Poly(ADP-Ribose) Polymerase Is Hyperactivated in Homologous Recombination–Defective Cells

Published OnlineFirst June 15, 2010; DOI: 10.1158/0008-5472.CAN-09-4716 Published OnlineFirst on June 15, 2010 as 10.1158/0008-5472.CAN-09-4716

Molecular and Cellular Pathobiology Cancer Research Poly(ADP-Ribose) Polymerase Is Hyperactivated in Homologous Recombination–Defective Cells

Ponnari Gottipati1, Barbara Vischioni1,2, Niklas Schultz3, Joyce Solomons1, Helen E. Bryant4, Tatjana Djureinovic3, Natalia Issaeva3, Kate Sleeth1, Ricky A. Sharma1, and Thomas Helleday1,3

Abstract Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) is activated by DNA single-strand breaks (SSB) or at stalled replication forks to facilitate DNA repair. Inhibitors of PARP efficiently kill breast, ovarian, or prostate tumors in patients carrying hereditary mutations in the homologous recombination (HR) genes BRCA1 or BRCA2 through synthetic lethality. Here, we surprisingly show that PARP1 is hyperactivated in replicating BRCA2- defective cells. PARP1 hyperactivation is explained by the defect in HR as shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 proteins, which we here show are all essential for efficient HR and also caused PARP hyperactivation and correlated with an increased sensitivity to PARP inhibitors. BRCA2-defective cells were not found to have increased levels of SSBs, and PAR polymers formed in HR-defective cells do not colocalize to replication protein A or γH2AX, excluding the possibility that PARP hyperactivity is due to increased SSB repair or PARP induced at damaged replication forks. Resistance to PARP inhibitors can occur through genetic reversion in the BRCA2 gene. Here, we report that PARP inhibitor–resistant BRCA2-mutant cells revert back to normal levels of PARP activity. We speculate that the reason for the sensitivity of HR-defective cells to PARP inhibitors is related to the hyperactivated PARP1 in these cells. Furthermore, the presence of PAR polymers can be used to identify HR-defective cells that are sensitive to PARP inhibitors, which may be potential biomarkers. Cancer Res; 70(13); OF1–10. ©2010 AACR.

Introduction PARP1 is an abundant nuclear protein that rapidly binds to DNA single-strand breaks (SSB) and catalyses the formation of The concept of synthetic lethality refers to a form of genetic PAR polymers from NAD+ (5), attached primarily on glutamic interaction in which the combination of two gene knockouts acid residues on acceptor proteins. The function of PARP1 in is lethal. This concept can be extended to suggest that pro- DNA repair is to rapidly attract SSB repair proteins, such as teins that are normally nonessential but are critical for survi- XRCC1, to the site of damage to catalyze subsequent repair val in cancer cells, owing to cancer-specific gene inactivation, (6). PARP1 has also been shown to have a distinct role in a can be targeted for “personalized” cancer therapy. Such treat- backup nonhomologous end joining pathway (B-NHEJ), active ment is potentially highly beneficial as it would selectively kill in cells defective in DNA-PK–catalyzed NHEJ (7, 8). Recently, cancer cells while sparing normal cells (1). The most striking PARP1 was shown to also have a role in restart of stalled rep- example of synthetic lethality is the treatment of homologous lication forks by attracting Mre11 to sites of stalled replication recombination (HR)–defective tumors with poly(ADP-ribose) forks (9, 10). Altogether, these findings suggest that PARP1 has (PAR) polymerase (PARP) inhibitors (2, 3). These agents have a ubiquitous role in several DNA repair pathways. been tested as monotherapy in patients with BRCA1- and Synthetic lethality between PARP and HR has been ex- BRCA2-mutated cancers with positive early results (4). plained by the role of PARP in SSB repair (2). It has been sug- gested that PARP inhibition decreases the repair of abundant Authors' Affiliations: 1Cancer Research UK-Medical Research Council, SSBs, which in turn increase the number of collapsed repli- Gray Institute for Radiation Oncology and Biology, University of Oxford, cation forks that require HR-mediated repair (11). This mo- Oxford, United Kingdom; 2Division of Radiation Oncology, European Institute of Oncology and University of Milan, Milan, Italy; and del is backed up by the observation that replication forks 3Department of Genetics Microbiology and Toxicology, Stockholm collapsed by SSBs are the primary endogenous substrates University, Stockholm, Sweden; and 4The Institute for Cancer Studies, for HR (12). However, the unprecedented sensitivity of HR- University of Sheffield, Sheffield, United Kingdom defective cells to PARP inhibitors exceeds that of any other Note: B. Vischioni and N. Schultz contributed equally to this work. class of compounds by at least one order of magnitude, sug- Corresponding Author: Thomas Helleday, Cancer Research UK-Medical gesting that other functions of PARP may also underlie the Research Council, Gray Institute for Radiation Oncology and Biology, University of Oxford, ORCRB, Roosevelt Drive, Oxford OX3 7DQ, United strong synthetic lethal interaction observed. Kingdom. Phone: 44-1865-617-337; Fax: 44-1865-617-334; E-mail: Although the use of PARP inhibitors in patients with can- [email protected]. cers arising from an inherited mutation in either BRCA1 or doi: 10.1158/0008-5472.CAN-09-4716 BRCA2 is being established, a potential barrier to the more ©2010 American Association for Cancer Research. widespread use of PARP inhibitors to treat sporadic cancer is

