MGMT expression predicts PARP-mediated resistance to temozolomide

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1 MGMT expression predicts PARP-mediated resistance to temozolomide Oihane Erice 1,2* , Michael P. Smith 1* , Rachel White 3 , Ibai Goicoechea 1,4 , Jorge Barriuso 1 , Chris Jones 5 , Geoffrey P. Margison 6 , Juan Carlos Acosta 3 , Claudia Wellbrock 1,8 and Imanol Arozarena 1,7,8 1 Manchester Cancer Research Centre, The University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK 2 Current address: Division of Hepatology and Gastroenterology, Biodonostia Research Institute, Calle Doctor Begiristain, San Sebastian, 20014, Spain 3 Edinburgh Cancer Research UK Centre and MRC Institute of Genetics and Molecular Medicine, Western General Hospital. University of Edinburgh, Crewe Road South, Edinburgh, EH4 2XR, UK 4 Current address: Oncology area, Biodonostia Research Institute, Calle Doctor Begiristain, San Sebastian, 20014, Spain 5 Divisions of Molecular Pathology and Cancer Therapeutics, Institute of Cancer Research, 15 Cotswold Road, Sutton, SM2 5NG, UK 6 Centre for Occupational and Environmental Health, The University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PL, UK 7 Current address: School of Applied Sciences. University of Huddersfield. HD1 3DH *These authors contributed equally to this work 8 Corresponding authors: Imanol Arozarena and Claudia Wellbrock. Email: [email protected], Tel: +44-148-4472722 Email: [email protected], Tel: +44-161-2755189 on March 24, 2015. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 16, 2015; DOI: 10.1158/1535-7163.MCT-14-0810

Transcript of MGMT expression predicts PARP-mediated resistance to temozolomide

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MGMT expression predicts PARP-mediated resistance to temozolomide

Oihane Erice1,2*, Michael P. Smith1*, Rachel White3, Ibai Goicoechea1,4, Jorge

Barriuso1, Chris Jones5, Geoffrey P. Margison6, Juan Carlos Acosta3, Claudia

Wellbrock1,8 and Imanol Arozarena1,7,8

1 Manchester Cancer Research Centre, The University of Manchester, Michael

Smith Building, Oxford Road, Manchester, M13 9PT, UK

2Current address: Division of Hepatology and Gastroenterology, Biodonostia

Research Institute, Calle Doctor Begiristain, San Sebastian, 20014, Spain

3Edinburgh Cancer Research UK Centre and MRC Institute of Genetics and

Molecular Medicine, Western General Hospital. University of Edinburgh, Crewe

Road South, Edinburgh, EH4 2XR, UK

4Current address: Oncology area, Biodonostia Research Institute, Calle Doctor

Begiristain, San Sebastian, 20014, Spain

5Divisions of Molecular Pathology and Cancer Therapeutics, Institute of Cancer

Research, 15 Cotswold Road, Sutton, SM2 5NG, UK

6Centre for Occupational and Environmental Health, The University of

Manchester, Stopford Building, Oxford Road, Manchester, M13 9PL, UK

7Current address: School of Applied Sciences. University of Huddersfield. HD1

3DH

*These authors contributed equally to this work

8Corresponding authors: Imanol Arozarena and Claudia Wellbrock.

Email: [email protected], Tel: +44-148-4472722

Email: [email protected], Tel: +44-161-2755189

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Running title: MGMT mediated response to temozolomide and PARP inhibitors

Key words: melanoma, temozolomide, DNA damage, repair, MGMT, PARP

List of abbreviations: TMZ: temozolomide; MGMT: methylguanine-DNA-

methyl-transferase; OLAP: olaparib; ABT: ABT-888 or veliparib; MMS:

methylmethanesulfonate; CRC: colorectal cancer; MM: malignant melanoma;

DDR: DNA damage response

Financial information:

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

(C11591/A10202). J. Barriuso is funded through a fellowship by Marie Curie

(FP7-PEOPLE-2012-IEF-329702). J. C. Acosta holds fellowships by Cancer

Research UK (C47559/A16243) and Medical Research Council (R42576 MRC -

Chancellors Fellowship).

Person to whom reprints should be sent: Imanol Arozarena, School of Applied

Sciences. University of Huddersfield. HD1 3DH

Conflict of Interest Statement: The authors declare no conflict of interest.

Word count: 5020 words

Figures: 6 main figures plus 5 supplementary figures.

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ABSTRACT

Melanoma and other solid cancers are frequently resistant to chemotherapies

based on DNA alkylating agents such as dacarbazine and temozolomide (TMZ).

