mgmt expression predicts parp-mediated resistance to temozolomide · companion diagnostics and...

Companion Diagnostics and Cancer Biomarkers

MGMT Expression Predicts PARP-MediatedResistance to TemozolomideOihane Erice1, Michael P. Smith1, Rachel White2, Ibai Goicoechea1, Jorge Barriuso1,Chris Jones3, Geoffrey P. Margison4, Juan C. Acosta2, Claudia Wellbrock1, andImanol Arozarena1

Abstract

Melanoma and other solid cancers are frequently resistant tochemotherapies based on DNA alkylating agents such as dacar-bazine and temozolomide. As a consequence, clinical responsesare generally poor. Such resistance is partly due to the ability ofcancer cells to use a variety of DNA repair enzymes to maintaincell viability. Particularly, the expression of MGMT has beenlinked to temozolomide resistance, but cotargeting MGMT hasproven difficult due to dose-limiting toxicities. Here, we showthat the MGMT-mediated resistance of cancer cells is profound-ly dependent on the DNA repair enzyme PARP. Both in vitro and

in vivo, we observe that MGMT-positive cancer cells stronglyrespond to the combination of temozolomide and PARP inhi-bitors (PARPi), whereas MGMT-deficient cells do not. In mel-anoma cells, temozolomide induced an antiproliferative senes-cent response, which was greatly enhanced by PARPi in MGMT-positive cells. In summary, we provide compelling evidence tosuggest that the stratification of patients with cancer upon theMGMT status would enhance the success of combinationtreatments using temozolomide and PARPi. Mol Cancer Ther;14(5); 1236–46. �2015 AACR.

IntroductionMelanoma is a form of skin cancer notoriously resistant to

current therapies. Before the arrival of targeted therapies againstthe BRAF and MEK components of the hyperactivated MAPKpathway, alkylating agent-based chemotherapy was the first-linetreatment for decades. In around 40%of all cases, patients qualifyfor BRAF and MEK inhibitors and targeted therapies provide agood clinical response. However, after 2 to 8 months, the vastmajority of patients relapse (1–3). For these patients, their hopeslie in chemotherapy or immunotherapy. The only FDA-approved

chemotherapeutic agent is the intravenously administered pro-drug dacarbazine, which is metabolically converted in the liver toMTIC, a toxic monofunctional DNA-alkylating agent (4, 5). Analkylating agent related to dacarbazine but with excellent oralbioavailability is temozolomide, which can cross the blood brainbarrier and therefore represents an alternative to dacarbazine formelanoma with brain metastases (6–8). Temozolomide is anattractive chemotherapy agent for patients with unresectablemetastatic melanoma. However, single-agent escalated dosetemozolomide compared with single-agent dacarbazine treat-ment did not improve overall survival or progression-free survival(9, 10), and overall response rates for both drugs were 15% to20% (5). Temozolomide is FDA approved for anaplastic astro-cytoma and glioblastoma, but again with poor overall responsesof around26.5%(11). This suggests that to improve the efficacy oftemozolomide, we need to enhance our understanding about themode of action of temozolomide as a DNA-damaging and cyto-toxic drug.

Temozolomide methylates DNA bases predominantly at oxy-gen in position 6 and nitrogen at position 7 in guanine andnitrogen at position 3 in adenine (O6-metG, N7-metG, and N3-metA, respectively). O6-metG is the most toxic and mutagenicDNA modification produced by temozolomide and is quicklyrepaired by the enzyme O6-methyl-guanine DNA methyl trans-ferase (MGMT). MGMT incorporates theO6-methyl adduct into acysteine and the enzyme is then degraded by proteolysis (4). Onthe other hand,N3-metA andN7-metG repair aremediated by thebase excision repair (BER) machinery in a process that involvesPARP, which ADP-ribosylate DNA and proteins. When acting onDNA, PARP proteins create a docking site for the incorporation ofother components of the BER machinery, which complete therepair process (4).

The most evident mechanism of resistance to both DTIC andtemozolomide is MGMT activity, and MGMT downregulationsensitizes cancer cells to temozolomide (12).Moreover, in elderly

1Manchester Cancer Research Centre, The University of Manchester,Michael Smith Building, Oxford Road, Manchester, United Kingdom.2EdinburghCancer ResearchUKCentre andMRC Institute of GeneticsandMolecular Medicine,Western General Hospital, University of Edin-burgh, Edinburgh,UnitedKingdom. 3Divisions ofMolecular PathologyandCancer Therapeutics, Institute of Cancer Research, Sutton, UnitedKingdom. 4Centre for Occupational and Environmental Health, TheUniversity of Manchester, Stopford Building, Manchester, UnitedKingdom.