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the identification of HR-defective tumors. In the study des- The secondary antibodies were AlexaFluor 555 donkey or goat cribed here, we find that HR-defective cells have hyperacti- anti-mouse IgG, AlexaFluor 488 donkey or goat anti-rabbit vated PARP activity. Furthermore, we show that PARP IgG, and AlexaFluor 555 or 594 goat anti-rat (all from Molec- inhibitor–resistant BRCA2-defective cells revert to nearly ular Probes). Antibodies were diluted in PBS containing normal PARP activity. These data challenge the current view 3% bovine serum albumin. Images were obtained with a Zeiss that the mechanism of action of PARP inhibitors relates pre- LSM 510 inverted confocal microscope using a planapochro- dominantly to SSB repair. Importantly, the data provide mat 63×/numerical aperture 1.4 oil immersion objective. evidence in favor of a potential biomarker to identify HR- Images were processed using Adobe PhotoShop (Abacus, defective tumors that will respond to PARP inhibitor therapy, Inc.). At least 300 nuclei were counted on each slide. a biomarker that may also be used to identify PARP inhibitor– Cell cycle analyses of PARP activity was made by correla- resistant BRCA1-orBRCA2-mutated cancers. ting the staining of the thymidine analogue EdU with the staining of PAR polymers. V-C8 cells were treated for Materials and Methods 30 minutes with 10 μmol/L EdU before fixation. Staining of EdU was performed according to the manufacturer's pro- Cell culture and isolation of PARP tocol (Click iT EdU Alexa Fluor 488 or 647, Molecular inhibitor–resistant cells Probes), and PAR polymers were stained with a rabbit poly- U2OS (human osteosarcoma) cells were obtained from clonal antibody. The mean fluorescence intensity for EdU American Type Culture Collection. Malgorzata Z. Zdzienicka and PAR was calculated for each individual nucleus by using (Department of Molecular Cell Genetics, N. Copernicus the ToPro-marked DNA as marker for the nuclei. Around University, Bydgoszcz, Poland) generously provided the 100 cells from four different images were analyzed with V-C8 and V-C8+B2 hamster cell lines (13). Pancreatic cancer the program ImageJ (17). cell lines CAPAN-1, BXPC3, and PARP inhibitor–resistant CAPAN1 clone C2-12 (14) were kindly provided by Dr. Measurements of NAD(P)H levels Toshiyasu Taniguchi (Department of Obstetrics and Gynecol- A water-soluble tetrazolium salt (5 mmol/L WST-8) was ogy, University of Washington, Seattle, WA), and U2OS cells used to monitor the amount of NAD(P)H through its reduction stably transfected with shRNA against WRN (15) were to a yellow formazan dye (18). Five thousand cells were plated provided by Dr. Vilhelm Bohr (Laboratory of Molecular at least in triplicate into wells of a 96-well plate and cultured in Gerontology, National Institute on Aging, Baltimore, MD). 100 μL normal growth medium for 4 hours at 37°C. CK8 buffer All cell lines in this study were grown in Dulbecco's modi- (Dojindo Molecular Technology), containing WST-8, was then fied Eagle's medium (DMEM) with 10% fetal bovine serum, added, and cells were incubated at 37°C. Reduction of WST-8 in 1× nonessential amino acids, and penicillin-streptomycin the presence of NAD(P)H was determined by measuring visible (100 units/mL) at 37°C under an atmosphere containing absorbance (OD450) after 4 hours. Relative levels of NAD(P)H in 5% CO2. different cells lines were calculated after 4-hour incubation in To isolate PARP inhibitor–resistant cells, 3 × 106 V-C8 cells CK8 buffer by comparing the cell lines. were treated with 60 μg/mL ethyl-methane sulfonate (EMS) in a 100-mm Petri dish. Upon reaching confluency, cells were PARP activity assay replated at 1 × 105 in 10% FCS DMEM to allow recovery and To assay cellular PARP activity in cells, cells were grown in phenotypic expression of mutagenesis for 4 days. The PARP 10-cm tissue culture dishes in medium alone or in medium inhibitor 1,8-naphthalimide (150 μmol/L) was added to the with 100 μmol/L ANI for 24 hours. Cells were washed once plates for 7 days before 26 colonies were individually selected with ice-cold PBS, lysed in 50 μL of PARP buffer containing and characterized. The BRCA2 gene was subsequently se- 0.5 mol/L NaCl, 1% (v/v) NP40, and Complete protease inhibi- quenced in six PARP inhibitor–resistant clones as described tors (Roche) on ice for 30 minutes with occasional vortexing. elsewhere (16). The lysates were clarified by centrifugation at 14,000 rpm, at 4°C for 10 minutes. The protein concentration of the extracts Chemicals was quantified using Bradford protein dye reagent (Bio-Rad), The PARP inhibitor 4-amino-1,8-naphthalimide (ANI) and and PARP activity was assayed using the Trevigen Universal hydroxyurea were purchased from Acros Organics. ANI and chemiluminescent PARP assay kit according to the manufac- hydroxyurea were dissolved in DMSO and PBS, respectively. turer's instructions, with modifications. The lysate (30 μg/well) was added in duplicates to the wells containing PARP buffer Immunofluorescence and PARP cocktail, followed by incubation at room tempera- Cells were plated onto coverslips and grown for 24 hours in ture for 1 hour. Activated DNA was added to the standards the presence or absence of treatments as indicated; cells were but was omitted from the extracts. The wells were washed fixed, stained, and mounted as previously described (10). The thrice with PBS and thrice with PBS and 0.1% Triton X-100, primary antibodies used were mouse monoclonal antibodies followed by incubation with a 1:1,000 dilution of streptavidin- against PADPR (clone-10H, Genetex) or rabbit polyclonal horseradish peroxidase in strep diluent buffer for 1 hour. The (clone 96-10-04, Alexis) γH2AX (clone JBW301, Millipore), wells were washed again thrice with PBS and thrice with PBS rabbit polyclonal antibodies against RAD51 (H-92, Santa Cruz and 0.1% Triton X-100. Chemiluminescent detection was per- Biotechnology), and rat monoclonal anti-RPA32 (Cell Signaling). formed per the manufacturer's instructions. The background

