ATRPathwayInhibitionIsSyntheticallyLethalinCancerCells...

Therapeutics, Targets, and Chemical Biology

ATRPathway Inhibition Is Synthetically Lethal in CancerCellswith ERCC1 Deficiency

Kareem N. Mohni, Gina M. Kavanaugh, and David Cortez

AbstractThe DNA damage response kinase ATR and its effector kinase CHEK1 are required for cancer cells to survive

oncogene-induced replication stress. ATR inhibitors exhibit synthetic lethal interactions, with deficiencies in theDNA damage response enzymes ATM and XRCC1 and with overexpression of the cell cycle kinase cyclin E. Here,we report a systematic screen to identify synthetic lethal interactions with ATR pathway–targeted drugs,rationalized by their predicted therapeutic utility in the oncology clinic. We found that reduced function in theATR pathway itself provided the strongest synthetic lethal interaction. In addition, we found that loss of thestructure-specific endonuclease ERCC1-XPF (ERCC4) is synthetic lethal with ATR pathway inhibitors. ERCC1-deficient cells exhibited elevated levels of DNA damage, which was increased further by ATR inhibition. Whentreated with ATR or CHEK1 inhibitors, ERCC1-deficient cells were arrested in S-phase and failed to completecell-cycle transit even after drug removal. Notably, triple-negative breast cancer cells and non–small cell lungcancer cells depleted of ERCC1 exhibited increased sensitivity to ATR pathway–targeted drugs. Overall, weconcluded that ATR pathway–targeted drugs may offer particular utility in cancers with reduced ATR pathwayfunction or reduced levels of ERCC4 activity. Cancer Res; 74(10); 2835–45. �2014 AACR.

IntroductionReplicating DNA is sensitive to a wide array of endogenous

and exogenous damaging agents, which can lead to replicationfork stalling. To accomplish error-free replication, stalledreplication forks need to be stabilized to prevent their collapseinto double strand breaks (DSB), which can lead to genomicrearrangements and/or apoptosis (1, 2). The DNA damageresponse kinase ATR coordinates many of the activities atstalled forks, including fork stabilization and restart (3). ATRactivation also slows the cell cycle to allow for DNA repairthrough the phosphorylation of CHK1 (3).The loss of one or more DNA damage response or repair

pathways drives tumorigenesis but also forces cancer cells tobe more reliant on other pathways, providing an opportunityfor the development of targeted therapeutics (4). For exam-ple, cancers with mutations in BRCA1/2 are sensitive toPARP inhibitors as the replication-associated DSBs causedby PARP inhibition cannot be repaired when the BRCA-dependent homologous recombination system is nonfunc-tional (5, 6). PARP inhibitors can also trap PARP on DNAcreating a toxic intermediate that requires a second DNA

repair pathway such as homologous recombination or post-replicative repair to remove the PARP–DNA complexes (7).The synthetic lethality between PARP inhibitors and muta-tions in BRCA1/2 provides a paradigm for combining DNArepair inhibitors with specific mutations or in combinationwith chemotherapy agents.

Oncogene activation often initiates an ATR-dependent rep-lication stress response that is needed for continued cellgrowth (8–10). Thus, ATR pathway inhibitors are being devel-oped as cancer therapeutics. For example, CHK1 inhibitorshave shown promise in preclinical models, and there areseveral ongoing and completed phase I and II clinical trials(11–13). Recently, specific ATR inhibitors have been describedby AstraZeneca (14), Vertex Pharmaceuticals (15, 16), and theFernandez-Capetillo laboratories (17). These inhibitors func-tion to inhibit the growth of cancer cell lines in vitro andsynergize with DNA-damaging agents such as cisplatin (15).Furthermore, ATR inhibition demonstrated efficacy in a xeno-graft mouse model of pancreatic cancer in combination withgemcitabine (18).

ATR inhibitors also exhibit synthetic lethal interactions withATM and XRCC1 deficiency as well as with cyclin E over-expression (15, 17, 19). To date, no systematic approach toidentify synthetic lethal interactions with ATR pathway–tar-geted drugs has been reported. This information could be usedin the clinic to design better clinical trials with ATR pathway–targeted drugs and improve patient outcomes.

The current study was initiated to identify genes that, whenlost, exhibit synthetic lethal relationships with ATR pathwayinhibitors. We conducted a synthetic lethal screen with DNArepair proteins and identified reduced ATR pathway functionand the ERCC1-XPF nuclease as synthetic lethal with ATR or

Authors' Affiliation: Department of Biochemistry, Vanderbilt UniversitySchool of Medicine, Nashville, Tennessee

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

Corresponding Author: David Cortez, Department of Biochemistry, Van-derbilt University School of Medicine, Nashville, TN 37232. Phone: 615-322-8547; Fax: 615-343-0704; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-3229

�2014 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 2835

on July 1, 2018. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 24, 2014; DOI: 10.1158/0008-5472.CAN-13-3229

http://cancerres.aacrjournals.org/

CHK1 inhibition. ERCC1-XPF functions in the repair of bulkyDNA adducts, double strand breaks, interstrand crosslinks(ICL), and separation of sister chromatids at fragile sites(20, 21). Importantly, ERCC1 is being explored as a potentialbiomarker in lung and other kinds of cancer (22–25). Low levelsof ERCC1 expression correlate with greater sensitivity tocisplatin and higher 5-year survival rates. Several phase IIclinical trials are also underway using ERCC1 protein levelsto determine whether to treat patients with platinum-basedchemotherapy (22, 23). Our data demonstrate that both triple-negative breast cancer and non–small cell lung cancer cell linesdepleted of ERCC1 exhibit increased sensitivity to ATR path-way inhibition. Thus, ERCC1 statusmay be a useful indicator ofsensitivity to ATR pathway–targeted drugs.

Materials and MethodsCells and reagents

U2OS and 293T cells were obtained from American TypeCulture Collection (ATCC) and maintained in Dulbecco'sModified Eagle's Media (DMEM) supplemented with 7.5% FBS.Triple-negative breast cancer cells lines BT549 and HCC1806and non–small cell lung cancer cells lines A549 and H157 wereobtained from ATCC and maintained in RPMI supplementedwith 10% FBS. The HCT-116–derived ATR flox/þ and ATRflox/� were previously described (26). XPF-deficient fibro-blasts XP2YO and XP2YO þ XPF were provided by OrlandoScharer in August 2013 and maintained as described (27). TheERCC1-null (clone 216) and complemented A549 cells (clone216þ 202) were provided by Jean Charles Soria, Ken Olaussen,

and Luc Friboulet in December 2013 and maintained asdescribed (25). All cells lines were thawed from early-passagestocks and were passaged less than 30 times before use. Nofurther cell line authentication was performed. The ATRinhibitor VE-821 (15) was synthesized by the Vanderbilt Insti-tute for Chemical Biology Chemical Synthesis Facility. TheCHK1 inhibitor AZD7762 was previously described (28).