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

O. Erice and M.P. Smith contributed equally to this article.

Current address for O. Erice: Division of Hepatology and Gastroenterology,Biodonostia Research Institute, Calle Doctor Begiristain, San Sebastian 20014,Spain; current address for I. Goicoechea: Oncology Area, Biodonostia ResearchInstitute, Calle Doctor Begiristain, San Sebastian 20014, Spain; and currentaddress for I. Arozarena: School of Applied Sciences, University of Huddersfield,Huddersfield HD1 3DH, United Kingdom.

Corresponding Authors: Imanol Arozarena, University of Huddersfield, Queen-sgate, HuddersfieldHD1 3DH,UnitedKingdom. Phone: 44-148-4472722; Fax: 44-148-4472182; E-mail: [email protected]; and Claudia Wellbrock,E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-14-0810

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 14(5) May 20151236

on February 18, 2021. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst March 16, 2015; DOI: 10.1158/1535-7163.MCT-14-0810

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glioblastoma patients MGMT status predicts clinical response totemozolomide (13) Unfortunately, therapies combining MGMTinhibitors with temozolomide in melanoma or colorectal cancerpatients have so far failed to improve outcome due to exacerbatedtreatment-related hematologic toxicity (14–16). More recently,several small-molecule inhibitors have been developed to targetother DNA repair mechanisms, with the aim to use them assynthetically lethal single agents or in combination therapieswith chemotherapeutic agents such as temozolomide. Notably,current clinical trials are testing PARP inhibitors (PARPi) asmonotherapy in BRCA-deficient breast cancer patients or incombination with DNA-alkylating agents (17, 18). Combinationtherapies ofDTICor temozolomide andPARPi are better toleratedthan those with MGMT inhibitors, but myelosuppression andliver toxicity still represent clinical concerns (19, 20), and overallsurvival rates were not improved compared with patients treatedwith alkylating alone (21–23). Therefore, further research iswarranted to identify markers that help stratify patients fortemozolomide-based therapies to improve clinical outcome. Ourstudy suggests that MGMT-mediated resistance to temozolomidein melanoma and other cancer types requires PARP and thatMGMT expression correlates with improved response to combi-nation of temozolomide with PARPi.

Materials and MethodsCell culture

Melanoma cell lines were provided by Prof. Richard Marais(Cancer Research UK, Manchester Institute, The University ofManchester, Manchester, United Kingdom) and Dr. Adam Hurl-stone (Faculty of Life Sciences, University of Manchester, Man-chester, United Kingdom) and have been described recently (24).Colorectal carcinoma cells were obtained from Prof. StephenTaylor, originally from ATCC. Glioblastoma cell lines have beendescribed recently (25). Cell stocks were expanded and vials keptin liquid nitrogen. New aliquots were thawed every 5 to 7 weeks.Melanoma cell lines were authenticated in house by short tandemrepeat profiling. Cells were cultured in DMEM or in RPMI-164medium (SIGMA) as previously indicated, supplemented with0.5% penicillin and streptomycin (SIGMA) and 10% bovine calfserum (PAA). Cells were grown at 37�C in a 5%CO2 environment.WM98.1-MGMT–expressing cellswere establishedby transfectionof a pcDNA3 vector encoding the cDNA for human MGMT.MGMT-expressing cells were selected using G418 as pcDNA3expresses the Neomycin resistance gene.

ReagentsTemozolomideandmethylmethanesulfonate (MMS)were from

SIGMA. Olaparib, veliparib (ABT-888), selumetinib (AZD-6244),and lomeguatrib were from Selleck Chemicals.

Proliferation assaysCells were plated in 96-well plates and treated with serial

increasing concentrations (range, 0.15 mmol/L to 1 mmol/L) ofdrugs as indicated. After 5 days, cells were fixed and stained withtoluidine blue, the concentration necessary to inhibit cell growthto a 50% (GI50) was calculated as previously described (24).

Colony formation assaysCells were plated in 12-well plates and treated, from the top-left

well, with DMSO, 5, 25, 75, 250, or 1000 mmol/L temozolomide

for 24 hours (Supplementary Fig. S1). Then cells were washed andfresh, drug-free medium was replaced. Cells were left to formcolonies for 10 to 15 days before being fixed and stained with asolution of 4%Formaldehyde (Fisher Scientific) and 0.5%Crystalviolet (SIGMA) in PBS. Remaining dyewas solubilized in PBS, 1%SDS (Fisher Scientific).