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reading was subtracted from the readings of the samples, and using Dharmafect-1 following the manufacturer's instruc- PARP activity was calculated using the standard curve tions. Cells were then cultured in normal growth medium obtained from readings of the standards. for 48 hours and then lysed for Western blot analysis as described above. shRNA treatment Small interfering RNA target sequences against RAD54: 5′- Alkaline comet assay GAATGATCTGCTTGAGTAT-3′, RAD52: 5′-AAAGACTACCT- The alkaline comet assay was performed as described pre- GAGATCACTA-3′,BLM:5′-GCTAGGAGTCTGCGTGCGA-3′, viously (19). Briefly, V-C8, V-C8+B2, and PARP inhibitor– WRN: 5′-TGAAGAGCAAGTTACTTGCTT-3′, and XRCC3: 5′- resistant V-C8 clones 1C and 2B were trypsinized, treated, AGAACGGCCTCCTTACACT-3′ were cloned into pENTR/ or mock treated in suspension with 30 μmol/L H2O2 for H1/TO vector using the BLOCK-iT Inducible H1 RNAi Entry 5 minutes on ice and embedded on a microscope slide in Vector Kit per the manufacturer's instructions. agarose (Bio-Rad). Cells were pretreated with 100 μmol/L The shRNAs were reverse transfected into the cells in ANI for 18 hours before PARP inhibitor treatment, as de- 96-well plates using Lipofectamine 2000 (Invitrogen) accor- scribed above. The slides were lysed in buffer containing ding to the manufacturer's instructions. Cells were then cul- 2.5 mol/L NaCl, 100 mmol/L EDTA, 10 mmol/L Tris-HCl tured in normal growth medium for 48 hours before (pH 10.5), 1% (v/v) DMSO, and 1% (v/v) Triton X-100 for trypsinization and replating for toxicity assays, or lysis and 1 hour at 4°C. The slides were then incubated in the dark extract preparation for Western blotting and Trevigen assay. for 30 minutes in cold electrophoresis buffer [300 mmol/L Depletion was confirmed by Western blotting. For the NaOH, 1 mmol/L EDTA, 1% (v/v) DMSO (pH 13)] to allow 24-hour treatment with PARP inhibitor, 100 μmol/L ANI the DNA to unwind before electrophoresis at 25 V for was added 24 hours after transfection. 25 minutes. After neutralization with 0.5 mol/L Tris-HCl (pH 8.0), the slides were stained with SYBR Gold (Invitrogen) Toxicity assay and analyzed using the Komet 5.5 image analysis software Two hundred cells were plated in duplicate into six-well (Kinetic Imaging Ltd.). plates overnight before the addition of increasing doses of PARP inhibitor for a continuous treatment. Seven to 12 days Flow cytometry later, when colonies could be observed, cells were fixed and U2OS cells expressing the DR-GFP substrate (20) were re- stained with methylene blue in methanol (4 g/L). Colonies verse transfected with shRNAs in 96-well plates as described consisting of >50 cells were subsequently counted. When above. Cells were then cultured in normal growth medium shRNA-depleted cells were used, they were transfected as for 48 hours and were then either transfected or not with above for 48 hours, then replated in the presence or absence 0.2 μg/well pCMV3xnlsI-SceI vector using 0.25 μg/well of of increasing doses of the PARP inhibitor. the transfection reagent polyethylenimine. Cells were then cultured in normal growth medium for a further 48 hours. Western blotting Cells were then trypsinized and analyzed for green fluores- Cells were lysed in radioimmunoprecipitation assay buffer cent protein (GFP) expression in 96-well plates using the inthepresenceof1×proteaseandphosphataseinhibitor BD FACSCalibur HTS system on a green (488/530 nm) versus cocktails (Sigma). Protein concentrations of the extracts were orange (633/661 nm) fluorescent plots. determined using Bradford protein dye reagent (Bio-Rad). An aliquot of 60 μg total protein was run on a SDS-PAGE Results gel and transferred to Hybond ECL membrane (Amersham Pharmacia). This membrane was immunoblotted with PARP1 is hyperactivated in BRCA2-defective cells mouse monoclonal antibody against PADPR (Clone 10H, PARP1-inhibited or knockout cells have an increased level Genetex), mouse polyclonal against PARP1 (Santa Cruz of HR, as seen by an increase in sister chromatid exchange Biotechnology), goat polyclonal against RAD54 (Santa Cruz (21), RAD51 foci (22), or HR (23). The hyper-HR phenotype is Biotechnology), rabbit polyclonal against RAD52 (Santa explained by the defect in SSB repair in PARP-inhibited cells, Cruz Biotechnology), goat polyclonal against BLM (Santa Cruz resulting in collapsed replication forks that trigger HR (2). Biotechnology), rabbit polyclonal against WRN (Santa Cruz Such a model predicts that HR acts downstream of PARP Biotechnology), mouse monoclonal against XRCC3 (Novus and hence would not be able to affect PARP activity. Biologicals), mouse monoclonal against β-actin (Millipore), To challenge this model, we stained BRCA2-defective V-C8 and mouse monoclonal against α-tubulin (Sigma), in 5% milk cells and the same cells complemented with the wild-type overnight. Immunoreactive protein was visualized using ECL BRCA2 gene (V-C8+B2) for PAR polymers using an antibody reagents (Roche) following the manufacturer's instructions. (Fig. 1A and B). We found an increase in the number of cells containing PAR foci, which represent sites of PARP activity PARP1 siRNA (Fig. 1C). To confirm the increased level of PAR polymers CAPAN1 cells were plated at a density of 100,000 per well in BRCA2-defective cells, we also determined the PAR level in a six-well plate and cultured in normal growth medium on Western blotting (Fig. 1D). We also blotted for the PARP1 overnight before transfection. Cells were then transfected protein and found that PARP1 protein levels are unchanged with 50 nmol/L of PARP1 siRNA (10) or nontargeting siRNA between HR-proficient and HR-defective cells, in line with

