducedmutation

Subclonedcoloniessequenced (#)

Estimatedallelefrequency

SUM149 CAA-CCTAGCCTTCCAAGAGAAGAAAAAGAAGAGAAACTAGAAACAG-TTAAAGTGTCTAATAATGCTGAAGACCCCAAAGATCTCATGTTAAG

N/A 20 1.00

SUM149.B1.S�

CAA-CCTAG——————————————————————————–TAAG 80-bp Deletion 20 0.66CAA-CCTAGCCTTCCAAGAGAAGAAAAAGAAGAGAAACTAGAAACAG-TTAAAGTGTCTAATAATGCTGAAGACCCCAAAGATCTCATGTTAAG

SUM149(2288delT)

0.33

Table 1. Sequencing of BRCA2 in CAPAN1.B2.S�

Cell lineReference sequence for BRCA2CAGCAAGTGGAAAATCTGTCCAGGTATCAGATGCT

CRISPR-Inducedmutation

Subcloned coloniessequenced (#)

Estimated allelefrequency

CAPAN-1 CAGCAAG-GGAAAATCTGTCCAGGTATCAGATGC N/A 20 1.00CAPAN-1.B2.S

�CAGCAAG-GGAAAATCTGTCCAGGTATCAGATGC CAPAN-1(6174delT) 20 0.59CAGCAAG-GGAAAAT—————CAGGTATCAGATGC 5-bp Deletion 0.41

Dr�ean et al.





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Olaparib exposure induces Darwinian selection favoring secondary BRCA mutants in vitro. A, Experimental schematic. Secondary mutant and parentalcells were mixed at a 1:20 ratio and then exposed to DMSO, olaparib, or cisplatin. After drug exposure, populations were analyzed for surviving fraction andsecondary mutant:parental proportions using ddPCR. B, Bar graph illustrating the effect of drug exposure on population surviving fraction in eitherCAPAN1.B2.S� :CAPAN1 or SUM149.B1.S� :SUM149 cocultures. C, Bar graph illustrating the increase in secondary mutant clone frequency following 14 days of drugexposure. D, Graphs showing the frequency of CAPAN1B2�S cells in CAPAN1/CAPAN1B2� cocultures exposed to 500 nmol/L olaparib. Clone frequency wasestimated by ddPCR at the time points shown. Error bars represent SEM from three independent measurements.






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PARPi-induced selectivity operates in BRCA2 isogenic DLD1 tumor cells. A, Dose–response curves illustrating 6-well clonogenic survival data for BRCA2isogenic cells, DLD1.BRCA2WT/WT and DLD1.BRCA2–/–, exposed to olaparib over 14 days. Error bars represent SEM from triplicate experiments. B, Bar graphillustrating the increase in DLD1.BRCA2WT/WT frequency in a 1:100 starting DLD1.BRCA2WT/WT:DLD1.BRCA2–/– ratio coculture following 13 days of drugexposure. C, Bar graph showing starting ratio of DLD1.BRCA2–/– to DLD1.BRCA2WT/WT influences time (days) for DLD1.BRCA2WT/WT cells to reach clonal dominance(75% of cell population). D and E, Graphs showing time required for DLD1.BRCA2WT/WT-GFP to reach clonal dominance (75%, dotted line) when exposed toeither 25 nmol/L or 100 nmol/L of olaparib (D) or 10 nmol/L of talazoparib (E) in DLD1.BRCA2WT/WT:DLD1.BRCA2–/– cocultures.

Dr�ean et al.





frequency of the secondary mutant clone compared with DMSO-exposed cultures, with the fraction of CAPAN1.B2.S�cells in eachPARPi exposed culture gradually increasing over time (Fig. 2D).We also found that the initial frequency of the secondary mutantclone prior to drug treatment influenced the ability of the sec-ondarymutant clone to eventually dominate the population (i.e.,>75% of the cell population) once cells were exposed to PARP

inhibitor, as might be expected of a Darwinian process (Fig. 2D,compare 1:1, 1:20, and 1:40 ratio cultures).