Synthetic lethal siRNA screenThe custom siRNA library targets 240 known DNA repli-

cation and DNA repair genes with 4 unique siRNAs per genein individual wells. The siRNA was plated into 96-well platesin replicates and frozen at —80�C. The plates were thawedand siRNA was resuspended in OptiMEM with Dharmafect1.U2OS cells were then added to each plate. The final siRNAconcentration was 10 nmol/L. Cells were split into two 96-well dishes 72 hours after transfection and incubated inmedia with or without ATR inhibitor at a final concentrationof 1 mmol/L for an additional 72 hours. Cell viability wasmeasured with alamar blue (Invitrogen). Nonfluorescentcell-permeable alamar blue dye is reduced to a moleculethat fluoresces red in metabolically active cells, which allowsfor a quantitative measure of viability. The percent viabilityof ATR inhibitor treated to untreated was determined foreach siRNA to take into account siRNA-specific effects oncell growth. The z-scores were calculated using the meanand SD of the log10(percent viability) values. The valuespresented in Fig. 1 are the mean z-scores from three inde-pendent transfections.

100

20

40

60

80

0

Perc

ent via

bili

ty

Untreated ATRi

siNTsiATR

2

1

−1

−2

−3

−4

−5

−6

−7

0

ATR

ATRIP

ERCC1

Mean Z-score

A

TR

i/untr

eate

d Z

-score

Non targeting

XPF

ClaspinRPA

HUS1Chk1

ATM

XPAXPC

96-well dish

Transfect cells

with siRNA

n = 3

Untreated +ATRi

Measure cell

viability

A B C

72 h

XRCC1

XPDXPB

100

20

40

60

80

120

0

E HCT116

ATR flox/+

ATR flox/−

ATR

CHK1

Pe

rce

nt

of

co

ntr

ol

HC

T116

ATR

flox

/−

ATR

flox

/+

100

20

40

60

80

120

0

ATRi concentration (μmol/L) ATRi concentration (μmol/L)

0.1 1 10 0.1 1 10

D shGFP

shATR

shCHK1

Pe

rce

nt

of

co

ntr

ol

shG

FP

shC

HK1

shAT

R

ATR

CHK1

Ku70

Figure 1. siRNA screen identifies synthetic lethal interactions with ATR inhibition. A, schematic of the siRNA screen. U2OS cells were transfected with siRNAsand then were left untreated or treated with 1 mmol/L ATRi. Cell viability was determined with alamar blue. B, percent viability of the nontargeting (NT) andATR siRNA controls from the screen. Values represent the mean � SD of three independent replicates. C, the z-scores of treated compared with untreatedcell viability for each gene is shown. Each data point represents the mean of three independent replicates and calculation of the z-scores is described inMaterials and Methods. D, U2OS cells were infected with lentiviruses expressing the indicated shRNA and selected with puromycin and then treated withincreasing doses of ATRi for 72 hours. E, HCT-116 wild-type, ATR flox/þ, and ATR flox/� cells were treated with increasing doses of ATRi for 72 hours.For D and E, cell viability was determined with alamar blue and reported as a percent of the untreated control cells.

Mohni et al.

Cancer Res; 74(10) May 15, 2014 Cancer Research2836




RNA interference transfection and sequencesAll siRNA transfections were done with 10 nmol/L siRNA

and Dharmafect 1 (Invitrogen). Cells were transfected withindividual siRNAs 72 hours before the start of an experiment.The following siRNA sequences were used: siERCC1-1 GAGAA-GAUCUGGCCUUAUG, siERCC1-2 CGACGUAAUUCCCGA-CUAU, siERCC1–4 CAGCGGACCUCCUGAUGGA, siXPF-2GUAGGAUACUUGUGGUUGA, siXPF-3 ACAAGACAAUCCGC-CAUUA, siXPC-2GGAGGGCGAUGAAACGUUU, siXPC-3GAG-GUGGACUCUCUUCUGA, siXPA-1 GAAAGACUGUGAUUUA-GAA, siXPA-2 GCCAUGAACUGACAUAUGA. The nontargetingsiRNA was Qiagen All Star Negative control. Constructionand use of lentiviruses expressing short hairpin RNA(shRNA) targeting GFP, ATR, and CHK1 has been previouslydescribed (29).

Cell viability assaysCells were plated in 96-well plates 72 hours after siRNA

transfection, the ATR or CHK1 inhibitor was added to themedia for an additional 72 hours and cell viability was mea-sured with alamar blue. Values represent themean (n¼ 6) anderror bars represent SD. For statistical analysis, normalizeddata were fitted to nonlinear regression curves and used todetermine IC50 values. These IC50 values were compared usingan F test in Prism version 6.

Western blot analysisWestern blot analysis was performed as previously

described (30). Primary antibodies ERCC1 (D-10) and PCNA(FL-261) were purchased from Santa Cruz; H2B (Ab1790) andKu70 (Ab3114) were purchased from Abcam; XPF (Bethyl LabsA301–315); XPC (Novus NB100-477); XPA (NeoMarkers MS-650-P0); cleaved PARP (Cell Signaling 9541); and GAPDH(Millipore MAB374).

Immunofluorescence analysisImmunofluorescence analysis was performed and quanti-

fied as previously described (30). The gH2AX (JBW301) anti-body was purchased from Millipore. Nuclei were stained with40,6-diamidino-2-phenylindole (DAPI).

Cell-cycle analysis and isolation of proteins on nascentDNAPropidium iodide (PI) staining and isolation of proteins on

nascent DNA (iPOND) were performed as previously described(30–32).

ResultsIdentification of synthetic lethal interactions with ATRinhibitionWe performed an siRNA synthetic lethal screen with an

ATR inhibitor (ATRi) to determine cancer contexts thatmight provide a therapeutic window for ATR pathway drugs.As ATR is required for the cellular response to many forms ofDNA damage during S-phase, we reasoned that ATR pathwayinhibition might be most useful in cancer cells lacking othergenome maintenance pathways. Therefore, we generated acustom library targeting 240 DNA replication and repair

genes with 4 individual siRNAs per gene. The library wastransfected into U2OS cells and then cells were left eitheruntreated or treated with ATRi for 72 hours, and cell viabilitywas measured with alamar blue dye (Fig. 1A). Cell viability ofeach ATRi-treated sample was scored and compared withthe same untreated siRNA to control for any siRNA-specificeffects. The dose of ATRi was optimized such that it had onlyminimal cell killing on cells expressing a control siRNA andhad maximal killing of cells depleted of ATR (Fig. 1B). Lowerdoses of ATRi are sufficient to kill cells depleted of ATRpresumably because less ATRi is needed to completelyinhibit all of the remaining ATR protein. This result alsosuggests that the ATR inhibitor does not work by trappingan inactive protein at the site of damage like PARP inhibi-tors. The percent viability was calculated for all genes in thelibrary and converted to a z-score as described in Materialsand Methods. Smaller z-scores indicate more confidentsynthetic lethal relationships to ATRi.