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Figure 1. PARP is hyperactivated in BRCA2-defective cells. Immunofluorescence staining for spontaneous PAR (green) and DNA (red) in BRCA2-defective V-C8 (A) and BRCA2-complemented V-C8+B2 (B) cells. Scale bar, 10 μm. C, quantification of the above immunofluorescence staining. D, Western blot analysis of PAR (top), and PARP1 and β-actin (bottom) in V-C8 and V-C8+B2 cells. E, PARP activity in the cellular extracts of V-C8 and V-C8+B2 cells measured as the amount of ribosylation on histone-coated plates. F, PARP activity in V-C8 and V-C8+B2 cells measured by the decrease of free NAD(P)H. G, Western blot analysis of PAR (top) and β-actin (bottom) in BRCA2-proficient BXPC3 and BRCA2-deficient CAPAN1 pancreatic cancer cells treated or not treated with 100 μmol/L of PARP inhibitor ANI for 24 hours. H, Western blot analysis of PARP1 (top), and PAR and β-actin (bottom) in CAPAN1 cells after PARP1 or nontarget (NT) siRNA depletion for 48 hours. The average and SD from at least three experiments are shown. Values marked with asterisks are statistically significant compared with control (**, P < 0.01; ***, P < 0.001).

previous observations that PARP levels and activity do not and that the amount of PAR polymers could be reduced by correlate (24). a PARP inhibitor (Fig. 1G), showing that this effect was not The most likely explanation for the increase in PAR poly- limited to the V-C8 cells. mers is an increase in PARP activity. To test if PARP is acti- Although there are at least 15 PARP proteins described, vated in BRCA2-defective cells, we used a modified in vitro most cellular PAR polymers are attributed to PARP1 activity. PARP activity assay. In this assay, cell lysates were incubated Here, we wanted to determine if the increase in PAR poly- in the presence of NAD+ and a conjugated histone acceptor mers is a consequence of PARP1 being hyperactivated. To protein, but in the absence of activated DNA, allowing us to test this, we performed PARP1 siRNA depletion in CAPAN1 determine already activated PARP in the cell lysates. Using cells and found that the increase in PAR polymers could be this assay, we found that BRCA2-defective cells have more entirely reversed by the depletion (Fig. 1H), showing that activated PARP compared with the same cells complemented PARP1 is hyperactivated in BRCA2-defective cells. WT with a BRCA2 gene (Fig. 1E). PARP activity can also be measured in cells by quantifying the level of free NAD(P)H, Loss of HR results in PARP hyperactivation and which is in equilibrium with NAD+ (18). Using this method, PARP inhibitor sensitivity we found that the total level of free NAD(P)H in BRCA2- Next, we wanted to determine if the increased PARP activity defective V-C8 cells is only two thirds of that in BRCA2- and PAR levels was a result of loss of HR in BRCA2-defective complemented V-C8+B2 cells (Fig. 1F), presumably because cells. To test this, we transiently depleted the levels of nones- a large portion of NAD+ is incorporated into PAR polymers. sential HR proteins RAD54, RAD52, BLM, and WRN as well as Although the V-C8 and V-C8+B2 cell pair is isogenic and XRCC3 in osteosarcoma U2OS cells using shRNA vectors, and only differs in BRCA2 status, we wanted to determine if the confirmed the reduced protein levels (Fig. 2A). First, we deter- effect was more widely applicable to other BRCA2-defective mined that depletion of these proteins indeed affects HR. The cells. The CAPAN1 pancreatic cancer cell line is defective in DR-GFP construct previously published (20) was integrated BRCA2 and is often used in combination with the BXPC3 into U2OS cells, and HR between the two nonfunctional GFP pancreatic cell line that is proficient in BRCA2 (25). We genes to generate a functional GFP gene can be triggered by found an increased level of PAR polymers present in transient transfection of the pCMV3xnlsI-SceI vector, which BRCA2-defective CAPAN1 cells compared with BXPC3 cells introduces a DNA double-strand break (DSB) in one of the