As a consequence, clinical responses are generally poor. Such resistance is partly

due to the ability of cancer cells to use a variety of DNA repair enzymes to

maintain cell viability. Particularly, the expression of MGMT has been linked to

TMZ resistance, but co-targeting MGMT has proven difficult due to dose limiting

toxicities. Here we show that the MGMT mediated resistance of cancer cells is

profoundly dependent on the DNA repair enzyme PARP. Both in vitro and in vivo

we observe that MGMT-positive cancer cells strongly respond to the combination

of TMZ and PARP inhibitors, while MGMT-deficient cells do not. In melanoma

cells TMZ induced an anti-proliferative senescent response, which was greatly

enhanced by PARP-inhibitors in MGMT-positive cells. In summary, we provide

compelling evidence to suggest that the stratification of cancer patients upon the

MGMT status would enhance the success of combination treatments using TMZ

and PARP inhibitors.

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INTRODUCTION

Melanoma is a form of skin cancer notoriously resistant to current therapies.

Before the arrival of targeted therapies against the BRAF and MEK components

of the hyper-activated MAPK pathway, alkylating agent-based chemotherapy was

the first line treatment for decades. In around 40% of all cases, patients qualify

for BRAF and MEK inhibitors and targeted therapies provide a good clinical

response. However, after 2-8 months, the vast majority of patients relapse (1-3).

For these patients their hopes lie in chemotherapy or immunotherapy. The only

FDA approved chemotherapeutic agent is the intravenously administered

prodrug dacarbazine, which is metabolically converted in the liver to MTIC, a

toxic monofunctional DNA alkylating agent (4, 5). An alkylating agent related to

dacarbazine but with excellent oral bioavailability is temozolomide (TMZ), which

can cross the blood brain barrier and therefore represents an alternative to

dacarbazine for melanoma with brain metastases (6-8). TMZ is an attractive

chemotherapy agent for patients with unresectable metastatic melanoma.

However single-agent escalated dose TMZ compared to single-agent dacarbazine

treatment did not improve overall survival or progression-free survival (9, 10),

and overall response rates for both drugs were 15-20% (5). TMZ is FDA

approved for anaplastic astrocytoma and glioblastoma, but again with poor

overall responses of around 26.5% (11). This suggests that in order to improve

the efficacy of TMZ, we need to enhance our understanding about the mode of

action of TMZ as a DNA damaging and cytotoxic drug.

TMZ methylates DNA bases predominantly at oxygen in position 6 and

nitrogen at position 7 in guanine and nitrogen at position 3 in adenine (O6-metG,

N7-metG and, N3-metA respectively). O6-metG is the most toxic and mutagenic

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DNA modification produced by TMZ and is quickly repaired by the enzyme 06-

methyl-guanine DNA methyl transferase (MGMT). MGMT incorporates the O6-

methyl adduct into a cysteine and the enzyme is then degraded by proteolysis

(4). On the other hand N3-metA and N7-metG repair are mediated by the base

excision repair (BER) machinery in a process that involves Poly-(ADP-ribose)-

polymerase proteins (PARP), which ADP-ribosylate DNA and proteins. When

acting on DNA, PARP proteins create a docking site for the incorporation of other

components of the BER machinery, which complete the repair process (4).

The most evident mechanism of resistance to both DTIC and TMZ is MGMT

activity, and MGMT down-regulation sensitises cancer cells to TMZ (12).

Moreover, in elderly glioblastoma patients MGMT status predicts clinical

response to TMZ (13) Unfortunately, therapies combining MGMT inhibitors with

TMZ in melanoma or colorectal cancer patients have so far failed to improve

outcome due to exacerbated treatment-related haematological toxicity (14-16).

More recently several small molecule inhibitors have been developed to target

other DNA repair mechanisms, with the aim to use them as synthetically lethal

single agents or in combination therapies with chemotherapeutic agents such as

TMZ. Notably, current clinical trials are testing PARP inhibitors as monotherapy

in BRCA-deficient breast cancer patients or in combination with DNA alkylating

agents (17, 18). Combination therapies of DTIC or TMZ and PARP inhibitors are

better tolerated than those with MGMT inhibitors, but myelosuppression and

liver toxicity still represent clinical concerns (19, 20), and overall survival rates

were not improved compared to patients treated with alkylating alone (21-23).

Therefore further research is warranted to identify markers that help stratify

patients for TMZ based therapies to improve clinical outcome. Our study

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suggests that MGMT mediated resistance to TMZ in melanoma and other cancer

types require PARP and that MGMT expression correlates with improved

response to combination of TMZ with PARP inhibitors.

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MATERIAL AND METHODS

Cell culture

Melanoma cell lines were provided by Prof. Richard Marais and Dr. Adam

Hurlstone and have been described recently (24). Colorectal carcinoma cells

were obtained from Prof. Stephen Taylor, originally from ATCC. Glioblastoma cell

lines have been described recently (25). Cell stocks were expanded and vials

kept in liquid nitrogen. New aliquots were thawed every 5-7 weeks. Melanoma

cell lines were authenticated in house by short tandem repeat profiling. Cells

were cultured in Dulbecco's Modified Eagle's Medium or in RPMI-164 medium

(SIGMA) as previously indicated, supplemented with 0.5% penicillin and

streptomycin (SIGMA) and 10% bovine calf serum (PAA, Yeovil, UK). Cells were

grown at 37ºC in a 5%CO2 environment. WM98.1-MGMT expressing cells were

established by transfection of a pcDNA3 vector encoding the cDNA for human

MGMT. MGMT expressing cells were selected using G418 as pcDNA3 expresses

the Neomycin resistance gene.