Genes in the ATR pathway were the largest family of DNArepair proteins to yield strong synthetic lethal relationshipswith ATR inhibition (Fig. 1C and Supplementary Table S1).While not a comprehensive library, every known ATR pathwaygene in the library was synthetic lethal (at least 3 of 4 siRNAs)with ATRi, including ATR, ATRIP, RPA, CHEK1, CLSPN, HUS1,RAD1, RAD17, TIMELESS, and TIPIN. ATR pathway genes aremutated or deleted in up to 25% of some cancer types (33),suggesting that reduced functionality of the ATRpathway itselfcould provide a therapeutic window for ATR-targeted thera-pies. To confirm that reduced expression of ATR pathwaygenes is synthetic lethal with ATRi, we depleted cells of ATR orCHK1 using shRNA and performed dose–response curves withATRi. Silencing of both ATR and CHK1 sensitized cells to ATRinhibition as compared with a control shRNA (Fig. 1D). Finally,we confirmed that even heterozygous mutation in ATR issufficient to hypersensitize cancer cells to ATR inhibitors usingHCT116 cells lacking one ATR allele (Fig. 1E; ref. 26). Theknown synthetic lethal interactions with ATM and XRCC1were also identified in the screen further validating the screen-ing procedure.

ERCC1-XPF–deficient cells are sensitive to ATR pathwayinhibition

One of the high scoring genes identified in the syntheticlethal screen was ERCC1. As described in the Introduction,ERCC1 has recently been suggested as an important bio-marker in lung and other kinds of cancer (22). To validateERCC1, and its binding partner XPF, deficiencies as synthet-ic lethal with ATR inhibition, we knocked down ERCC1 andXPF with 2 individual siRNAs and performed dose–responsecurves with ATRi. Both siRNAs for each gene sensitized cellsto ATRi by as much as one order of magnitude (Fig. 2A andB). To test whether the synthetic lethality was specific toATR or more generalizable to the ATR pathway, we alsotreated knockdown cells with a CHK1 inhibitor (CHK1i).Both ERCC1 and XPF knockdown also sensitized cells toCHK1i, suggesting that inhibition of the ATR pathway andnot only ATR is synthetic lethal in ERCC1-XPF–deficientcells. ERCC1 knockdown always yielded greater synthetic

ATR Inhibition Is Synthetic Lethal with Loss of ERCC1

www.aacrjournals.org Cancer Res; 74(10) May 15, 2014 2837




lethality with ATRi than XPF, although knockdown of eithersignificantly reduced the IC50 values of ATRi and CHK1i ascompared with control cells. This difference may derive fromthe differences in viability caused by the ERCC1 or XPFknockdown on their own. In all siRNA experiments pre-

sented, knockdown of XPF by itself reduces the viability ofcells more than knockdown of ERCC1. For example, in U2OScells, knockdown of ERCC1 or XPF results in 80% and 60%viability, respectively. Thus, it is harder to observe largelosses in viability with the addition of ATR pathway

100

20

40

60

80

120

0Perc

ent of contr

ol

ATRi concentration (μmol/L)0.1 1 10

A

B

D

siNT

siERCC1-1

siERCC1-2

ERCC1

XPF

GAPDH

XPC

1 2 2 23 3

siERCC1

siXPF

siXPC

siNT

XP+XPF

ERCC1-null+ERCC1

ERCC1null

+ERCC1

GAPDH

XPF

ERCC1

XP +XPF

GAPDH

XPF

XPA

GAPDH

100

20

40

60

80

120

0

CHK1i concentration (μmol/L)

0.01 0.1 1

100

20

40

60

80

120

0Perc

ent of contr

ol

ATRi concentration (μmol/L)



0.1 1 10

siNT

siXPF-2

siXPF-3

100

20

40

60

80

120

0




0.01 0.1 1

100

20

40

60

80

120

0Perc

ent of contr

ol

0.1 1 10

siNT

siXPC-2

siXPC-3

100

20

40

60

80

120

00.01 0.1 1

100

20

40

6080

140

0Perc

ent of contr

ol

Perc

ent of contr

ol

Perc

ent of contr

ol

Perc

ent of contr

ol

Perc

ent of contr

ol

Perc

ent of contr

ol

Perc

ent of contr

ol

Perc

ent of contr

ol

0.1 1 10

siNT

siXPA-1

siXPA-2100

20

40

6080

140

00.01 0.1 1

120 120

NT A-1 A-2

100

20

40

60

80

0

100

20

40

60

80

0

ATRi CHK1i CisE H

G

F

siNTsiERCC1-1

siXPF-2

siXPC-3

siXPA-2

120

ATRi CHK1i Cis

100

20

40

60

80

0

C120

ATRi CHK1i Cis

***

*

**

**

**

**

**

Figure 2. ATR pathway inhibition is synthetic lethal with ERCC1/XPF deficiency. A–F, U2OS cells were transfected with the indicated siRNAs and then treatedwith the ATR or CHK1 inhibitor. NT represents the nontargeting siRNA and the numbers after specific siRNAs refer to the sequences described in Materialsand Methods. Cell viability was determined with alamar blue and reported as a percent of the untreated control cells for ERCC1 (A), XPF (B), XPC (D),and XPA (E). All knockdowns were done simultaneously and separated into individual graphs for ease in comparison, and the control siRNA values areidentical in each graph. Knockdown of ERCC1 or XPF yielded significant reductions in IC50 values as compared with control cells (P < 0.001) whereasknockdown of XPC or XPA did not. Statistics are described in Materials and Methods. C, U2OS cells transfected with the indicated siRNAs were treatedwith 1 mmol/L ATRI, 0.05 mmol/L CHK1i, or 1 mmol/L cisplatin for 24 hours, and surviving colonies were scored 14 days later. �, P < 0.001, as comparedwith the control siRNA for each treatment. E and F, Western blot analyses of the cells used in A–E. G and H, XPF-deficient patient fibroblast (XP) and cellscomplemented with XPF (þXPF; G) and ERCC1-null A549 cells and cells complemented with ERCC1 (þERCC1; H) were either left untreated or treated with 1mmol/L ATRI, 0.05 mmol/L CHK1i, or 1 mmol/L cisplatin for 24 hours and surviving colonies were scored 14 days later. �, P < 0.001, as comparedwith the complemented cell line for each treatment. Western blot analyses show ERCC1 and XPF levels in the null and complemented cell lines.