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two GFP genes. HR proficiency can be determined by the num- was not as profound as reported in BRCA2 knockout or ber of cells expressing the GFP protein (Fig. 2B). Depletion of mutated cells, but similar to that previously obtained using RAD54, RAD52, BLM, WRN, or XRCC3 all decreased HR fre- siRNA strategies (2, 23), possibly on account of the inability quency, showing that all of these proteins are indeed required of shRNA to completely deplete the HR protein levels for efficient HR (Fig. 2C). (Fig. 2A) and the transient nature of the depletion. Interestingly, we found a marked increase in PAR polymer formation in all cells depleted for any of the HR proteins, Decreased PARP activity in BRCA2-defective cells which could be reverted by the addition of a PARP inhibitor selected for PARP inhibitor resistance (Fig. 3A). Acquired resistance to cisplatin and PARP inhibitors in We next determined if PARP is activated after depletion of BRCA2-defective cancer cells overlaps and involves reactiva- HR using the modified in vitro PARP activity assay described tion of HR through secondary mutations in the BRCA2 gene above. Using this assay, we found that HR-depleted cells have (14, 26). Such acquired resistance may explain why some pa- more activated PARP than control-transfected cells (Fig. 3B). tients carrying BRCA1 or BRCA2 mutations do not respond We also wanted to determine if the increased PARP activity to PARP inhibitor treatment (4). Here, we wanted to test if in HR-depleted cells also correlated with a decreased survival BRCA2-defective cells that acquire resistance through gene- to a PARP inhibitor (ANI). Unsurprisingly, we found that all tic reversion also revert their high PARP activity back to low cells depleted for HR proteins showed an increased sensitivity PARP activity. To test this, we determined the PAR polymer to PARP inhibitors (Fig. 3C), in line with previous reports levels in C2-12 clone of the CAPAN1 cells, which is resistant (2, 3). The magnitude of sensitivity in shRNA-depleted cells to cisplatin due to reversion of the BRCA2 gene (14). We

Figure 2. Depletion of HR proteins impair HR in the DR-GFP recombination reporter. A, Western blot analysis of RAD54, RAD52, BLM, WRN, or XRCC3 depletion in U2OS cells when transiently transfected with the respective shRNAs for 48 hours. β-Actin is used as a loading control. B, DR-GFP HR assay, investigating the restoration of a functional GFP gene from two defective GFP genes through HR was used to determine HR proficiency in shRNA-depleted U2OS cells. Transient transfection of the pCMV3xnlsI-SceI vector (+I-SceI) introduces a site-specific DSB in one of the GFP copies that can then be repaired by HR to restore a functional GFP gene that is scored by fluorescence- activated cell sorting analysis. C, quantification of GFP-positive U2OS cells following pCMV3xnlsI-SceI transfection and depletion of HR proteins by shRNA. The average and SD (error bars) from four experiments are depicted. Asterisks designate statistical significance in t test (***, P < 0.001).