Reagents

Temozolomide and methylmethanesulfonate were from SIGMA. Olaparib,

veliparib (ABT-888), selumetinib (AZD-6244) and lomeguatrib were from

Selleck Chemicals, Newmarket, UK.

Proliferation assays

Cells were plated in 96-wel plates and treated with serial increasing

concentrations (range: 0.15M to 1mM) of drugs as indicated. After five days

cells were fixed and stained with toluidine blue, the concentration necessary to

inhibit cell growth to a 50% (GI50) was calculated as previously described (24).

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Colony formation assays

Cells were plated in 12-well plates and treated, from the top-left well, with DMSO,

5, 25, 75, 250 or 1000M TMZ for 24 hours (Supplementary Figure 1). Then cells

were washed and fresh, drug-free medium was replaced. Cells were left to form

colonies for 10-15 days before being fixed and stained with a solution of 4%

Formaldehyde (Fisher Scientific) and 0.5% Crystal violet (SIGMA) in PBS.

Remaining dye was solubilised in PBS, 1% Sodium Dodecyl Sulphate (SDS, Fisher

Scientific).

Xenograft Assay

Animal studies described within were approved by The University of Manchester

Ethical Review Board and performed according to UK Home Office regulations.

Briefly GFP-expressing human melanoma cells were injected into the pericardial

cavity of 48 hr post-fertilization zebrafish embryos. Embryos were treated at 1

day post-injection with DMSO (SIGMA), TMZ, olaparib or the combination of both.

On day 1 and 5 pictures were taken and relative growth quantified as previously

described (26).

Western blotting

Cell lysates were prepared using RIPA buffer and analysed as described (27).

Primary antibodies used were: MGMT (Thermo Fisher Scientific), phospho-

Thre68-CHK-2 and Ser345-CHK-1, MLH-1, MSH-2, MSH-6 and cleaved caspase 3

(Cell Signalling), ERK2 and beta-tubulin (Santa Cruz Biotechnologies).

MGMT activity assays

Briefly, increasing amounts of PARP or MGMT inhibitors were incubated with

recombinant MGMT (~6 fmoles) for 1h at room temperature, then substrate

DNA was added and incubation continued for 1h. After incubation, assay samples

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were processed as described previously in (28) and radioactivity quantified by

scintillation counting.

FACS analysis

100000 cells were treated as indicated for 3 days, fixed in 80% ice-cold ethanol

in PBS. Cells were then washed in PBS and incubated in a solution containing PBS,

RNase A and Propidium Iodide (SIGMA) at 37ºC for 1 hour. The analysis was

performed using FACS-Calibur (Becton Dickinson).

Senescence-associated β-galactosidade (SA-β-gal) assay

After 5 days of treatment cells were fixed in 0.5% glutaraldehyde and stained

overnight with X-gal solution: 0.12mM K3Fe(CN)6, 0.12mM K4Fe(CN)6, 1mM

MgCl2 and 1mg/ml 5-Bromo-4-cloro-3-indolyl--D-galactopiranoside in PBS pH

6.0. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Bright

field and UV images were taken using an inverted fluorescence microscope. The

percentage of SA-β-Gal-positive cells is determined upon counting of at least 100

cells.

High Content Analysis of the senescent response.

For the High Content Analysis, cells where plated in 96-well plates and stained

by immunofluorescence. Two fluorescence images corresponding to DAPI (to

identify individual cells) and gene-primary antibody/AlexaFluor488-secondary

antibody were acquired and analysed as previously described (29). Each well

was scored a percentage positive of reference channel to the total cells.

Antibodies used were: anti--H2AX (Millipore), anti-BrdU (Invitrogen), anti-

p21Cip1 (Sigma), anti-p53 (Santa Cruz) and anti-phospho-(Ser/Thr)Q (Cell

Signaling).

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Statistical analysis

Unless indicated otherwise, data are from assays performed in triplicate, with

error bars to represent standard deviations or errors from the mean. Statistics

used were: predominately Student t-test and One-way ANOVA with Dunnett's

Multiple Comparison Test performed using GraphPad Prism version 4.00 for Mac

OS, GraphPad Software, www.graphpad.com.