Xenograft assayAnimal studies described within were approved by The Uni-

versity of Manchester (Manchester, United Kingdom) EthicalReview Board and performed according to UK Home Officeregulations. Briefly GFP-expressing human melanoma cells wereinjected into the pericardial cavity of 48 hours post-fertilizationzebrafish embryos. Embryos were treated at 1 day after injectionwith DMSO (SIGMA), temozolomide, olaparib, or the combina-tion of both. On days 1 and 5, pictures were taken and relativegrowth quantified as previously described (26).

Western blottingCell lysates were prepared using RIPA buffer and analyzed as

described (27). Primary antibodies used were: MGMT (ThermoFisher Scientific), phospho-Thre68-CHK-2 and Ser345-CHK-1,MLH-1, MSH-2, MSH-6, and cleaved caspase-3 (Cell SignalingTechnology), ERK2, and b-tubulin (Santa Cruz Biotechnologies).

MGMT activity assaysBriefly, increasing amounts of PARP or MGMT inhibitors were

incubated with recombinant MGMT (�6 fmoles) for 1 hour atroomtemperature, then substrateDNAwas added and incubationcontinued for 1 hour. After incubation, assay samples wereprocessed as described previously in (ref. 28) and radioactivityquantified by scintillation counting.

FACS analysisOne lakh cells were treated as indicated for 3 days, fixed in 80%

ice-cold ethanol in PBS. Cells were then washed in PBS andincubated in a solution containing PBS, RNase A, and propidiumiodide (SIGMA) at 37�C for 1 hour. The analysis was performedusing FACS-Calibur (Becton Dickinson).

Senescence-associated b-galactosidade (SA-b-gal) assayAfter 5 days of treatment, cells were fixed in 0.5% glutaral-

dehyde and stained overnight with X-gal solution: 0.12 mmol/LK3Fe(CN)6, 0.12 mmol/L K4Fe(CN)6, 1 mmol/L MgCl2, and1 mg/mL 5-Bromo-4-cloro-3-indolyl-b-D-galactopiranoside inPBS pH 6.0. Cell nuclei were stained with DAPI. Bright field andUV images were taken using an inverted fluorescence micro-scope. The percentage of SA-b-Gal–positive cells is determinedupon counting of at least 100 cells.

High content analysis of the senescent responseFor the high content analysis, cells were plated in 96-well plates

and stained by immunofluorescence. Two fluorescence imagescorresponding to DAPI (to identify individual cells) and gene-primary antibody/AlexaFluor488-secondary antibody wereacquired and analyzed as previously described (29). Each wellwas scored a percentage positive of reference channel to thetotal cells. Antibodies used were: anti-g-H2AX (Millipore), anti-BrdU (Invitrogen), anti-p21Cip1 (Sigma), anti-p53 (Santa CruzBiotechnology), and anti-phospho-(Ser/Thr)Q (Cell SignalingTechnology).

MGMT-Mediated Response to Temozolomide and PARP Inhibitors

www.aacrjournals.org Mol Cancer Ther; 14(5) May 2015 1237




Statistical analysisUnless indicated otherwise, data are from assays performed in

triplicate, with error bars to represent SD or errors from themean.Statistics used were: predominately Student t test and one-wayANOVA with a Dunnett multiple comparison test performedusing GraphPad Prism version 4.00 for Mac OS, GraphPadSoftware, www.graphpad.com.

ResultsPARP inhibition specifically sensitizesMGMT-proficient cells totemozolomide