We also used a different model system, isogenic DLD1 tumorcell lines with or without targeted mutations in BRCA2 (DLD1.BRCA2WT/WT and DLD1.BRCA2–/– (29, 30), to validate theseobservations. We labelled DLD1.BRCA2WT/WT cells with a GFPand DLD1.BRCA2–/– cells with a red fluorescent protein (RFP)

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PARPi-induced Darwinian selection of BRCA-proficient tumor cells in vivo.A, Experimental schematic of mixed CAPAN1:CAPAN1.B2.S� xenografts treated witholaparib. B, Bar chart illustrating CAPAN-1 (white) and CAPAN1.B2.S� (red) cell frequency in tumor xenografts prior to drug treatment. Values shown for eachanimal were derived from three tumor sections with mean � SEM shown. C, Bar graph illustrating the increase in secondary mutant clone frequency intumors following 28-day olaparib treatment (n ¼ 6, mean � SEM shown). P values were calculated by Student t test. D, Correlation between fold increase in thefrequency of the secondary mutant clone and the periodicity of olaparib administration.






Figure 5.

PARPi-resistant, secondary mutant clones and parental tumor cells are sensitive to AZD-1775 in vitro and in vivo. A, Waterfall plot comparing AUC valuescollated from a 5-day exposure to AZD-1775 from 146 cancer cell lines. B, Dose–response survival curves illustrating 6-well clonogenic survival data inCAPAN1, CAPAN1.B2.S� , SUM149, SUM149.B1.S�, MCF-10A, and MCF-12A cells exposed to AZD-1775. C, Bar graph illustrating (Continued on the following page.)

Dr�ean et al.





marker to enable detection and monitoring of coculture popula-tions via FACS (Supplementary Fig. S5A). We found that in theabsence of drug exposure, theDLD1.BRCA2WT/WT cells exhibited aselective advantage over DLD1.BRCA2–/–cells, as previouslyshown (ref. 29; Supplementary Fig. S5B), and that these cellsexhibit more than a 10-fold difference in olaparib sensitivity(Fig. 3A). We then mixed DLD1.BRCA2WT/WT cells into DLD1.BRCA2–/– cells in vitro at starting ratios of 1:1, 1:10, 1:100, and1:1,000, exposed these cocultures to either olaparib or talazo-parib, andmonitored the temporal evolutionof the population inresponse to PARPi (Supplementary Fig. S5A). Similar to theCAPAN1 and SUM149 isogenic models, we observed that ola-parib and talazoparib both selected for DLD1.BRCA2WT/WT cellsover DLD1.BRCA2–/– cells in a Darwinian fashion (Fig. 3B). Forexample, both olaparib and talazoparib exposure resulted in a3-fold increase in DLD1.BRCA2WT/WT cells compared with theDMSO-exposed cell population after 13 days of drug exposure(Fig. 3B). In addition, we noticed that the time taken for theDLD1.BRCA2WT/WT clone to reach clonal dominance was less incell populations that had higher starting proportion of DLD1.BRCA2WT/WT cells (Fig. 3C–E), as observed in the CAPAN1 cocul-ture model.

Darwinian selection of secondary mutant tumor cells alsooperates in vivo

Wealso assessedwhether aDarwinian process influenced the invivo response to PARPi treatment. To do this, we generated cohortsof mice bearing subcutaneous xenografts consisting of a mixtureof CAPAN1parental andCAPAN1.B2.S� secondarymutant tumorcells (Fig. 4A). We found that inoculating 5 � 106 tumor cells ata 1:1 CAPAN1:CAPAN1.B2.S� ratio reproducibly generated100mm3 xenografts 10 days after innoculation, where each clonewas present in equal proportion (Fig. 4B). When tumors reached100 mm3, tumor-bearing mice were randomized into the follow-ing treatment cohorts to assess the selective pressure of PARPitreatment in vivo: (i) olaparib (50mg/kg) administered once daily,(ii) olaparib (50 mg/kg) administered every other day, (iii)olaparib (50 mg/kg) administered twice a week on days 1 and4, (iv) drug vehicle administered daily. In addition, sentinel micewere sacrificed prior to treatment so that the CAPAN1:CAPAN1.B2.S� ratio in tumors prior to therapy could be confirmed (Fig. 4A;Supplementary Fig. S6A). We found that 50 mg/kg olaparibtreatment, administered daily, every other day, or twice weekly,though well-tolerated, did not decrease tumor growth comparedwith the vehicle (P > 0.05 ANOVA for tumor volume in eacholaparib treatment cohort versus vehicle; Supplementary Fig. S6Band S6C). We hypothesized that the absence of overall antitumorefficacy in this particular casemight be due to failure to inhibit thePARPi secondary mutant clone in xenografts. To test this, we