RESULTS

PARP inhibition specifically sensitises MGMT proficient cells to TMZ

To assess the ability of PARP inhibitors (PARPi) to sensitise melanoma cells to

TMZ we treated a panel of 12 melanoma cell lines with increasing concentrations

of TMZ in the absence or presence of the PARP inhibitor olaparib. We used

olaparib at a concentration of 0.25M, which did not affect cell proliferation

alone (Supplementary Figure 2A), but is reported to synergise with alkylating

agents to inhibit growth (30, 31). Olaparib significantly sensitised melanoma

cells to a five-day treatment with TMZ as seen by the GI50 values for TMZ

(Figure 1A and Supplementary Figure 1B). However, the cell lines segregated

into two defined groups: one with high GI50 values for TMZ, which showed

sensitisation to olaparib, and one with no significant difference (Figure 1A). We

therefore tested for differences in DNA repair regulators and found that all cell

lines belonging to the "high-GI50" group expressed enhanced levels of MGMT;

only one MGMT-expressing cell line segregated in the "low-GI50" group (Figure

1B). Expression data was complemented with methylation-specific PCR, where

MGMT promoter methylation was detectable in all MGMT-ve cells (Figure 1B).

When we grouped the cell lines upon MGMT status (MGMT+ve and MGMT-ve),

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this revealed that PARP inhibition only sensitized to TMZ in MGMT+ve

melanoma cells (Figure 1C). Indeed, the Potentiation Factor 50 (PF50, ratio of

the GI50 of TMZ in mono-therapy compared to the GI50 of TMZ in the presence

of PARP inhibitor) of olaparib was significantly higher in MGMT+ve cells than in

MGMT-ve cells where the PF50 was close to 1, indicating no potentiation (Figure

1D, Supplementary Figure 2B). Similar results were obtained from colony

formation assays where cells are tested for they capacity to repair DNA damage

induced by TMZ during just 24 hours. These assays confirmed that only MGMT

expressing cells responded to the combination of TMZ and olaparib (Figure 1E

and Supplementary Figure 2C).

Lastly we analysed whether the observed differential sensitivity to TMZ in the

presence of PARP inhibitors occurred also in vivo. To do so we used a human

melanoma xenograft assay in which we injected GFP-expressing A375

(MGMT+ve) or WM266-4 (MGMT-ve) cells into the pericardial cavity of zebrafish

embryos. This approach allows fast and efficient assessment of drug combination

treatments of tumour cells in an in vivo setting, which can be recapitulated in

other pre-clinical models (26, 32). Embryos were treated with DMSO or TMZ in

the presence or absence of olaparib and after 4 days xenograft volume was

assessed (Figure 1F). Again, A375 (MGMT+ve) xenografts responded to the

combination of olaparib and TMZ (Figure 1G), but did not affect xenograft

growth in WM266-4 (MGMT-ve) derived xenografts (Figure 1H). Together, our

data suggest that in human melanoma cells MGMT expression correlated with

synergistic action of TMZ and PARP inhibitors.

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MGMT is active in TMZ resistant cells

To establish whether MGMT expression correlated with its activity we used the

MGMT inhibitor lomeguatrib. We treated cell lines for 5 days with TMZ in the

absence or presence of 20M lomeguatrib (LOM), which only sensitised MGMT

expressing melanoma cells to TMZ. Accordingly the average PF50 for MGMT+ve

cells was significantly higher than in MGMT-ve cells (Figure 2A-C). As expected

the GI50 for MGMT+ve cells was significantly higher than for MGMT-ve cells.

Colony formation assays provided very similar results (Figure 2D-F). These data

confirmed that MGMT expression and activity are correlated in our panel of

melanoma cell lines, demonstrating that MGMT activity provides resistance to

TMZ and correlates with TMZ and PARP inhibitor synergy. Importantly, every

melanoma cell line expressed detectable levels of the Mismatch Repair (MMR)

proteins MSH-6, MSH-2 and MLH-1 (Supplementary Figure 3). This result,

together with the responses to TMZ and olaparib or lomeguatrib previously

observed, strongly suggested that all our melanoma cells are MMR proficient and

the observed differences are not related to MMR deficiency.

The MGMT-dependent response to TMZ-PARPi treatment is also seen in

other cancer types

To determine whether the impact of MGMT expression on the PARPi response is

specific for melanoma cells or cancer type-independent, we assessed the

response to TMZ and olaparib in cells from three cancer types where TMZ is

used as a therapeutic option: Glioblastoma, Pancreatic Neuroendocrine tumours

(NETs) and colorectal carcinoma (11, 33, 34). We treated seven glioblastoma cell

lines (four MGMT+ve and three MGMT-ve cell lines) known to be MMR+ve ((25))

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and observed that MGMT expression correlated with the response to TMZ

combined with olaparib (Figure 3A-C). Similarly, whereas the NET cell line BON,

which expresses high levels of MGMT responded to olaparib (and lomeguatrib)

co-treatment, QGP-1 NET cells, which lack MGMT, were unaffected by PARP

inhibition (Figure 3D-E). Again both NET cell lines where found to be MMR+ve

(Supplementary Figure 4A). Furthermore, a panel of seven MGMT expressing

colorectal cancer cell lines of known MMR status (35) responded to olaparib co-

treatment. (Figure 3F-G). Importantly, when grouped by MMR status the PF50

for olaparib was similar for both groups (Figure 3H). (Details of GI50s and PF50s

for each cell line can be found in Supplemental Figure 4B).