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found that these cells displayed lower levels of PAR polymers compared with the CAPAN1 cells (Fig. 4A), suggesting that the genetic reversion was accompanied with a reversion of the PARP hyperactivity back to normal levels. To investigate this mechanism of resistance further, we treated BRCA2-defective V-C8 cells with EMS before selection with a PARP inhibitor to isolate independent PARP inhibitor– resistant V-C8 clones. Parental V-C8 cells have two nonsense mutations in different alleles of the BRCA2 gene, one in exon 15 and the other in exon 16, resulting in a premature stop codon and a nonfunctional protein (16). We sequenced the BRCA2 cDNA in the five PARP inhibitor–resistant V-C8 clones, and all clones showed the same mutation, restoring the correct reading frame for BRCA2 and at the same time introducing a mutation within a highly conserved region in exon 15 (Fig. 4B). This mutation affects a highly conserved arginine that was also identified in a family with breast and ovarian cancers (27) and was previously described in mito- mycin C–resistant V-C8 cells (16). Thus, the reverted BRCA2 still has a defective ssDNA domain in the COOH-terminal part of the protein, as described earlier (14, 26). We tested the sensitivity of PARP inhibitor–resistant clones to another PARP inhibitor (ANI) and found that the clones had lost their sensitivity to PARP inhibitors (Fig. 4C). To determine if HR is restored in PARP inhibitor–resistant V-C8 clones, we investigated RAD51 foci formation as a hall- mark of ongoing HR in response to PARP inhibitor treatment. Although V-C8 cells were unable to form RAD51 foci also after hydroxyurea treatment (data not shown), we found that revertants had restored ability to form RAD51 foci in response to hydroxyurea (Fig. 4D and E), showing that the resistance mechanism described here is similar to that previously reported (14, 26). To test if the PARP inhibitor–resistant clones also reverted their PARP activity, we stained for the presence of PAR polymers in the revertant clones using Western blotting (Fig. 4F). We also measured PAR formation on the histone substrate as described above in two of the five clones and found a lower PARP activity level compared with the BRCA2-defective cells (Fig. 4G), which confirmed that the reversion to resistance is also accompanied with a reduced PAR formation.

PARP hyperactivation in HR-defective cells is not a result of an increase in DNA SSBs or stalled replication forks Figure 3. Depletion of HR proteins results in PARP hyperactivity and PARP1 has numerous functions in DNA metabolism, such correlates with survival following treatment with a PARP inhibitor. To test if PARP hyperactivation is related to reduced viability in HR-defective as transcription, chromatin organization, replication, and re- cells, we depleted nonessential HR proteins in U2OS cells. A, Western pair, and is activated primarily by DNA strand breaks (28). blot analysis of PAR (top) and β-actin (bottom) in U2OS cells transiently Here, we wanted to gain insights into the reason for PARP transfected with shRNAs against RAD54, RAD52, BLM, WRN, or XRCC3 being hyperactivated in HR-defective cells. HR is involved μ for 48 hours either treated or not treated with 100 mol/L of the PARP in the repair of DSBs, arising either as a result of collapsed inhibitor ANI for 24 hours. B, PARP activity (measured as the amount of ribosylation on histone-coated plates using the Trevigen assay) in the replication forks (11) or those induced after DNA replication cellular extracts of U2OS cells shRNA-depleted for RAD54, RAD52, BLM, (29, 30). There is a possibility that the number of SSBs and/or WRN, or XRCC3 in the presence or absence of 100 μmol/L PARP DSBs is higher in HR-defective cells, which could explain the inhibitor ANI. The average and SD from three experiments are shown. increase in PARP activity after loss of HR. To test this hy- C, clonogenic survival fraction of U2OS cells shRNA-depleted for RAD54, RAD52, BLM, WRN, or XRCC3 treated for 10 days with increasing pothesis, we measured the total amount of all DNA strand doses of PARP inhibitor ANI. The average and SD from four breaks (SSB+DSB) using the alkaline comet assay (Fig. 5A). experiments are shown. We found no increase in amount of DNA damage between

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Figure 4. PARP inhibitor and cisplatin-resistant BRCA2-defective cells resume near-normal PARP activity. A, Western blot analysis of PAR (top) and α-tubulin (bottom) in pancreatic cancer cell lines CAPAN1 defective in BRCA2, BXPC3 proficient in BRCA2, and the cisplatin-resistant clone of CAPAN1 (C2-12; 14). B, secondary genetic changes in mutated BRCA2 in PARP inhibitor–resistant V-C8 clone 2B as depicted by BRCA2 sequence analysis of BRCA2-defective V-C8 (top) and PARP inhibitor–resistant V-C8 clone 2B (VC8 PIR 2B; bottom). C, clonogenic survival in BRCA2-defective V-C8, BRCA2-complemented V-C8+B2, and V-C8 PARP inhibitor–resistant clones following treatment with increasing concentrations of the PARP inhibitor ANI. D, immunofluorescence staining for RAD51 and γH2AX foci formation in PARP inhibitor–resistant V-C8 cells following 1 mmol/L hydroxyurea treatment for 24 hours. Scale bar, 10 μm. E, quantification of hydroxyurea-induced RAD51 foci induced in PARP inhibitor–resistant V-C8 cells. F, Western blot analysis of PAR (top) and α-tubulin (bottom) in BRCA2-defective V-C8, BRCA2-complemented V-C8+B2, and PARP inhibitor–resistant V-C8 clones. G, PARP activity in cellular extracts of BRCA2-defective V-C8, BRCA2-complemented V-C8+B2, and PARP inhibitor–resistant V-C8 clones 1C and 2B, measured as the amount of ribosylation on histone-coated plates using the PARP activity assay. The average and SD from at least three experiments are shown. Values marked with asterisks are significantly different from control (*, P < 0.05; **, P < 0.01).