To assess the ability of PARPi to sensitize melanoma cells totemozolomide, we treated a panel of 12 melanoma cell lineswith increasing concentrations of temozolomide in the absenceor presence of the PARPi olaparib. We used olaparib at aconcentration of 0.25 mmol/L, which did not affect cell prolif-eration alone (Supplementary Fig. S2A), but is reported tosynergize with alkylating agents to inhibit growth (30, 31).Olaparib significantly sensitized melanoma cells to a 5-daytreatment with temozolomide as seen by the GI50 values fortemozolomide (Fig. 1A and Supplementary Fig. S1B). However,the cell lines segregated into two defined groups: one with highGI50 values for temozolomide, which showed sensitization toolaparib, and one with no significant difference (Fig. 1A). Wetherefore tested for differences in DNA repair regulators andfound that all cell lines belonging to the "high-GI50" groupexpressed enhanced levels of MGMT; only oneMGMT-expressingcell line segregated in the "low-GI50" group (Fig. 1B). Expressiondata were complemented with methylation-specific PCR, whereMGMT promoter methylation was detectable in all MGMT� cells(Fig. 1B). When we grouped the cell lines upon MGMT status(MGMTþ and MGMT�), this revealed that PARP inhibition onlysensitized to temozolomide in MGMTþ melanoma cells (Fig.1C). Indeed, the Potentiation Factor 50 (PF50, ratio of the GI50 oftemozolomide in monotherapy compared with the GI50 oftemozolomide in the presence of PARPi) of olaparib was sig-nificantly higher in MGMTþ cells than in MGMT� cells where thePF50 was close to 1, indicating no potentiation (Fig. 1D andSupplementary Fig. S2B). Similar results were obtained fromcolony formation assays where cells are tested for their capacityto repair DNA damage induced by temozolomide during just 24hours. These assays confirmed that only MGMT-expressing cellsresponded to the combination of temozolomide and olaparib(Fig. 1E and Supplementary Fig. S2C).

Finally, we analyzed whether the observed differential sensi-tivity to temozolomide in the presence of PARPi occurred also invivo. To do so, we used a human melanoma xenograft assay inwhich we injected GFP-expressing A375 (MGMTþ) or WM266-4(MGMT�) cells into the pericardial cavity of zebrafish embryos.This approach allows fast and efficient assessment of drug com-bination treatments of tumor cells in an in vivo setting, which canbe recapitulated in other preclinical models (26, 32). Embryoswere treated with DMSO or temozolomide in the presence orabsence of olaparib and after 4 days, xenograft volume wasassessed (Fig. 1F). Again, A375 (MGMTþ) xenografts respondedto the combination of olaparib and temozolomide (Fig. 1G), butdid not affect xenograft growth in WM266-4 (MGMT�)-derivedxenografts (Fig. 1H). Together, our data suggest that in humanmelanoma cells, MGMT expression correlated with synergisticaction of temozolomide and PARPi.

MGMT is active in temozolomide-resistant cellsTo establish whether MGMT expression correlated with its

activity, we used the MGMT inhibitor lomeguatrib. We treatedcell lines for 5 dayswith temozolomide in the absence or presenceof 20 mmol/L lomeguatrib (LOM), which only sensitized MGMT-expressing melanoma cells to temozolomide. Accordingly theaverage PF50 for MGMTþ cells was significantly higher than inMGMT� cells (Fig. 2A–C). As expected, the GI50 for MGMTþ cellswas significantly higher than for MGMT� cells. Colony formationassays provided very similar results (Fig. 2D–F). These dataconfirmed that MGMT expression and activity are correlated inour panel of melanoma cell lines, demonstrating that MGMTactivity provides resistance to temozolomide and correlates withtemozolomide and PARPi synergy. Importantly, everymelanomacell line expressed detectable levels of themismatch repair (MMR)proteins MSH-6, MSH-2, and MLH-1 (Supplementary Fig. S3).This result, together with the responses to temozolomide andolaparib or lomeguatrib previously observed, strongly suggestedthat all our melanoma cells are MMR proficient and the observeddifferences are not related to MMR deficiency.

The MGMT-dependent response to temozolomide–PARPitreatment is also seen in other cancer types

To determine whether the impact of MGMT expression on thePARPi response is specific for melanoma cells or cancer typeindependent, we assessed the response to temozolomide andolaparib in cells from three cancer types where temozolomide isused as a therapeutic option: glioblastoma, pancreatic neuroen-docrine tumors (NET), and colorectal carcinoma (11, 33, 34). Wetreated seven glioblastoma cell lines (4 MGMTþ and 3 MGMT�

cell lines) known to be MMRþ (25) and observed that MGMTexpression correlated with the response to temozolomide com-bined with olaparib (Fig. 3A–C). Similarly, although the NET cellline BON, which expresses high levels of MGMT responded toolaparib (and lomeguatrib) cotreatment, QGP-1NET cells, whichlack MGMT, were unaffected by PARP inhibition (Fig. 3D and E).Again, both NET cell lines were found to be MMRþ (Supplemen-tary Fig. S4A). Furthermore, a panel of seven MGMT-expressingcolorectal cancer cell lines of known MMR status (35) respondedto olaparib cotreatment. (Fig. 3F and G). Importantly, whengrouped by MMR status, the PF50 for olaparib was similar forboth groups (Fig. 3H; details of GI50 and PF50 values for each cellline can be found in Supplementary Fig. S4B).