MGMT over-expression confers PARP dependent resistance to TMZ

Our data suggest that MGMT expression provides resistance to TMZ in a PARP

dependent manner. To further confirm these findings, we ectopically expressed

MGMT in MGMT-ve melanoma cells (WM98-1) (Fig. 4A). As expected, MGMT

over-expression conferred resistance to TMZ but lomeguatrib sensitised WM98-

1-MGMT clones to TMZ induced growth inhibition (Fig. 4B). Crucially, the MGMT-

produced resistance could be overcome by treatment with olaparib (Fig.4C).

Similar results were obtained using veliparib (Supplemental figure 5A-B).

Furthermore, we observed that when MGMT activity is inhibited, PARP

inhibitors no longer sensitised to TMZ (Figure 4E-F). We show that PARP is

required for the MGMT mediated resistance to TMZ. As mentioned above TMZ

produces N3- and N7-methyl adducts, which are repaired by PARP mediated

BER. To assess the requirement of PARP in this repair, we used the alkylating

agent Methylmethanesulfonate (MMS), which exclusively produces N3- and N7-

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methyl adducts (36). Both olaparib and veliparib significantly sensitised WM98-

1 and WM98-1-MGMT cells to MMS (Figure 4G and Supplemental Figure 5C).

Similar results were obtained in a panel of melanoma cells confirming that PARP

is required for efficient BER in melanoma cells, irrespective of their MGMT status

(Figure 4H and Supplemental Figure 5D). Finally, to rule out the possibility that

PARP inhibitors non-specifically interfere with MGMT activity, we performed in

vitro MGMT activity assays and confirmed that, unlike lomeguatrib, even high

concentrations of olaparib or veliparib failed to inhibit MGMT (Supplementary

Figure 5E).

PARP inhibition potentiates TMZ induced cell cycle arrest in MGMT

proficient cells

To further understand the action of TMZ and PARP inhibition in the context of

the MGMT expression status, we studied the induction of DNA single and double

strand breaks measuring histone H2AX phosphorylation (H2AX) after a 16h

treatment. PARP inhibition only synergised with TMZ to induce DNA damage in

MGMT+ve cells (Figure 5A, B). Analysis of CHK-1 and 2 phosphorylation, (Thr68

and Ser345 respectively), at 24h of treatment revealed that in MGMT-ve

WM266-4 cells TMZ activated CHK1/2 at lower concentrations than in MGMT+ve

A375 cells (Figure 5C-D). While PARP inhibition did not enhance the activation

of CHK2 by TMZ in WM266-4 cells, in A375 cells TMZ-induced CHK-2

phosphorylation was potentiated by olaparib. On the other hand CHK-1

phosphorylation did not correlate consistently with olaparib co-treatment.

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We next analysed the cell cycle profile of A375 and WM266-4 cells after 72 hours

of treatment. In WM266-4 cells sub-optimal concentrations of TMZ (5M, unable

to affect cell cycle progression) failed to cooperate with olaparib to promote a

cell cycle arrest while 20M TMZ (GI50= 73.26M), produced a marked G2/M

arrest (Figure 5E). In contrast, in A375 cells (GI50= 439.65M) combining

olaparib with a sub-optimal concentration of TMZ (50M) produced a dramatic

arrest in the G2/M phase of the cell cycle (Figure 5E). Strikingly we were not able

to detect the appearance of a sub-G1 phase, which is associated with cell death

(37). In agreement with the absence of a sub-G1 population, we did not observe

any caspase3 cleavage in response to TMZ in melanoma cells, whereas this was

detectable in colorectal cancer cells (Figure 5F). Moreover, co-treatment of A375

cells with concentrations of TMZ and OLAP that potently affect cell proliferation

failed to induce caspase3 cleavage (Figure 5F, lower panel). On the other hand

we observed that melanoma cells treated with TMZ displayed a very flat

appearance with a marked increase in cell size/surface (Figure 5G). Because

these are common traits of a senescent response, we decided to gain more

insight into the role of senescence in the response of melanoma cells to TMZ (38).

Characterisation of the senescent response induced by the combination of

TMZ and PARP inhibition

A 5day treatment with growth inhibitory concentrations of TMZ induced

Senescence Associated -galactosidase (SA--gal) staining in MGMT-ve WM98-1

as well as MGMT+ve A375 and WM98-1-MGMT cells (Figure 6, A to C),

confirming previous observations suggesting that TMZ induces senescence

rather than apoptosis in melanoma cells (39). However, in MGMT+ve cells

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treated with concentrations of TMZ that induced only a weak SA--gal activity,

PARP inhibition strongly enhanced the senescent response (Figure 6B-C). High

Content Analysis of A375 cells, used to assess the expression of several known

markers and mediators of senescence (29), confirmed that the combination of

TMZ and PARPi induced a potent DNA damage response (DDR, maintained over

5 days of treatment), as indicated by ATM and ATR phosphorylation (ST/Q) and

a H2AX signal (Figure 6D). The activation of the DDR was accompanied by a

significant increase in p53 levels and the cell cycle inhibitor p21 (Figures 6E).