Mohni et al.





inhibitors on top of the lethality caused by XPF knockdownalone. To control for this, we tested synthetic lethality bycolony-forming assays, which are not influenced by differentrates of cell growth. We observe that both ERCC1 and XPFknockdown significantly sensitize cells to ATRi and CHK1itreatment (Fig. 2C). As expected, knockdown of ERCC1 andXPF also sensitized cells to cisplatin.As ERCC1-XPF regulates sensitivity to cisplatin via the

nucleotide excision repair (NER) and ICL repair pathways,we wished to determine whether loss of NER sensitized cellsto ATR pathway inhibition. Neither the core NER genes XPCand XPA nor the TFIIH components XPB and XPD weresynthetic lethal with ATRi in our screen (Fig. 1C). Whentested individually, XPC and XPA depletion sensitized cellsto cisplatin but did not sensitize cells to ATRi or CHK1i ineither short-term growth/viability or colony-forming assays(Fig. 2C–E) validating the screen results and suggesting that

NER deficiency does not sensitize cells to ATR pathwayinhibition. Protein knockdown for all siRNAs was confirmedby Western blotting (Fig. 2E and F). As expected, knockdownof ERCC1 also knocked down XPF and vice versa with siRNAtargeting ERCC1, more efficiently reducing the levels of bothproteins as compared with siRNA targeting XPF (34, 35). Oneof the siRNAs targeting ERCC1 also mildly reduced the levelof XPC, but as the other siRNA did not knockdown XPC andas XPC knockdown had no effect on synthetic lethality, thispotential off-target effect cannot explain the observed syn-thetic lethality. The dose–response curves are presented aspercent of untreated controls and values above 100% for lowdoses of ATRi and CHK1i are likely due to increased rates ofcell growth associated with partial ATR pathway inhibition.

To further confirm the specificity of the synthetic lethalitywith ERCC1/XPF, we tested it in more well-defined geneticsystems to eliminate the possibility of any off-target effects

1

10

100

ATRi − + − +

siNT + ATRi

siERCC1-1siNT

siERCC1-1 + ATRi

γH2A

X inte

nsity

(Arb

itra

ry u

nits)

γH2AX γH2AXDAPI DAPI

100

20

40

60

80

0

positiv

e c

ells

C

1 2 2 23 3

NT ERCC1 XPF XPC

100

20

40

60

80

0

positiv

e c

ells

D

A B

NT ERCC1 XPF XPC

Perc

ent

γH2A

X

Perc

ent

γH2A

X

No Foci Foci Pan-nuclear

No Treatment ATRi


Foci

Pan-

nuclear

* * * * * *

100

20

40

60

80

0

Perc

ent

γH2A

X

positiv

e c

ells

E

1 2 2 23 3

NT ERCC1 XPF XPC

CHK1i


* *

1 2 2 3 2 3

Figure 3. ERCC1/XPF knockdowncauses gH2AX elevation, which isfurther increased in the presence ofATR and CHK1 inhibitors. U2OScells were transfected with theindicated siRNAs and then treatedwith 1 mmol/L ATRi or 0.05 mmol/LCHK1i for 24 hours. Cells were thenfixed and stained for gH2AX. A,representative images ofnontargeting (NT) or ERCC1siRNA-transfected cells treated with theATR inhibitor. Both cells withgH2AX foci and pan-nuclear gH2AXare indicated with arrows. B,quantification of gH2AX intensity ofcells shown in A. C–E, thepercentage of gH2AX in foci(>10 foci per cell) or in a pan-nuclearstaining pattern was scored foruntreated (C), ATRi-treated (D), andCHK1i-treated (E) cells. At least 200cellswere scored for eachconditionbetween two independentexperiments. �, P < 0.05 for eachsiRNA sample compared with thecontrol cells for each condition.






of siRNA knockdowns. XPF-mutant patient cells were moresensitive to ATRi, CHK1i, and cisplatin as compared with thecomplemented cells (Fig. 2G). In addition, A549 lung cancercells in which ERCC1 was specifically disrupted were moresensitive to ATRi, CHK1i, and cisplatin as compared with thecomplemented cells (Fig. 2H). These 2 isogenic cell lines withdeficiencies in XPF or ERCC1 further validate the specificityof this genetic interaction and demonstrate that loss ofERCC1 and XPF are required for the observed syntheticlethality.

ERCC1 deficiency causes elevated DNA damage that isfurther increased when the ATR pathway is inhibited

In a previous screen, knockdown of ERCC1 and XPF bothcaused significant increases in the amount of gH2AX sug-gesting the induction of DNA damage in their absence (36).We confirmed this observation by immunofluorescence ofcontrol and ERCC1-depleted cells either untreated or treatedwith ATRi. The dose of ATRi used does not cause a notice-able increase in the amount of gH2AX in control cells;however, ERCC1 knockdown causes a significant increasein the amount of gH2AX in untreated cells, which is furtherincreased in ATRi-treated cells (Fig. 3A and B). Of note, in

untreated cells, ERCC1 knockdown results in gH2AX foci,but when ATRi is added, the staining changes to a combi-nation of foci and pan-nuclear gH2AX, with the pan-nuclearstaining being much brighter than the staining of gH2AX infoci (Fig. 3A). Pan-nuclear gH2AX is often associated withhigh levels of replication stress (30, 37).

To further quantify this data, we scored the percentages ofcells with no gH2AX, gH2AX in foci, and gH2AX in a pan-nuclear staining pattern in untreated, ATRi, and CHK1i-treated cells. ERCC1 and XPF knockdown both caused astatistically significant increase in the amount of gH2AX fociin untreated cells (Fig. 3C). The amount of these foci wasincreased, as where the percentage of cells exhibiting pan-nuclear gH2AX with the addition of ATRi (Fig. 3D). Asexpected, CHK1i treatment of control cells caused a largeincrease in the amount of gH2AX foci and a small percent ofcells with pan-nuclear gH2AX (Fig. 3E) (38). ERCC1 knock-down further increased these phenotypes with as many as50% to 70% of cells exhibiting pan-nuclear gH2AX (Fig. 3E).As observed previously, the increases in gH2AX were largerin ERCC1-depleted cells than in XPF-depleted cells, likelydue to the slower growth rate of XPF-depleted cells. XPCknockdown exhibited the same phenotypes as control

100

20

40

60

80

−− ATRi

siERCC1-1siNT

S G2–M

ATRi CHK1iCHK1i

G1Sub G1siERCC1-1siNT

Mock

AT

Ri

CH

K1i

DNA content

B C

siRNA

knockdown

ATRi or CHK1i

treatment (5 h)