MGMT overexpression confers PARP-dependent resistance totemozolomide

Our data suggest that MGMT expression provides resistance totemozolomide in a PARP-dependent manner. To further confirmthese findings, we ectopically expressed MGMT in MGMT� mel-anoma cells (WM98-1; Fig. 4A). As expected, MGMT overexpres-sion conferred resistance to temozolomide but lomeguatrib sen-sitizedWM98-1-MGMT clones to temozolomide-induced growthinhibition (Fig. 4B). Crucially, the MGMT-produced resistancecould be overcome by treatment with olaparib (Fig. 4C). Similarresults were obtained using veliparib (Supplementary Fig. S5Aand S5B). Furthermore, we observed that when MGMT activityis inhibited, PARPi no longer sensitized to temozolomide(Fig. 4D–F). We show that PARP is required for the MGMT-mediated resistance to temozolomide. As mentioned above,temozolomide produces N3- and N7-methyl adducts, which are

Erice et al.

Mol Cancer Ther; 14(5) May 2015 Molecular Cancer Therapeutics1238




repaired by PARP-mediated BER. To assess the requirement ofPARP in this repair, we used the alkylating agent MMS, whichexclusively produces N3- and N7-methyl adducts (36). Botholaparib and veliparib significantly sensitized WM98-1 andWM98-1-MGMT cells to MMS (Fig. 4G and Supplementary Fig.S5C). Similar results were obtained in a panel of melanoma cellsconfirming that PARP is required for efficient BER in melanomacells, irrespective of their MGMT status (Fig. 4H and Supplemen-tary Fig. S5D). Finally, to rule out the possibility that PARPinonspecifically interfere with MGMT activity, we performed invitro MGMT activity assays and confirmed that, unlike lomegua-trib, even high concentrations of olaparib or veliparib failed toinhibit MGMT (Supplementary Fig. S5E).

PARP inhibition potentiates temozolomide-induced cell-cyclearrest in MGMT-proficient cells

To further understand the action of temozolomide and PARPinhibition in the context of the MGMT expression status, westudied the induction of DNA single- and double-strand breaksmeasuring histone H2AX phosphorylation (gH2AX) after a 16-hour treatment. PARP inhibition only synergized with temozo-lomide to induce DNA damage in MGMTþ cells (Fig. 5A and B).Analysis of CHK-1 and 2 phosphorylation, (Thr68 and Ser345,respectively), at 24 hours of treatment revealed that in MGMT�

WM266-4 cells, temozolomide activated CHK1/2 at lower con-centrations than inMGMTþ A375 cells (Fig. 5C and D). AlthoughPARP inhibition did not enhance the activation of CHK2 by

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melanomacells. A,melanomacell lineswere treated with temozolomide withor without 0.25 mmol/L olaparib(OLAP). The graph shows the effect ofPARP inhibition on the average GI50for temozolomide. Each pointcorresponds to an individual cell line.B, top,Western blot analysis forMGMTexpression in melanoma cell lines.ERK2 was used as a loading control.Bottom, methylation-specific PCR forunmethylated (U) and methylated (M)promoter sequences. C and D, celllines were grouped upon their MGMTstatus, GI50 results from A reanalyzedand Potentiation Factor 50 calculated.E, Potentiation Factor results fromcolony formation assays on MGMTþ

and MGMT� cells treated as indicated.Student t test was used to comparethe effect of each treatment.F, pictures from A375 and WM266-4cells injected in zebra fish embryosand treated as indicatedfor 5 days. G and H, quantification ofthe in vivo effect of PARP inhibition ontemozolomide (500 mmol/L)-treatedxenografts. One-way ANOVA wasused to compare the effect of eachtreatment with DMSO (D)-treatedanimals. � ,P <0.05; �� ,P <0.01; n.s, notsignificant; �� , P < 0.001.






temozolomide in WM266-4 cells, in A375 cells temozolomide-inducedCHK-2phosphorylationwaspotentiated byolaparib.Onthe other hand, CHK-1 phosphorylation did not correlate con-sistently with olaparib cotreatment.