BRCA2-defective or BRCA2-proficient cells (Fig. 5B), sugges- the total number of strand breaks despite treating cells with ting that there is no overall increased level of SSBs in these a PARP inhibitor (Fig. 5B). Overall, our data show that the cells. Hydrogen peroxide was used as a positive control for increase in PARP activity in HR-defective cells was unrelated the induction of strand breaks. to repair of DNA strand breaks. There is a possibility that increased PARP activity denotes We recently reported that PARP is activated at stalled rep- an increase in SSB repair rate in HR-defective cells, which lication forks after hydroxyurea or thymidine treatments (10). would not be reflected as an increased level of overall SSBs. HR-defective cells often show poor growth rates as a result of If this is the case, inhibition of PARP would result in reduced a slow progression through the S phase of the cell cycle. The repair rates, and thus accumulation of SSBs. To test if there is poor growth phenotype of HR-defective cells can be reverted an increase in SSB repair rate in HR-defective V-C8 cells, we by complementing cells with a functional HR gene (31). Re- treated the cells with a PARP inhibitor (ANI) for 18 hours be- cently, it is also reported that HR proteins (i.e., BLM, Mre11, fore analysis. Our data show that there were no increase in XRCC3, and RAD51) are required for efficient restart of

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stalled replication forks (10, 32, 33). Thus, there is a possibility we fixed and stained for PAR polymers (Fig. 6C). We found that stalled replication forks accumulate in HR-defective cells that the amount of ongoing replication (EdU staining) cor- and that PARP binds to and is activated at these stalled forks. relates well with the intensity of PAR staining (Fig. 6D; To test this hypothesis, we induced PARP activity in U2OS R2 = 0.70), showing that PAR foci formation is linked with cells by stalling replication forks with hydroxyurea or follo- ongoing replication. In BRCA2-defective V-C8 cells, PAR wing depletion of WRN by shRNA (Fig. 6A and B). Replication foci did not costain with either RPA or γH2AX (Fig. 6E forks stalled with hydroxyurea accumulate ssDNA regions and F), suggesting that the lesion triggering PAR is not a that can be stained with the ssDNA binding protein replica- damaged replication fork. tion protein A (RPA). We previously showed that PAR foci colocalize to RPA-coated ssDNA regions at stalled replication forks (10), which was also seen here in U2OS cells treated Discussion with hydroxyurea (Fig. 6A). Depletion of the WRN protein using shRNA did not result in accumulation of spontaneous PARP inhibitors are an important novel class of anticancer RPA foci, and the PAR foci induced by the depletion of the drugs, and there are now more than 40 clinical trials ongoing WRN protein did not colocalize to RPA (Fig. 6A). or in development with PARP inhibitors in the treatment of HR proteins are involved in various processes at replica- cancer. Given the huge interest in PARP inhibitors, it is tion forks (33). To test if the increase of PARP activity is important to understand the mechanism of action of these related to replication, we incorporated the fluorescent thy- novel drugs and to be able to select patient populations that midine analogue EdU in BRCA2-defective V-C8 cells, which will respond to the treatment. Here, we show a marked increase in PARP activity in BRCA2-mutated V-C8 cells compared with the same cell line complemented with a functional BRCA2 gene, using four different methods. We also show that BRCA2-defective CAPAN1 cells have an increased PARP activity compared with the BXPC3 cells often used as a control. Because BRCA2-defective cells are hypersensitive to PARP inhibitors (2, 3), these data suggest that increased PARP activity in BRCA2-defective cells may be important for the selective cytotoxicity to these cells. Furthermore, the high level of PARP activity and in particular thepresenceofPARpolymersmaybeusefulinidentifying cancers that will respond to PARP inhibitor therapy. It is likely that HR proteins other than BRCA1 and BRCA2 are tumor suppressors. For instance, HR genes are mutated in non–Hodgkin's lymphoma, colon cancer (RAD54B, CtIP; refs. 34, 35), lipoma, uterine leiomyoma (RAD51B; ref. 36), skin basal and squamous cell, osteosarcoma (RECQL4; ref. 37), and in other cancers (BLM, WRN, Nbs1; refs. 37–39). Furthermore, several cancer cell lines are defective in HR (40). Thus, there is a possibility that PARP inhibitors may be useful also in treatment of other HR-defective tumors. Here, we show that shRNA depletion of RAD54, RAD52, BLM, WRN, and XRCC3 is associated in all cases with an increase in PARP activity and PAR polymer formation, which correlated with an increased sensitivity to a PARP inhibitor. Thus, PAR polymers may be useful as biomarkers to identify HR-defective cancers that would respond to PARP inhibitors. Unfortunately, not all BRCA-defective cancers seem to res- Figure 5. BRCA2-defective cells do not have increased levels of DNA pond to PARP inhibitor monotherapy (4), which may relate SSBs. Total DNA strand breakage was measured in BRCA2-defective to acquired resistance to cisplatin, which can occur through V-C8, BRCA2-complemented V-C8+B2, and PARP inhibitor–resistant genetic reversion of either the BRCA1 (41) or BRCA2 gene V-C8 clones 1C and 2B by alkaline comet assays with untreated cells (14, 26). Here, we show that the C2-12 cisplatin-resistant (control), with cells immediately after treatment with 30 μmol/L H O for 2 2 CAPAN1 clone, which reverted the BRCA2 mutation (14), 5 minutes on ice (H2O2), and with control and H2O2-treated cells pretreated with 100 μmol/L of the PARP inhibitor ANI for 18 hours. also reverted the PARP hyperactivity found in CAPAN1 cells. A, distribution of DNA strand breakage in the comet tails of control (top) We found that reversion of PARP hyperactivity is also true and H2O2-treated (bottom) V-C8 and V-C8+B2 cells. B, quantification of for V-C8 cells that reverted to PARP inhibitor resistance the total DNA strand breakage as percentage of tail DNA from the comet assay as described above. The percentage of tail DNA from at through BRCA2 reversion. These data suggest that low least 100 cells is quantified in each experiment. The average and SEM PAR polymer levels can predict resistance to PARP inhibitors from four experiments are shown. in BRCA2-mutated cancers.