Release into

Nocodazole (18 h)

A

Perc

ent of cells

in

phases o

f cell

cyle

0

D

ERCC1

PCNA

H2B

Thd Chase (1 h)ATRi (2 h)HU (2 h)

0.1% Input Click reaction

−−

−−−

−−−

−−− −

− −− −

No c

lick

No c

lick

E

1 8 9 10765432

0

ATRi

siNT siERCC1-1

Cleaved

PARP

GAPDH

ERCC1

24 48 Cis 0 24 48 Cis

ATRi

+

+

+

++ +

++

Figure 4. ERCC1 knockdowncauses an S-phase arrest in ATRi-and CHK1i-treated cells. U2OScells were transfected with theindicated siRNAs, treated with 1mmol/L ATRi or 0.05 mmol/L CHK1ifor 5 hours, and released intonocodazole for 18hours. Cellswerethen fixed and stained with PI andanalyzed by flow cytometry. A,diagram of experimental design.B, histograms of DNA content. C,quantification of the percent of cellsin each phase of the cell cycle.At least 10,000 live cells wereanalyzed for each condition. D,U2OS cells transfected with theindicated siRNAs were treated with1 mmol/L ATRi for the indicatednumber of hours or treated with1 mmol/L cisplatin for 48 hours andWestern blot analysis was done forcleaved PARP, ERCC1, andGAPDH. E, 293T cells labeled for10 minutes with EdU were chasedinto low-dose thymidine, HU, or HUand ATRi as described in Materialsand Methods. Western blotanalyses were done for ERCC1,PCNA, and H2B.

Mohni et al.





knockdown cells, further confirming our observation thatthe synthetic lethality is independent of NER. Together,these experiments suggest that the ATR pathway is neededto repair DNA damage caused by loss of ERCC1-XPF.

The ATR pathway is required for S-phase progression inERCC1-deficient cellsTo determine the mechanism for synthetic lethality with

ATRi in ERCC1-deficient cells, we knocked down ERCC1,treated cells with either ATRi or CHK1i, and analyzed theirprogression through the cell cycle. Control cells left untreat-ed or treated with either inhibitor completed DNA replica-tion and proceeded to M phase of the cell cycle where theyarrested at the mitotic checkpoint in the presence of noco-dazole (Fig. 4A and B). Conversely, ERCC1-deficient cellstreated with ATRi accumulated in S-phase and did notcomplete DNA synthesis (Fig. 4B). The ERCC1-deficient cellpopulation exhibits a large enrichment in S-phase and areduction in cells with 4N DNA content (Fig. 4C). Further-more, ATRi-treated, ERCC1-deficient cells exhibit higherlevels of cleaved PARP at both 24 and 48 hours after additionof ATRi as compared with control ATRi-treated control cells(Fig. 4D). This is also consistent with the increase in cellswith less than 2N DNA content in ERCC1-deficient cellsobserved by flow cytometry (Fig. 4B and C). Thus, ERCC1-deficient cells undergo apoptosis after addition of ATRpathway–targeted drugs.

As ERCC1-deficient cells accumulate in S-phase, we rea-soned that ERCC1 may be necessary for DNA replication incancer cells. To test this, we purified replication forks usingiPOND (31, 32) and identified ERCC1 at replication forks (lane7, Fig. 4E). Briefly, cells were labeled with the thymidine analogEdU for a short amount of time to label active replication forks.The EdU was then purified and proteins co-purifying with itwere analyzed by Western blot analysis. ERCC1 was alsopresent at stalled [hydroxyurea (HU)-treated] and collapsedforks (HU- and ATRi-treated; lanes 9 and 10, Fig. 4E). Itsassociation with replication forks is not due to a generalinteraction with chromatin, as ERCC1 is not associated withbulk chromatin that is not adjacent to a replication fork, as itcan be chased away with thymidine (lane 8, Fig. 4E). Theseresults are also consistent with the prior identification of XPFat replication forks using iPOND (39).

ATR pathway inhibition does not increase micronucleiformation in ERCC1-deficient cells

After DNA replication, ERCC1 plays a role in proper segre-gation of chromosomes, and knockdown of ERCC1 causes anincrease in micronuclei formation as chromosomes fail tosegregate properly (40, 41). As expected, we observed a signif-icant increase in the amount of micronuclei formation whenwe knocked down ERCC1 (Fig. 5A and B). This induction wasnot increased further with the addition of ATRi or CHK1i.These data suggest that ATRpathway inhibition does not affectthe postreplicative functions of ERCC1 such as chromosomesegregation.

ERCC1 deficiency sensitizes cancer cell lines to ATRpathway–targeted drugs

To test how generalizable our observations were, weextended our study to include 2 triple-negative breast cancercell lines and 2 non–small cell lung cancer cell lines. Dose–response curves of ATRi and CHK1i were completed oncancer cell lines depleted of ERCC1 with two specific siRNAs(Fig. 6). In all cases, cells depleted of ERCC1 were moresensitive to ATRi and CHK1i treatment than control cells.We did not observe a strict correlation between the degree ofERCC1 knockdown and the observed synthetic lethalityperhaps due to changes in cell growth rates or unknownoff-target effects. Nonetheless, these data indicate thatERCC1 protects cancer cells from ATR pathway–targeteddrugs in multiple cancer types.

DiscussionATR pathway inhibitors are currently in clinical trials and

have shown promise in combination with platinum drugs andgemcitabine (11, 15, 18). The current study was initiated todetermine whether any synthetic lethal interactions existbetween DNA repair proteins and ATR pathway–targeteddrugs. Our siRNA screen identified ERCC1-XPF deficiency asa potent sensitizer to ATR pathway inhibition in cancer cells.Loss of ERCC1 or XPF causes an increase in the number ofgH2AX foci in untreated cells, and addition of the ATR or CHK1inhibitors results in pan-nuclear gH2AX, suggestive of large

siNT siERCC1-1

No micronucleiMicronuclei

100

20

40

60

80

0

Pe

rce

nt

of

ce

lls

B

ATRi ATRi

siNT

CHK1i

siERCC1-1

CHK1i

siERCC1-2

CHK1i

A

nsns

P = 0.03

__ ATRi_

Figure 5. ERCC1 knockdown–induced micronuclei formation is notincreased in ATRi- or CHK1i-treated cells. U2OS cells were transfectedwithnontargetingor ERCC1siRNAand then treatedwith1mmol/LATRi or0.05 mmol/L CHK1i for 24 hours. Cells were then fixed and stained withDAPI to score micronuclei formation. A, representative images ofmicronuclei formation in ERCC1-depleted cells. Arrows, micronuclei. B,quantification of micronuclei formation. At least 200 cells were scored foreach condition between two independent experiments. P values areindicated on the graph; ns, not significant.






amounts of replication stress. ATR and CHK1 inhibitors causean S-phase arrest in ERCC1-deficient cells and these cells fail toenter mitosis even when the inhibitors are removed. ERCC1-XPF are often lost in many types of cancers (22, 23, 42), and wereport that depletion of ERCC1-XPF in both triple-negativebreast cancer and non–small cell lung cancer sensitize thesecancer cells to killing with ATR pathway–targeted drugs.Together, our data indicated that ERCC1 status in cancer mayhelp guide the choice of an ATR pathway–targeted drug fortreatment.