We next analyzed the cell-cycle profile of A375 and WM266-4cells after 72 hours of treatment. In WM266-4 cells, suboptimalconcentrations of temozolomide (5 mmol/L, unable to affectcell-cycle progression) failed to cooperate with olaparib topromote a cell-cycle arrest while 20 mmol/L temozolomide(GI50 ¼ 73.26 mmol/L), produced a marked G2–M arrest(Fig. 5E). In contrast, in A375 cells (GI50 ¼ 439.65 mmol/L)combining olaparib with a suboptimal concentration of temo-zolomide (50 mmol/L) produced a dramatic arrest in the G2–Mphase of the cell cycle (Fig. 5E). Strikingly, we were not able todetect the appearance of a sub-G1 phase, which is associatedwith cell death (37). In agreement with the absence of a sub-G1

population, we did not observe any caspase-3 cleavage inresponse to temozolomide in melanoma cells, whereas this wasdetectable in colorectal cancer cells (Fig. 5F). Moreover, cotreat-ment of A375 cells with concentrations of temozolomide andOLAP that potently affect cell proliferation failed to induce

caspase-3 cleavage (Fig. 5F, bottom). On the other hand, weobserved that melanoma cells treated with temozolomide dis-played a very flat appearance with a marked increase in cell size/surface (Fig. 5G). Because these are common traits of a senescentresponse, we decided to gain more insight into the role ofsenescence in the response of melanoma cells to temozolomide(38).

Characterization of the senescent response induced by thecombination of temozolomide and PARP inhibition

A 5-day treatment with growth-inhibitory concentrations oftemozolomide induced SA-b-gal staining in MGMT� WM98-1 aswell as MGMTþ A375 and WM98-1-MGMT cells (Fig. 6A–C),confirming previous observations suggesting that temozolomideinduces senescence rather than apoptosis inmelanoma cells (39).However, in MGMTþ cells treated with concentrations of temo-zolomide that induced only a weak SA-b-gal activity, PARPinhibition strongly enhanced the senescent response (Fig. 6B andC). High content analysis of A375 cells, used to assess theexpression of several knownmarkers andmediators of senescence(29), confirmed that the combination of temozolomide and

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Figure 2.Characterization of melanoma cell linesupon MGMT status. A, representativeexamples of dose–response curves forMGMTþ and MGMT� cells treatedwith temozolomide (TMZ) with orwithout lomeguatrib (LOM). B andC, graphs show the average GI50 fortemozolomide in the presence orabsence of lomeguatrib (B) and thePF50 for each cell line (C). D, colonyformation assay on four MGMTþ

and MGMT� cells treated withtemozolomide � LOM. E, PotentiationFactor 50 from D. F, representativeexamples of colony formationassays using A375 (MGMTþ) andWM9 (MGMT�) cells. D, DMSO.�� , P < 0.01; ns, not significant.

Erice et al.





PARPi induced a potent DNA damage response (DDR, main-tained over 5 days of treatment), as indicated by ATM and ATRphosphorylation (ST/Q) and a gH2AX signal (Fig. 6D). Theactivation of the DDR was accompanied by a significant increasein p53 levels and the cell-cycle inhibitor p21 (Fig. 6E). Finally, asexpected from a long-term cell-cycle arrest, we observed a dra-matic reduction in the number of cells undergoingDNA synthesis,as revealed by bromodeoxyuridine (BrdUrd) incorporation assay(Fig. 6F). In summary, when PARP is inhibited in MGMT-expres-sing melanoma cells, temozolomide-induced DNA damage trig-gers a senescence response that correlates with p53-mediatedupregulation of p21.

DiscussionBecause melanomas are notoriously resistant to chemotherapy,

we assessed the potential of combining temozolomide and PARPinhibition in melanoma cells. Strikingly, we observed that PARP,required for BER, plays a role in MGMT-mediated resistance totemozolomide. In MGMT� cells, PARP function appears to bedispensable for the repair of temozolomide-inducedDNAdamage,because olaparib does not enhance the temozolomide-inducedeffects on growth.On the other hand, inMGMTþ cells, theMGMT-mediated resistance to temozolomide-induced DNA damage isdependentonPARP, as evidencedby the synergyobservedbetweenPARPi and temozolomide to inhibit proliferation.

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Figure 3.Temozolomide (TMZ)–PARPi synergyin other cancer types. A andB, effect oftemozolomide with or withoutolaparib on seven glioblastoma celllines grouped upon their MGMT statusas determined by Western blotanalysis (B). C, Potentiation Factor50 of olaparib for each cell line.D, dose–response assay onneuroendocrine pancreatic BON(MGMTþ) and QGP-1 (MGMT�) cellstreated with serial increasingconcentrations of temozolomide inthe absence (solid lines) or presence(dotted lines) of olaparib. E, MGMTexpression in BON and QGP-1 cellsassessed byWestern blot analysis andpromoter methylation. F, MGMT levelson seven colorectal cancer cell lines.G, colony formation assay on sevencolorectal cancer cell lines groupedupon MMR status and treated asindicated. H, olaparib PotentiationFactor 50 for each cell line used in G.Student t test was used for statisticalcomparisons. � , P < 0.05; �� , P < 0.001;ns, not significant.