isolated tumor DNA from olaparib-treated mice (after 28-daytreatment) and assessed the relative ratio of parental versussecondary mutant clones by ddPCR. In mice that received drugvehicle alone, the ratio of parental versus secondary mutantclones remained approximately 50% (Supplementary Fig.S6A). However, in mice that received olaparib treatment, therelative frequency of CAPAN1.B2.S� cells increased in responseto therapy (Fig. 4C). This increase in CAPAN1.B2.S� frequency,in preference to the parental clone, was dependent upon theperiodicity of PARPi administration, for example, daily admin-istration of olaparib caused the greatest increase in CAPAN1.B2.S� enrichment, followed by every other day treatment andthen biweekly administration (Fig. 4C and D). This suggestedthat PARPi administration also selected for secondary mutanttumor cell clones in vivo and that the degree of secondarymutant clone selection was related to the extent of selectivepressure applied.

AZD-1775, a WEE1 kinase inhibitor, targets both parental andsecondary BRCA-mutant clones in vitro and in vivo

The coculture model systems described above allowed us toestablish that PARPi resistance, when driven by secondarymutations in BRCA1 or BRCA2, can operate along Darwinianprinciples. We also assessed whether we could identify thera-peutic vulnerabilities that would allow targeting of both paren-tal and secondary mutant tumor cell clones as a means tominimize the impact of secondary mutation. We assessedwhether small-molecule WEE1 cell-cycle checkpoint kinaseinhibitors (WEE1i; ref. 19) might have utility in this regard.We focused on WEE1 inhibitors for a number of reasons. WEE1prevents premature mitotic entry by phosphorylating and inhi-biting cyclin-dependent kinases such as cyclin dependentkinase 1 (CDK1; refs. 31, 32). This activity is particularly criticalin tumor cells with p53 pathway defects; p53 defects oftencooccur with BRCA mutations, and although secondary muta-tions in BRCA1/2 drive PARPi resistance, resistant tumors andcell lines remain p53 mutant (13). CAPAN1.B2.S� andSUM149.B1.S� clones retained the p53 mutations present inCAPAN1 and SUM149 parental tumor cell clones (Supplemen-tary Figs. S7 and S8). We also found that in an analysis of in vitrosensitivity to the clinical WEE1 kinase inhibitor, AZD-1775, in apanel of tumor cell lines, CAPAN1.B2.S� and SUM149.B1.S�

were among the most sensitive of 146 lines profiled (Fig. 5A).We confirmed this AZD-1775 sensitivity in subsequent clono-genic survival experiments and found that, when comparedwith nontumor breast epithelial cell lines (MCF10A andMCF12A), both CAPAN1 and SUM149-derived secondarymutant tumor cell clones retained profound sensitivity toAZD-1775 seen in parental tumor cells (average 22-fold