Impaired ATR pathway function sensitizes to ATRinhibitors

In addition to ERCC1-XPF, our screen also identified theknown synthetic lethal interactions between ATM (15) and

XRCC1 (19), suggesting the validity of our approach and screendesign. In addition, we also observed strong synthetic lethalitywith all of the known ATR pathway genes that were present inthe siRNA library. ATR pathway genes are mutated or deletedin up to 15% to 25% of samples from some cancer types,suggesting that reduced ATR pathway functionality may be auseful criteria for selecting patients for ATR-targeted drugs(33). Furthermore, the screening strategy is also a usefulapproach to identify novel ATR pathway proteins, some ofwhich may prove to be druggable targets, such as the recentlyreported replication protein A inhibitors (43, 44).

Notably, not all DNA repair pathways are synthetic lethalwith ATR inhibition. For example, neither nucleotide excisionrepair nor mismatch repair deficiencies cause hypersensitiza-tion to ATR inhibitors. This study represents the first

100

20

40

60

80

120

0

A

100

20

40

60

80

120

0

B

100

20

40

60

80

120

00.01 0.1 1

0.1 1

0.1 1

0.1 1

100

20

40

60

80

120

00.01

HCC1806

BT549

100

20

40

60

80

120

0

C100

20

40

60

80

120

00.01

H157

100

20

40

60

80

120

00.1 1 10

0.1 1 10

0.1 1 10

0.1 1 10

D100

20

40

60

80

120

00.01

siNT

siERCC1-1

siERCC1-4

A549

siNT

siERCC1-1

siERCC1-2

siNT

siERCC1-1

siERCC1-2

siNT

siERCC1-1

siERCC1-4

ERCC1

GAPDH

ERCC1

GAPDH

ERCC1

GAPDH

ERCC1GAPDH

NT 1-1 1-2

NT 1-1 1-4

NT 1-1 1-4

NT 1-1 1-2

Perc

ent of contr

ol

Perc

ent of contr

ol

Perc

ent of contr

ol

Perc

ent of contr

ol

Perc

ent of contr

ol

Perc

ent of contr

ol

Perc

ent of contr

ol

Perc

ent of contr

ol









Figure 6. ERCC1 depletionsensitizes triple-negative breastcancer and non–small cell lungcancer cell lines to ATRi andCHK1i. BT549 (A), HCC1806 (B),H157 (C), and A549 (D) weretransfected with the indicatedcontrol or ERCC1 siRNA and thentreated with the ATR or CHK1inhibitor. Cell viability wasdetermined with alamar blue andreported as a percent of theuntreated control cells.Knockdown of ERCC1 yieldedsignificant reductions in IC50 valuesfor both siERCC1 siRNAs ascompared with control cells(P < 0.001). Statistics are describedin Materials and Methods. Westernblot analyses of cells afterknockdown are shown in theinsets.

Mohni et al.





systematic search for synthetic lethal interactions with ATRpathway–targeted drugs. Performing this screen with theCHK1 inhibitor or in a whole-genome siRNA library may revealadditional genetic relationships that can be exploited forcancer therapy.

ERCC1-XPF synthetic lethality with ATR inhibitionWe present 2 possible explanations for the observed syn-

thetic lethality between ERCC1-XPF deficiency and ATR inhi-bition. First, we and others have observed ERCC1-XPF atreplication forks and it is possible that in their absence,replication forks become unstable and require ATR to main-tain them. Second, defects in the repair of ICLs and replicationof genomic fragile sites when ERCC1-XPF are inactivated (20,21) may create an increased need for ATR signaling. The firstpossible explanation is unlikely, as it does not appear thatERCC1 loss affects the inherent stability of stalled replicationforks. ERCC1-depleted cells are not sensitive to HU, a forkstalling agent, and are able to resume DNA synthesis quicklyafter HU removal (ref. 45 and data not shown).We favor the second explanation that incomplete repair and

replication processes induced by loss of ERCC1-XPF are thesource of ATR dependency. The amount of endogenous DNAdamage is increased in the absence of ERCC1-XPF as indicatedby the increase in gH2AX. This gH2AX is likely caused asincomplete repair intermediates collapse into DSBs. Repair ofthese breaks and completion of DNA replication require ATR,explaining the observed synthetic lethality. This explanation isalso consistent with our observation that ERCC1-XPF–defi-cient cells do not complete S-phase when ATR is inhibited.Importantly, the repair requirement for ERCC1-XPF that issynthetic lethal with ATR inhibition is not standard nucleotideexcision repair as other NER genes are not synthetic lethal withATRi.We did observe some differences between knockdown of

ERCC1 and XPF in some assays. We suspect this difference islargely due to differences in knockdown efficiencies and result-ing cell growth rates. The similar results seen in the colony-forming assays, which are less influenced by cell growth ratesthan the alamar blue assay, are consistent with this explana-tion. However, it is also possible that some differences are dueto XPF-independent functions of ERCC1 (46).Loss of ERCC1-XPF is also synthetic lethal with PARP

inhibition although the mechanism is likely to be differentthan the synthetic lethality observed with ATR. ERCC1-defi-cient cells treated with PARP inhibitors undergo a prolongedG2–M arrest accompanied by activated checkpoint signaling(45, 47). The authors of these studies suggest that in the contextof PARP inhibition, the essential role of ERCC1 is in homol-ogous recombination, as ERCC1 loss yields the same pheno-type as BRCA2 loss. This contrasts with the strong S-phasearrest seen in ERCC1-deficient cells treated with ATRi. Our

data suggest that ERCC1 is also functioning during DNAreplication, presumably to repair endogenous sources of DNAdamage that stall the replication fork and in the resolution ofreplication intermediates at fragile sites.