We suggest that, upon temozolomide treatment, a threshold ofN3-,N7-methylationneeds to be reachedbefore PARP-dependentDNA repair becomes relevant to the antiproliferative effect oftemozolomide. In the absence of MGMT, 06-meG is not repaired(4), and the immediate toxicity of 06-meG prevents this thresholdto be reached. Alternatively, tolerance to 06-meG due to deficientMMR might allow to reach this threshold, which would explainprevious results, obtained inMMR�/MGMT� leukemia cells (40).

So far the combination of MGMT inhibitors with dacarbazineor temozolomide has failed in the clinic, but our data suggest thatthe MGMT status could be a predictive marker for response totherapies combining temozolomide and PARPi. To date, early-phase clinical trials using temozolomide andveliparib inCNSandprostate cancer patients have shownmodest activity (21, 41). Onthe other hand, one phase II trial in patients with melanomashowed an increase in progression-free survival over historical

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Figure 4.MGMT reexpression restores PARPi–temozolomide (TMZ) synergy. A,Western blot analysis for MGMTexpression in lysates from WM98-1melanoma cell clones expressingeither an empty vector or MGMT. B,graphs show the average GI50 forthree empty vector and fourMGMT-expressing clones treated withtemozolomide with or withoutlomeguatrib or olaparib. C, PF50 ofolaparib on cells treated in B. D, colonyformation assay on WM98-1-MGMTcells treated with temozolomide withor without olaparib in the presence orabsence of constitutive inhibition ofMGMT. E, representative colonyformation assay from C. F,Potentiation factor for olaparib oncells treated as in D. G and H, averageGI50 for MMS in the presence orabsence of olaparib in WM98-1 cellsexpressing MGMT or empty vector (G)or in a panel of MGMTþ andMGMT� melanoma cells lines (H).��, P < 0.01; ��, P < 0.001; ns, notsignificant.

Erice et al.





records (23). Although trials are in progress, it is clear that there isstill a need for biomarkers predicting response to specific therapiesto increase clinical response. Indeed the upcoming clinical trial inglioblastoma combining temozolomide and veliparib in patientswith MGMT promoter methylation (A071102, NCT02152982,www.clinicaltrials.gov) will hopefully shedmore light on the roleof MGMT as a marker.

Because we observe a role for MGMT in the temozolomi-deþPARPi mediated growth inhibition in cell lines from fourdifferent cancer types, our data suggest a common mechanism ofresponse to temozolomide. Indeed Palma and colleaguesobserved that a first cycle of temozolomide and veliparib wasmost effective in MGMTþ tumors from different origin. Initially,MGMT� tumors did not benefit from PARPi cotreatment while asecond cycle demonstrated efficacy in tumors that underwentreexpression of MGMT (42). On the other hand, cancer-type

specific responses cannot be ruled out as it has been suggestedthat only MGMT� leukemia and glioblastoma multiforme cellswould respond to temozolomide combined with the PARPiveliparib (40, 43, 44). It is nevertheless important to considerthat variables such as differences in PARPi action, bioavailability,or the applicationof particular treatment regimes need to be takeninto account to correlate in vitro and in vivo efficacy of temozo-lomide and PARPi combinations as well as clinical efficacy(31, 42–45). Indeed, to synergize with temozolomide, both drugswork through catalytic PARP inhibition and trapping PARP–DNAcomplexes. Although olaparib has been shown to be more effec-tive to trap PARP–DNA complexes than veliparib, we showevidence that the role of PARP in MGMT-induced resistance canbe overcome by both inhibitors (31). This could be explained bythe different concentrations at which olaparib and veliparib wereused (0.25 and 5 mmol/L, respectively).