(Continued.) the increase in secondary mutant clone frequency following 14 days of drug exposure. D, Graph showing the frequency of CAPAN1B2�S cellsin CAPAN1/CAPAN1B2� cocultures exposed to AZD-1775. Clone frequency was estimated by ddPCR and the time points shown. Error bars represent SEMfrom three independent measurements. This experiment was conducted alongside the experiment described in Fig. 2D; to allow comparison, the responseto olaparib, and DMSO exposure from Fig. 2D is replotted here. E, Western blot analysis for CAPAN1 and CAPAN1.B2.S� cells lysates probed for pCDC2(Y15),g-H2AX (a DNA damage marker), and cleaved PARP1 (a marker of apoptosis). Tubulin was used as a loading control. F, Experimental schematic of mixedCAPAN1:CAPAN1.B2.S� xenografts treated with olaparib or AZD-1775. G, Bar chart illustrating CAPAN-1 (white) to CAPAN-1.B2.S� (red) clone ratio in intumour xenografts prior to drug treatment. Values shown from six sentinel animalswithmean� SEM shown.H, Tumor volumeplotted against length of treatment forindividual xenografts comprised of CAPAN1:CAPAN1.B2.S� mixed tumor cells over 150 days (n ¼ 18 total, n ¼ 6 in each cohort). I, Survival curves usingmaximum tumor size (1,500mm3) as a surrogate for survival from the experiment shown in E. J,Bar chart showing proportion of CAPAN1.B2.S� tumor cells followingtreatment from the experiment shown in E (n ¼ 6, mean � SEM). P values were calculated by Student t test.






difference in AUC, P < 0.0001 vs. MCF10A or MCF12A,ANOVA; Fig. 5B). We confirmed these observations usingcoculture systems and found that at SF50 concentrations (con-centration required to inhibit 50% of cells) of either olaparib orAZD-1775, olaparib exposure increased the relative frequencyof the secondary mutant clones, but AZD-1775 did not (Fig.5C). This observation was confirmed when we used ddPCR tomonitor the frequency of the secondary mutant clone over timein cocultures exposed to AZD-1775 (Fig. 5D). We also observedthat parental and secondary mutant SUM149 and CAPAN1clones were sensitive to additional small-molecule cell-cyclecheckpoint inhibitors including PF-477736, a CHK1 inhibitor(33, 34), and VX-970, an ATR inhibitor (35) when comparedwith nontumor epithelial cells (Supplementary Fig. S9A andS9B). This suggested that even when partial BRCA1 or BRCA2protein function was restored by secondary mutation, vulner-ability to small-molecule inhibitors that target cell-cycle check-points still existed. These effects did not appear to represent arelatively nonspecific sensitivity to cytotoxic agents in theparental and secondary mutant tumor cells, as these did notdisplay an overtly distinct level of sensitivity to paclitaxel,capecitabine, or gemcitabine when compared with MCF10 orMCF12A cells (Supplementary Fig. S9C–S9F).

Previous studies have shown thatWEE1 inhibitors cause tumorcell cytotoxicity by reducing the extent of CDC2 phosphorylationat Y15 (36). We found that in both CAPAN1 and CAPAN1.B2.S�

cells, AZD-1775 exposure caused a decrease in CDC2 Y15 phos-phorylation, an effect that was enhanced with prolonged drugexposure (Fig. 5E). We noted that AZD-1775 exposure caused anincrease inH2AXphosphorylation (gH2AX), a biomarker ofDNAdamage, in both CAPAN1 and secondary mutant CAPAN1.B2.S�

cells (Fig. 5E). This increase in gH2AXwas commensurate with anincrease in PARP cleavage, ameasure of apoptosis (Fig. 5E). UsingFACS profiling, we found that AZD-1775 exposure had a verysimilar effect on cell-cycle fractions in both CAPAN1 andCAPAN1.B2.S� cells, both of which demonstrated a profoundreduction in the fraction of cells in active S-phase, with a com-mensurate increase in the proportion of cells in nonreplicating Sphase (Supplementary Fig. S10). In CAPAN1 cells, AZD-1775exposure caused a reduction in the active S-phase fraction from25.9% to 3.4% (with a 3.9%–52.1% increase in nonreplicatingS-phase), while CAPAN1.B2.S� cells showed a reduction in activeS-phase from 27.8% to 4.2% (with a 3.3%–51.4% increase innonreplicating S-phase). These observations were reminiscent ofthose seen in H3K36me3-deficient cells, where WEE1 inhibitionalso caused a severe reduction in the active S-phase fraction (37).This suggested that WEE1 inhibition targeted CAPAN1 cells in S-phase, regardless of whether BRCA2 was dysfunctional (as inCAPAN1) or somewhat reconstituted by the presence of a sec-ondary BRCA2 mutation (as in CAPAN1.B2.S�).