ERCC1 as a predictive biomarker for ATR pathway–targeted therapies

ERCC1 is a recognized modulator of the response toplatinum-based chemotherapies (22, 25). As such, mucheffort has been devoted to evaluating ERCC1 levels in tumorsamples to guide treatment. While there has been contro-versy in the best methods to detect ERCC1 in tumors due tothe inability of PCR and antibody-based detection methodsto discriminate between the functional and nonfunctionalERCC1 isoforms (22, 25), several laboratories are working todevelop appropriate tumor screening methods. Thus, clin-ical evaluation of ERCC1 levels in tumors is a viable diag-nostic option before the initiation of chemotherapy. Deple-tion of ERCC1 in every cancer cell line we have testedsensitizes the cells to ATR pathway inhibition. Therefore,tumors with low levels of ERCC1 may prove more sensitiveto ATR pathway inhibitors and provide a patient selectionstrategy. We are also excited about the recent disclosure ofERCC1-XPF small-molecule inhibitors (48). These inhibitors,combined with ATR pathway–targeted drugs, could be usedin combination therapies to treat cancer cells and improveoutcomes.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K.N. Mohni, D. CortezDevelopment of methodology: K.N. MohniAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.N. Mohni, G.M. KavanaughAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.N. MohniWriting, review, and/or revision of the manuscript: K.N. Mohni, G.M.Kavanaugh, D. CortezStudy supervision: D. Cortez

AcknowledgmentsThe authors thank Orlando Scharer for providing the XPF-mutant patient

cells and Jean Charles Soria, Ken Olaussen, and Luc Friboulet for providing theERCC1-null cells.

Grant SupportThis work was supported by a Breast Cancer Research Foundation and NIH

CA102792 grants to D. Cortez, T32CA093240 to K.N.Mohni, and F32GM103254 toG.M. Kavanaugh.

The costs of publication of this article were defrayed in part 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 November 11, 2013; revised January 27, 2014; accepted February 22,2014; published OnlineFirst March 24, 2014.

References1. Aguilera A, Gomez-Gonzalez B. Genome instability: a mechanistic

view of its causes and consequences. Nat Rev Genet 2008;9:204–17.2. Branzei D, Foiani M. Maintaining genome stability at the replication

fork. Nat Rev Mol Cell Biol 2010;11:208–19.






3. Cimprich KA, Cortez D. ATR: an essential regulator of genome integ-rity. Nat Rev Mol Cell Biol 2008;9:616–27.

4. Curtin NJ. DNA repair dysregulation from cancer driver to therapeutictarget. Nat Rev Cancer 2012;12:801–17.

5. BryantHE, Schultz N, ThomasHD, Parker KM, Flower D, Lopez E, et al.Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 2005;434:913–7.

6. Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB,et al. Targeting the DNA repair defect in BRCA mutant cells as atherapeutic strategy. Nature 2005;434:917–21.

7. Murai J, Huang SY, Das BB, Renaud A, Zhang Y, Doroshow JH, et al.TrappingofPARP1andPARP2byclinical PARP inhibitors. CancerRes2012;72:5588–99.

8. Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, et al. DNAdamage response as a candidate anti-cancer barrier in early humantumorigenesis. Nature 2005;434:864–70.

9. Gorgoulis VG, Vassiliou LV, Karakaidos P, Zacharatos P, Kotsinas A,Liloglou T, et al. Activation of the DNA damage checkpoint andgenomic instability in human precancerous lesions. Nature 2005;434:907–13.

10. Bartek J, Bartkova J, Lukas J. DNA damage signalling guards againstactivated oncogenes and tumour progression. Oncogene 2007;26:7773–9.

11. Carrassa L, Damia G. Unleashing Chk1 in cancer therapy. Cell Cycle2011;10:2121–8.

12. Garrett MD, Collins I. Anticancer therapy with checkpoint inhibitors:what, where and when? Trends Pharmacol Sci 2011;32:308–16.

13. MaCX, Janetka JW, Piwnica-Worms H. Death by releasing the breaks:CHK1 inhibitors as cancer therapeutics. Trends Mol Med 2011;17:88–96.

14. Foote KM, Blades K, Cronin A, Fillery S, Guichard SS, Hassall L, et al.Discovery of 4-{4-[(3R)-3-Methylmorpholin-4-yl]-6-[1-(methylsulfonyl)cyclopropyl]pyrimidin-2-y l}-1H-indole (AZ20): a potent and selectiveinhibitor of ATR protein kinase with monotherapy in vivo antitumoractivity. J Med Chem 2013;56:2125–38.

15. Reaper PM, Griffiths MR, Long JM, Charrier JD, Maccormick S,Charlton PA, et al. Selective killing of ATM- or p53-deficientcancer cells through inhibition of ATR. Nat Chem Biol 2011;7:428–30.

16. Charrier JD, Durrant SJ, Golec JM, Kay DP, Knegtel RM, MacCormickS, et al. Discovery of potent and selective inhibitors of ataxia telangi-ectasia mutated and Rad3 related (ATR) protein kinase as potentialanticancer agents. J Med Chem 2011;54:2320–30.

17. Toledo LI, Murga M, Zur R, Soria R, Rodriguez A, Martinez S, et al. Acell-based screen identifies ATR inhibitors with synthetic lethal prop-erties for cancer-associated mutations. Nat Struct Mol Biol 2011;18:721–7.

18. Fokas E, Prevo R, Pollard JR, Reaper PM, Charlton PA, Cornelissen B,et al. Targeting ATR in vivo using the novel inhibitor VE-822 results inselective sensitization of pancreatic tumors to radiation. Cell DeathDis2012;3:e441.

19. Sultana R, Abdel-Fatah T, Perry C, Moseley P, Albarakti N, Mohan V,et al. Ataxia telangiectasia mutated and Rad3 related (ATR) proteinkinase inhibition is synthetically lethal in XRCC1 deficient ovariancancer cells. PLoS One 2013;8:e57098.

20. Friedberg EC,Walker GC, SiedeW,Wood RD, Schultz RA, EllenburgerT. DNA repair and mutagenesis. 2nd ed.Washington DC: ASM Press;2006.

21. NaimV,Wilhelm T, DebatisseM, Rosselli F. ERCC1andMUS81-EME1promote sister chromatid separation by processing late replicationintermediates at common fragile sites during mitosis. Nat Cell Biol2013;15:1008–15.

22. Besse B, Olaussen KA, Soria JC. ERCC1 and RRM1: ready for primetime? J Clin Oncol 2013;31:1050–60.

23. Kirschner K, Melton DW. Multiple roles of the ERCC1-XPF endonu-clease in DNA repair and resistance to anticancer drugs. AnticancerRes 2010;30:3223–32.

24. McNeil EM, Melton DW. DNA repair endonuclease ERCC1-XPF as anovel therapeutic target to overcome chemoresistance in cancertherapy. Nucleic Acids Res 2012;40:9990–10004.

25. Friboulet L,OlaussenKA, Pignon JP, ShepherdFA, TsaoMS,GrazianoS, et al. ERCC1 isoform expression and DNA repair in non-small-celllung cancer. N Engl J Med 2013;368:1101–10.