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Figure 5.Melanoma cell response totemozolomide (TMZ) and PARPinhibition. A and B, quantificationof gH2AX expression inWM266-4 and A375 cellstreated as determined byimmunofluorescence. C and D,Western blot analysis of phosphoThr68-CHK1 (ppCHK1), phosphoSer345-CHK2 (ppCHK2) inWM266-4 and A375 cells treatedas indicated. E, effects on the cellcycle: distribution in G1, S, andG2–M phases of the cell cycle ofWM266-4 and A375 cells treatedfor 3 days with temozolomide withorwithout olaparib. F, top,Westernblot analysis of cleaved caspase-3of RKO colorectal cells (CRC) orA375melanoma cells (MM) treatedwith 500mmol/L temozolomide for3 days. Bottom, cleaved caspase-3expression in A375 cells treated asindicated for 3 days. AZD,selumetinib (AZD-6244: 2mmol/L).G, bar and whisker graph showingthe increase in cell size upontreatment of MGMT� (WM98-1)and MGMT þ (A375 and WM98-1-MGMT) cells with temozolomidefor 5 days as determined usingImageJ. F and G, induction ofb-galactosidase activity upontreatment of WM98-1 (F) or A375(G) cells with temozolomide withor without olaparib for 5 days.�� , P < 0.01; �� , P < 0.001; ns, notsignificant.






Our data provide compelling evidence of MGMT as a potentialmarker for temozolomideþ PARPi-based therapies. If confirmedclinically, this has the potential for improving clinical responsesand reducing toxicity (41).

Importantly, MGMT status assessment remains a challenge andalthough MGMT promoter methylation has already entered clin-ical guidelines as predictive biomarker in elderly patients withglioblastoma, it has not been fully validated across cancer types(13, 46). We observed that some MGMT-expressing melanomacell lines and, notably, also someMGMT-expressing gliobastomacell lines are in fact positive for promoter methylation (Fig. 1B;ref. 25), suggesting that apositive signal for promotermethylationdoes not always correlate with MGMT expression. Thus, theMGMT promoter status alone might not be sufficient as markerin a clinical setting. As alreadydescribed for glioblastomaandNETtumors, we propose that this issue could be overcome by com-bining promoter methylation with IHC (47, 48).

Notably, we observed that temozolomide and olaparib failedto induce apoptosis in melanoma cells, but rather induced senes-cence, which ismost probably regulated through the p53/p21axis(39, 49). Whether treatment with monoalkylating agents inhibitsmechanisms of cell death, or whether the apoptotic machinery is

turned off as cells get arrested in the G2–M phase of the cell cyclewarrant further investigation.

In summary, our data provide strong evidence that, specificallyin cells expressingMGMT, the combination of temozolomide andPARPi might be of benefit and improve responses.

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

Authors' ContributionsConception and design: I. ArozarenaDevelopment of methodology: O. Erice, I. Goicoechea, J.C. Acosta,C. Wellbrock, I. ArozarenaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.P. Smith, R. White, J. Barriuso, C. Jones,G.P. Margison, J.C. Acosta, I. ArozarenaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): O. Erice, M.P. Smith, R. White, G.P. Margison,J.C. Acosta, I. ArozarenaWriting, review, and/or revision of the manuscript: O. Erice, I. Goicoechea,J. Barriuso, G.P. Margison, J.C. Acosta, C. Wellbrock, I. ArozarenaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G.P. Margison, I. ArozarenaStudy supervision: I. Arozarena

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Figure 6.Characterization of temozolomide(TMZ)/PARPi-induced senescence. A–C, induction of senescence-associatedb-galactosidase activity upontreatment of WM98.1 (A), WM98-1-MGMT (B), or A375 (C) cells asindicated for 5 days. D, quantificationof the DDR using antibodies againstthe consensus sequencephosphorylated by ATM and ATR(S/TQ) and gH2AX. E, temozolomideand olaparib cooperate to significantlyincrease p53 and p21 protein levels.F, temozolomide and olaparibsynergize to block DNA synthesis asdetermined by quantification ofBrDUrd incorporation. One-wayANOVA was used to compare theeffect of the double treatment withDMSO or temozolomide alone.�� , P < 0.001; ns, not significant.


Erice et al.




Grant SupportC. Wellbrock is a recipient of a program grant by Cancer Research UK

(C11591/A10202). J. Barriuso was funded through a fellowship byMarie Curie(FP7-PEOPLE-2012-IEF-329702). J.C. Acosta holds fellowships by CancerResearch UK (C47559/A16243) and Medical Research Council (R42576 MRC,Chancellors Fellowship).

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

Received September 22, 2014; revised February 10, 2015; accepted March 8,2015; published OnlineFirst March 16, 2015.

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2015;14:1236-1246. Published OnlineFirst March 16, 2015.Mol Cancer Ther Oihane Erice, Michael P. Smith, Rachel White, et al. TemozolomideMGMT Expression Predicts PARP-Mediated Resistance to

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