To investigate whether WEE1 inhibitor sensitivity in PARPi-sensitive and resistant clones also operated in vivo, we assessed theeffect of AZD-1775 treatment on mice bearing mixed CAPAN1/CAPAN1.B2.S� xenografts (each clone present at a 1:1 ratio, Fig.5F). Mice with established tumors were treated with eitherAZD-1775, olaparib, or drug vehicle. Sentinel mice sacrificedprior to treatment showed the CAPAN1:CAPAN1.B2.S� ratio intumors prior to therapy was 1:1 (Fig. 5G). We used the time takenfor tumors to reach 1,500 mm3 as a surrogate measure of survival(Fig. 5H) and found that while olaparib treatment had minimalbenefit (P ¼ 0.86, log-rank Mantel–Cox test compared with

vehicle), AZD-1775 treatment led to a significant survival benefit(P ¼ 0.011, log-rank Mantel–Cox test compared witholaparib; Fig. 5I). Consistent with these observations, ddPCRanalysis of tumors at the end of treatment showed that olaparibtherapy caused a relative enrichment in the frequency of thesecondary mutant clone (P ¼ 0.058 compared with vehicle,Student t test) while AZD-1775 did not (P ¼ 0.43, comparedwith vehicle, Student t test; Fig. 5J).

DiscussionIn this study, we used CRISPR-generated BRCA1 or BRCA2

secondary mutant daughter clones alongside isogenic parentalcell lines todemonstrate that PARPi exposure selects for secondarymutant clones in aDarwinianmanner, both in vitro and in vivo.Wefound that the extent of selection for secondarymutant cloneswasinfluenced by the frequency of drug administration. In micebearing tumors comprised of an equal proportion of BRCA2-mutant and BRCA2 secondary mutant tumor cells, olaparib hadminimal effects on tumor growth, but did preferentially select forthe secondarymutant daughter clone over the parental tumor cell.It would be reasonable to infer that high frequencies of secondarymutant cells hinder the therapeutic effectiveness of PARP inhibi-tors. We also found that a WEE1 inhibitor, AZD-1775, had agreater therapeutic effect on mixed parental/secondary mutanttumors than olaparib. This example suggests that therapeuticvulnerabilities might still exist in tumors that have a high fre-quency of secondary mutant clones. Our data also suggest thatsecondary mutant and parental tumor cells also show sensitivityto other cell cycle/DNAdamage repair inhibitors, includingCHK1and ATR inhibitors (Supplementary Fig. S9). It seems possiblethat while secondary BRCA1 or BRCA2 gene mutations restoresomeHR function, these are unlikely to reverse the complex set ofgenomic rearrangements, aneuploidy, and p53 mutations foundin BRCA1 or BRCA2 mutant tumors prior to treatment (38). Wehypothesize that these latter characteristics sensitize tumor cells todrugs such as WEE1 inhibitors, perhaps explaining why AZD-1775 targets both parental and secondary mutant clones. Thishypothesis remains to be tested, but the observation that second-ary mutant tumor cells are sensitive to AZD-1775 raises thepossibility that therapeutic vulnerabilities still exist in PARPi-resistant tumors.

In clinical studies, the MTD for single-agent AZD-1775 wasidentified as 225mg twice per dayorally over 2.5days perweek for2 weeks per 21-day cycle, a dosing regimen sufficient to elicit anumber of antitumor responses (39). In our in vivo studies (Fig. 5)we used 30mg/kg AZD-1775 twice-daily treatments for the entireduration of the study (150 days). This treatment approach waswell tolerated in mice and based on prior mouse-based experi-ments using this WEE1 inhibitor (40). Nevertheless, it is possiblethat a similar constant dosing approachmay not bewell-toleratedin humans. Subsequent work might assess the potential of usingintermittent WEE1 inhibitor dosing schedules to assess whetherthese also elicit a survival benefit in experiments similar to thoseshown in Fig. 5.