Finally, as expected from a long-term cell cycle arrest we observed a dramatic

reduction in the number of cells undergoing DNA synthesis, as revealed by BrdU

incorporation assay (Figure 6F). In summary, when PARP is inhibited in MGMT

expressing melanoma cells TMZ induced DNA damage triggers a senescence

response that correlates with p53-mediated up-regulation of p21.

DISCUSSION

Because melanomas are notoriously resistant to chemotherapy, we assessed the

potential of combining TMZ and PARP inhibition in melanoma cells. Strikingly,

we observed that PARP, required for Base Excision Repair, plays a role in MGMT

mediated resistance to TMZ. In MGMT-ve cells PARP function appears to be

dispensable for the repair of TMZ induced DNA damage, because olaparib does

not enhance the TMZ induced effects on growth. On the other hand, in MGMT+ve

cells the MGMT mediated resistance to TMZ induced DNA damage is dependent

on PARP, as evidenced by the synergy observed between PARP inhibitors and

TMZ to inhibit proliferation.

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We suggest that, upon TMZ treatment, a threshold of N3-, N7-methylation needs

to be reached before PARP-dependent DNA repair becomes relevant to the anti-

proliferative effect of TMZ. In the absence of MGMT, 06-meG is not repaired (4),

and the immediate toxicity of 06-meG prevents this threshold to be reached.

Alternatively, tolerance to 06-meG due to deficient MMR might allow to reach this

threshold, which would explain previous results, obtained in MMR-ve/MGMT-ve

leukemia cells (40).

So far the combination of MGMT inhibitors with dacarbazine or TMZ has failed in

the clinic, but our data suggest that the MGMT status could be a predictive

marker for response to therapies combining temozolomide and PARP inhibitors.

To date, early phase clinical trials using temozolomide and veliparib in CNS and

prostate cancer patients have shown modest activity (21, 41). On the other hand

one Phase II trial in melanoma patients showed an increase in progression-free

survival over historical records (23). Although trials are in progress it is clear

that there is still a need for biomarkers predicting response to specific therapies

in order to increase clinical response. Indeed the upcoming clinical trial in

glioblastoma combining TMZ and veliparib in patients with MGMT promoter

methylation (A071102, NCT02152982, www.clinicaltrials.gov) will hopefully

shed more light on the role of MGMT as a marker.

Because we observe a role for MGMT in the TMZ+PARPi mediated growth

inhibition in cell lines from four different cancer types our data suggest a

common mechanism of response to TMZ. Indeed Palma et al. 2009 observed that

a first cycle of TMZ and veliparib was most effective in MGMT+ve tumours from

different origin. Initially, MGMT-ve tumours did not benefit from PARP inhibitor

co-treatment while a 2nd cycle demonstrated efficacy in tumours that underwent

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18

re-expression of MGMT (42). On the other hand cancer type specific responses

cannot be ruled out as it has been suggested that only MGMT-ve leukemia and

glioblastoma multiforme cells would respond to TMZ combined with the PARP

inhibitor veliparib (40, 43, 44). It is nevertheless important to consider that

variables such as differences in PARPi action, bioavailability or the application of

particular treatment regimes need to be taken into account to correlate in vitro

and in vivo efficacy of TMZ and PARP inhibitor combinations as well as clinical

efficacy (31, 42-45). Indeed, in order to synergise with TMZ, both drugs work

through catalytic PARP inhibition and trapping PARP-DNA complexes. Although

olaparib has been shown to be more effective to trap PARP-DNA complexes than

veliparib, we show evidence that the role of PARP in MGMT induced resistance

can be overcome by both inhibitors (31). This could be explained by the different

concentrations at which olaparib and veliparib were used (0.25M and 5M,

respectively).

Our data provide compelling evidence of MGMT as a potential marker for

TMZ+PARPi based therapies. If confirmed clinically, this has the potential for

improving clinical responses and reducing toxicity (41).

Importantly, MGMT status assessment remains a challenge and although MGMT

promoter methylation has already entered clinical guidelines as predictive

biomarker in elderly patients with glioblastoma it has not been fully validated

across cancer types (13, 46). We observed that some MGMT expressing

melanoma cell lines and, notably, also some MGMT expressing gliobastoma cell

lines are in fact positive for promoter methylation (Fig. 1B and (25)), suggesting

that a positive signal for promoter methylation does not always correlate with

MGMT expression. Thus, the MGMT promoter status alone might not be

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19

sufficient as marker in a clinical setting. As already described for glioblastoma

and NET tumours we propose that this issue could be overcome by combining

promoter methylation with immunohistochemistry (47, 48).