26. Cortez D, Guntuku S, Qin J, Elledge SJ. ATR and ATRIP: partners incheckpoint signaling. Science 2001;294:1713–6.

27. Orelli B, McClendon TB, Tsodikov OV, Ellenberger T, Niedernhofer LJ,Scharer OD. The XPA-binding domain of ERCC1 is required fornucleotide excision repair but not other DNA repair pathways. J BiolChem 2010;285:3705–12.

28. Zabludoff SD,DengC,GrondineMR,SheehyAM,Ashwell S, CalebBL,et al. AZD7762, a novel checkpoint kinase inhibitor, drives checkpointabrogation and potentiates DNA-targeted therapies. Mol Cancer Ther2008;7:2955–66.

29. Mohni KN, Dee AR, Smith S, Schumacher AJ, Weller SK. Efficientherpes simplex virus 1 replication requires cellular ATR pathwayproteins. J Virol 2013;87:531–42.

30. Couch FB, Bansbach CE, Driscoll R, Luzwick JW, Glick GG, Betous R,et al. ATR phosphorylates SMARCAL1 to prevent replication forkcollapse. Genes Dev 2013;27:1610–23.

31. Sirbu BM, Couch FB, Feigerle JT, Bhaskara S, Hiebert SW, Cortez D.Analysis of protein dynamics at active, stalled, and collapsed replica-tion forks. Genes Dev 2011;25:1320–7.

32. Sirbu BM,Couch FB, Cortez D.Monitoring the spatiotemporal dynam-ics of proteins at replication forks and in assembled chromatin usingisolation of proteins on nascent DNA. Nature protocols 2012;7:594–605.

33. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al.The cBio cancer genomics portal: an open platform for exploringmultidimensional cancer genomics data. Cancer Discov 2012;2:401–4.

34. Gaillard PH, Wood RD. Activity of individual ERCC1 and XPF subunitsin DNA nucleotide excision repair. Nucleic Acids Res 2001;29:872–9.

35. Arora S, Kothandapani A, Tillison K, Kalman-Maltese V, Patrick SM.Downregulation of XPF-ERCC1 enhances cisplatin efficacy in cancercells. DNA Repair (Amst) 2010;9:745–53.

36. Paulsen RD, Soni DV, Wollman R, Hahn AT, Yee MC, Guan A, et al.A genome-wide siRNA screen reveals diverse cellular processesand pathways that mediate genome stability. Mol Cell 2009;35:228–39.

37. Bansbach CE, Betous R, Lovejoy CA, Glick GG, Cortez D. Theannealing helicase SMARCAL1 maintains genome integrity at stalledreplication forks. Genes Dev 2009;23:2405–14.

38. Syljuasen RG, Sorensen CS, Hansen LT, Fugger K, Lundin C, Johans-son F, et al. Inhibition of human Chk1 causes increased initiation ofDNA replication, phosphorylation of ATR targets, and DNA breakage.Mol Cell Biol 2005;25:3553–62.

39. Gilljam KM, Muller R, Liabakk NB, Otterlei M. Nucleotide excisionrepair is associated with the replisome and its efficiency dependson a direct interaction between XPA and PCNA. PLoS One 2012;7:e49199.

40. Melton DW, Ketchen AM, Nunez F, Bonatti-Abbondandolo S,Abbondandolo A, Squires S, et al. Cells from ERCC1-deficient miceshow increased genome instability and a reduced frequency of S-phase-dependent illegitimate chromosome exchange but a normalfrequency of homologous recombination. J Cell Sci 1998;111:395–404.

41. Niedernhofer LJ, Odijk H, Budzowska M, van Drunen E, Maas A, TheilAF, et al. The structure-specific endonuclease Ercc1-Xpf is required toresolveDNA interstrand cross-link-induceddouble-strandbreaks.MolCell Biol 2004;24:5776–87.

42. Ozkan C, Gumuskaya B, Yaman S, Aksoy S, Guler G, Altundag K.ERCC1 expression in triple negative breast cancer. J BUON 2012;17:271–6.

43. Glanzer JG, Carnes KA, Soto P, Liu S, Parkhurst LJ, Oakley GG. Asmall molecule directly inhibits the p53 transactivation domain frombinding to replication protein A. Nucleic Acids Res 2013;41:2047–59.

44. Patrone JD, Kennedy JP, Frank AO, FeldkampMD, Vangamudi B, PelzNF, et al. Discovery of protein-protein interaction inhibitors of replica-tion protein A. ACS Med Chem Lett 2013;4:601–5.


Mohni et al.




45. Postel-Vinay S, Bajrami I, Friboulet L, Elliott R, Fontebasso Y, DorvaultN, et al. A high-throughput screen identifies PARP1/2 inhibitors as apotential therapy for ERCC1-deficient non-small cell lung cancer.Oncogene 2013;32:5377–87.

46. Rageul J, Fremin C, Ezan F, Baffet G, Langouet S. The knock-down ofERCC1 but not of XPF causes multinucleation. DNA Repair (Amst)2011;10:978–90.

47. ChengH,ZhangZ,BorczukA,PowellCA,BalajeeAS,LiebermanHB,etal.PARP inhibition selectively increases sensitivity to cisplatin in ERCC1-lownon-small cell lung cancer cells. Carcinogenesis 2013;34:739–49.

48. Jordheim LP, Barakat KH, Heinrich-Balard L, Matera EL, Cros-PerrialE, Bouledrak K, et al. Small molecule inhibitors of ERCC1-XPF protein-protein interaction synergize alkylating agents in cancer cells. MolPharmacol 2013;84:12–24.






2014;74:2835-2845. Published OnlineFirst March 24, 2014.Cancer Res Kareem N. Mohni, Gina M. Kavanaugh and David Cortez ERCC1 DeficiencyATR Pathway Inhibition Is Synthetically Lethal in Cancer Cells with

Updated version

10.1158/0008-5472.CAN-13-3229doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2014/03/24/0008-5472.CAN-13-3229.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/74/10/2835.full#ref-list-1

This article cites 47 articles, 15 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/74/10/2835.full#related-urls

This article has been cited by 9 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/74/10/2835To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-13-3229

http://cancerres.aacrjournals.org/content/suppl/2014/03/24/0008-5472.CAN-13-3229.DC1

http://cancerres.aacrjournals.org/content/74/10/2835.full#ref-list-1

http://cancerres.aacrjournals.org/content/74/10/2835.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerres.aacrjournals.org/content/74/10/2835


ATRPathwayInhibitionIsSyntheticallyLethalinCancerCells...

Documents

Transcript of ATRPathwayInhibitionIsSyntheticallyLethalinCancerCells...