One implication of this work is that the detection of secondaryBRCA1 or BRCA2 mutations in patients could be important ininfluencing the choice of therapy. At present, secondarymutationsin BRCA1 or BRCA2 can be detected by Sanger DNA sequencing(14–16) or by targeted DNA capture and deep sequencing (13).Circulating tumor DNA and circulating tumor cells might also


Dr�ean et al.




display some of the secondary BRCA1 or BRCA2mutations foundin solid tumors. Detecting secondary mutations in such liquidbiopsies might allow the early emergence of secondarymutationsto be identified as a biomarker predicting the eventual clinicalmanifestation of PARPi resistance. One avenue we will nowexplore is to utilize the in vivo system we have described here toassess this possibility. A key quality of the model systemsdescribed here is that they allow the construction of coculturesand xenografts where the frequency and identity of secondarymutants is known. This will hopefully facilitate experiments thataim to examine further principles that govern clonal evolutionand influence drug resistance in BRCA1- or BRCA2-mutant can-cers. Alongside these models, we also note that the first PDX withPARPi resistance-causing mutations have been recently described(41). These provide another system in which to assess how theclonal structure of tumors evolve in response to therapy. Thecombined use of engineered systems, such as that described here,alongside PDX systems will be critical in establishing what factorsdetermine the response to treatment, and importantly, whattherapeutic approaches could be taken to minimize the impactof secondary BRCA1/2 gene mutations.

Disclosure of Potential Conflicts of InterestN.C. Turner reports receiving commercial research support fromAstraZeneca

and is a consultant/advisory board member for AstraZeneca. A. Ashworthprovided expert testimony for AstraZeneca. C.J. Lord has received speakersbureau honoraria from AstraZeneca and Vertex and is a consultant/advisoryboardmember for Vertex and SunPharma. A. Ashworth andC.J. Lord are namedinventors on patents describing the use of PARP inhibitors and stand to gainfrom their use as part of the ICR "Rewards to Inventors" scheme. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: A. Dr�ean, C.T. Williamson, A. Ashworth, C.J. LordDevelopment of methodology: A. Dr�ean, C.T. Williamson, I. Garcia-Murillas,C.J. LordAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Dr�ean, R. Brough, I. Brandsma, M. Menon,A. Konde, H.N. Pemberton, J. Frankum, N. Badham, C.J. LordAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Dr�ean, C.T. Williamson, A. Konde, I. Garcia-Murillas, H.N. Pemberton, J. Campbell, A. Gulati, N.C. Turner, C.J. LordWriting, review, and/or revisionof themanuscript:A.Dr�ean, C.T.Williamson,N.C. Turner, S.J. Pettitt, A. Ashworth, C.J. LordAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Menon, A. Konde, R. Rafiq, C.J. LordStudy supervision: C.T. Williamson, A. Ashworth, C.J. Lord

AcknowledgmentsThis work was funded by Breast Cancer Now and Cancer Research UK as

part of Programme Grants (to C.J. Lord). We thank the Tumour ProfilingUnit (TPU) at the Institute of Cancer Research for carrying out DNA sequenceanalysis. We acknowledge NIHR funding to the Royal Marsden BiomedicalResearch Centre.

Grant SupportThis work was supported by Breast Cancer Now (CTR-Q4-Y2; to C.J. Lord)

and Cancer Research UK (CRUK/A14276; to C.J. Lord).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 30, 2017; revised May 2, 2017; accepted June 5, 2017;published OnlineFirst June 15, 2017.

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2017;16:2022-2034. Published OnlineFirst June 15, 2017.Mol Cancer Ther Amy Dréan, Chris T. Williamson, Rachel Brough, et al.

-Mutant CancersBRCA1/2Modeling Therapy Resistance in

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