Notably, we observed that TMZ and olaparib failed to induce apoptosis in

melanoma cells, but rather induced senescence, which is most probably

regulated through the p53/p21axis (39, 49). Whether treatment with mono-

alkylating agents inhibits mechanisms of cell death, or if the apoptotic machinery

is turned-off as cells get arrested in the G2/M phase of the cell cycle warrants

further investigation.

In summary, our data provide strong evidence that, specifically in cells

expressing MGMT, the combination of TMZ and PARP inhibitors might be of

benefit and improve responses.

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49. Mirzayans R, Andrais B, Scott A, Murray D. New insights into p53 signaling and cancer cell response to DNA damage: implications for cancer therapy. J Biomed Biotechnol. 2012;2012:170325. Figure Legends

Figure 1. PARP inhibition synergises with TMZ only in MGMT+ve melanoma

cells

A, Melanoma cell lines were treated with TMZ with or without 0.25M olaparib

(OLAP). The graph shows the effect of PARP inhibition on the average GI50 for

TMZ. Each point corresponds to an individual cell line. B. Top, Western-blot for

MGMT expression in melanoma cell lines. ERK2 was used as a loading control.

Bottom, Methylation-specific PCR for un-methylated (U) and methylated (M)

promoter sequences. C-D, Cell lines were grouped upon their MGMT status, GI50

results from A re-analysed and Potentiation Factor 50 calculated. E, Potentiation

Factor results from colony formation assays on MGMT+ve and –ve cells treated

as indicated. Student’s t test was used to compares the effect of each treatment.

*p<0.05, **p<0.01. n.s: not significant. F, Pictures from A375 and WM266-4 cells

injected in zebra fish embryos and treated as indicated for 5 days. G-H,

Quantification of the in vivo effect of PARP inhibition on TMZ (500M) treated

xenografts. One-way Anova was used to compare the effect of each treatment

with DMSO (D) treated animals. ***p<0.001.

Figure 2. Characterization of melanoma cell lines upon MGMT status

A, Representative examples of dose-response curves for MGMT+ve and MGMT-

ve cells treated with TMZ with or without lomeguatrib (LOM). B-C, Graphs show

the average GI50 for TMZ in the presence or absence of lomeguatrib (B) and the

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Potentitation Factor 50 for each cell line (C). D, Colony formation assay on four

MGMT+ve and –ve cells treated treated with TMZ +/- LOM. E, Potentiation Factor

50 from D. F, Representative examples of colony formation assays using A375

(MGMT+ve) and WM9 (MGMT-ve) cells. D: DMSO.

Figure 3. TMZ-PARPi synergy in other cancer types

A-B, Effect of TMZ with or without olaparib of seven glioblastoma cell lines

grouped upon their MGMT status as determined by western-blot (B). C,

Potentiation Factor 50 of olaparib for each cell line. D, Dose-response assay on

neuroendocrine pancreatic BON (MGMT+ve) and QGP-1 (MGMT-ve) cell treated

with serial increasing concentrations of TMZ in the absence (full lines) or

presence (dotted lines) of olaparib. E, MGMT expression in BON and QGP-1 cells

assessed by western-blot and promoter methylation. F, MGMT levels on seven

colorectal cancer cell lines. G, Colony formation assay on 7 colorectal cancer cell

lines grouped upon MMR status and treated as indicated. H, Olaparib

Potentiation Factor 50 for each cell line used in G. Student’s t test was used for

statistical comparisons. * p<0.05, **p<0.001.

Figure 4. MGMT re-expression restores PARPi-TMZ synergy.

A, Western-blot for MGMT expression in lysates from WM98-1 melanoma cell

clones expressing either an empty vector or MGMT. B, Graphs show the average

GI50 for 3 empty vector and 4 MGMT-expressing clones treated with TMZ with

or without lomeguatrib or olaparib. C, Potentiation Factor 50 of olaparib on cells

treated in B. D, Colony formation assay on WM98-1-MGMT cells treated with

TMZ with or without olaparib in the presence or absence of constitutive

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inhibition of MGMT. E, Representative colony formation assay from C. F,

Potentiation factor for olaparib on cells treated as in D. G-H, Average GI50 for

methylmethane-sulfonate (MMS) in the presence or absence of olaparib in

WM98-1 cells expressing MGMT or empty vector (G) or in a panel of MGMT+ve

and –ve melanoma cells lines (H).

Figure 5. Melanoma cell response to TMZ and PARP inhibition

A-B, Quantification of H2AX expression in WM266-4 and A375 cells treated as

determined by immunofluorescence. C-D, Western-blot of phospho Thr68-CHK1

(ppCHK1), phospho Ser345-CHK2 (ppCHK2), of WM266-4 and A375 cells treated

as indicated. E, Effects on the cell cycle: Distribution in G1, S and G2/M phases of

the cell cycle of WM266-4 and A375 cells treated for 3 days with TMZ with or

without olaparib. F, upper panel: Western-blot of cleaved caspase 3 of RKO

colorectal cells (CRC) or A375 melanoma cells (MM) treated with 500M TMZ for

3 days. Lower panel, cleaved caspase 3 expression in A375 cells treated as

indicated for 3 days. AZD: selumetinib (AZD-6244: 2M). G, Barr and whisker

graph showing the increase in cell size upon treatment of MGMT-ve (WM98-1)

and +ve (A375 and WM98-1-MGMT) cells with TMZ for 5 days as determined

using ImageJ. F-G, Induction of beta-galactosidase activity upon treatment of

WM98-1 (F) or A375 (G) cells with TMZ with or without olaparib for 5 days.