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Hyperactivated PARP in Recombination-Defective Cells

Figure 6. PAR polymers formed in HR-defective cells are not associated with RPA foci. A, immunofluorescence staining for PAR and RPA foci formation in U2OS cells treated or not treated with 0.5 mmol/L of hydroxyurea for 24 hours or shRNA-depleted for WRN. PAR foci formed after hydroxyurea treatment colocalize with RPA foci; however, PAR polymers formed when HR protein WRN is depleted do not colocalize with RPA, suggesting that they are different from PAR foci formed at stalled replication forks. B, quantification of PAR foci in U2OS cells treated or not treated with 0.05 mmol/L of hydroxyurea (HU) for 24 hours or shRNA-depleted for WRN. The average and SD from three experiments are shown. C, immunofluorescence staining for PAR and EdU in V-C8 cells treated with 10 μmol/L of EdU for 30 minutes showing an S-phase dependence of PAR polymer formation. Scale bar, 10 μm. D, correlation between mean intensity of EdU and PAR staining for 100 nuclei (R2 = 0.70). E, immunofluorescence staining for PAR and RPA in V-C8 cells. Scale bar, 10 μm. F, immunofluorescence staining for PAR and γH2AX in V-C8 cells. Scale bar, 5 μm.

HR and PARP are both involved in the repair of DNA Here, we speculate that DNA lesions accumulate in HR- strand breaks and replication lesions. The synthetic lethality defective cells that required PARP-mediated repair and that between strand breaks and replication lesions is likely to be this is an important mechanism of action for the selective related to these repair pathways. We found that increased killing of HR-defective cells. PARP activity in HR-defective cells is not explained by accu- In conclusion, we show that PARP is hyperactivated in HR- mulation of DNA lesions known to trigger PARP activity (i.e., defective cells. We propose that this finding may be useful in DNA strand breaks or damaged replication forks). The le- identifying HR-defective cancers that will respond to PARP sions that trigger HR in mitotic mammalian cells are poorly inhibitor therapy or in identifying HR-defective tumors that characterized, and we know even less of putative lesions ac- are resistant to PARP inhibitor therapy. cumulating in HR-defective cells. PARP is involved in a vari- Disclosure of Potential Conflicts of Interest ety of DNA repair pathways [i.e., SSB repair (5, 6), B-NHEJ (7, 8), and replication repair (9, 10)], and it is likely that it A patent has been filed by Cancer Research Technology Ltd. based on the data is involved in yet undefined replication repair pathways. presented in this report, with T. Helleday and N. Schultz as named inventors.

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Gottipati et al.

Grant Support The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Swedish Cancer Society, Swedish Children's Cancer Foundation, Swedish Research Council, Swedish Pain Relief Foundation, Yorkshire Cancer Research, Received 12/31/2009; revised 04/07/2010; accepted 05/08/2010; published Cancer Research UK, and Medical Research Council. OnlineFirst 06/15/2010.

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OF10 Cancer Res; 70(13) July 1, 2010 Cancer Research

Poly(ADP-Ribose) Polymerase Is Hyperactivated in Homologous Recombination−Defective Cells

Ponnari Gottipati, Barbara Vischioni, Niklas Schultz, et al.

Cancer Res Published OnlineFirst June 15, 2010.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-09-4716

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