Figure 6. Characterisation of TMZ/PARPi induced senescence.

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A-B-C. Induction of Senescence-associated -galactosidase activity upon

treatment of WM98.1 (A), WM98-1-MGMT (B) or A375 (C) cells as indicated for

5 days. D. Quantification of the DNA damage response using antibodies against

the consensus sequence phosphorylated by ATM and ATR (S/TQ) and H2AX. E,

TMZ and olaparib cooperate to significantly increase p53 and p21 protein levels.

F, TMZ and olaparib synergise to block DNA synthesis as determined by

quantification of BrDU incorporation. One-way Anova was used to compare the

effect of the double treatment with DMSO or TMZ alone.

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Figure 1.

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Figure 2.

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200

400

600

800

TM

Z G

I50 (m

M) *

ns

Figure 3.

MGMT

β-tub

A B

D

Glioblastoma cancer cells

F

TMZ

GI5

0 (

μM

)

MGMT+ve MGMT-ve

Pancreatic neuroendocrine cancer cells

rela

tive c

ell

num

ber

(%)

log[TMZ] (mM)

G H

C

Glioblastoma cells

Glioblastoma cells

MGMT

ERK2

BON-1CQGP

MGMT

ERK2

BON QGP1

MGMT

ERK2

BON-1CQGP

U M U M

BON QGP-1

E

MGMT

β-tub

Colorectal cancer cells

Colorectal cancer cells Colorectal cancer cells

D OLAP D OLAP0

500

1000

1500

TM

Z C

FI5

0 (

mM

)

MMR +VE MMR -VE

*

ns

**

HT29

SW

480

SW

837

RKO

LOVO

DLD

-1

HCT-1

16

All +v

e

All -v

e0

2

4

6

8

10

12

14

16

18

20

ola

parib P

F50

MMR+veMMR-ve

ns

on March 24, 2015. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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Figure 4.

A B

C D

E F

G

MGMT

ERK2

MGMT+ve clones

1 2 3 4

WM98.1

D OLAP D OLAP0

50

100

150

MM

S G

I50 (

mM

)

MGMT+ve MGMT-ve

*** ***

D LOM0

2

4

6

8

10

ola

parib P

F50

**

H

TMZ

GI5

0 (

μM

)

DLO

M

OLA

P DLO

M

OLA

P

0

200

400

600

800

**ns

***

vector MGMT

TM

Z C

FI5

0 (

mM

)

WM98.1

vector

1

vector

2

vector

3

MGM

T.1

MGM

T.2

MGM

T.3

MGM

T.4

All ve

ctor

All M

GM

T0

2

4

6

8

10

12

14

ola

parib P

F50

MGMT-veMGMT+ve

**

WM98.1

TMZ

GI5

0 (

μM

)

WM98.1-MGMT

Melanoma cells

WM98.1-MGMT

D

OLA

P D

OLA

P D

OLA

P

0

50

100

150

200

MM

S C

FI5

0 (

mM

)

vector MGMT c1 MGMT c2

*** *** ***

WM98.1

MM

S G

I50

M)

on March 24, 2015. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 16, 2015; DOI: 10.1158/1535-7163.MCT-14-0810

Figure 5.

A B

C WM266.4 (MGMT-ve) A375 (MGMT+ve)

E F

G

D

- + - + TMZ CRC MM

cleaved caspase 3

ERK2

ppCHK2

ppCHK1

ERK2

- + - + - + - + OLAP D 20 50 200 μM TMZ

- + - + - + - + OLAP

ppCHK2

ppCHK1

ERK2

D 5 20 50 μM TMZ

WM266.4 (MGMT-ve) A375 (MGMT+ve)

cleaved caspase 3

A375 - O - O - O - O - AZD

- T20 T50 T200 D

β-tub

DT100 D

T750 D

T500

0

10

20

30

40

50

Cell

siz

e (

A.U

.)

WM98.1 A375WM98.1 MGMT

*** **

***

D

OLA

P T5

T5 +

OT20 D

OLA

PT50

T50 +

O

0

25

50

75

100

GO/G1

S

G2/M

WM266.4 A375

% c

ells

on March 24, 2015. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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Figure 6.

A B

C D

E F

A375 (MGMT+ve)

WM98.1-vector (MGMT-ve)

WM98.1- clon 3 (MGMT+ve)

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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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on March 24, 2015. © 2015 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 16, 2015; DOI: 10.1158/1535-7163.MCT-14-0810