Werner Syndrome Helicase Has a Critical Role in DNA Damage Responses in the Absence of a Functional Fanconi Anemia Pathway

Home , FANCA, FANCD2, FANCG, Ku70, RAD51, Werner syndrome helicase

Published OnlineFirst July 18, 2013; DOI: 10.1158/0008-5472.CAN-12-2975

Cancer Therapeutics, Targets, and Chemical Biology Research

Monika Aggarwal1, Taraswi Banerjee1, Joshua A. Sommers1, Chiara Iannascoli3, Pietro Pichierri3, Robert H. Shoemaker2, and Robert M. Brosh, Jr.1

Abstract Werner syndrome is genetically linked to mutations in WRN that encodes a DNA helicase-nuclease believed to operate at stalled replication forks. Using a newly identiﬁed small-molecule inhibitor of WRN helicase (NSC 617145), we investigated the role of WRN in the interstrand cross-link (ICL) response in cells derived from patients with Fanconi anemia, a hereditary disorder characterized by bone marrow failure and cancer. In FA-D2 / cells, NSC 617145 acted synergistically with very low concentrations of mitomycin C to inhibit proliferation in a WRN-dependent manner and induce double-strand breaks (DSB) and chromosomal abnormalities. Under these conditions, ataxia–telangiectasia mutated activation and accumulation of DNA-dependent protein kinase, catalytic subunit pS2056 foci suggested an increased number of DSBs processed by nonhomologous end-joining (NHEJ). Rad51 foci were also elevated in FA-D2 / cells exposed to NSC 617145 and mitomycin C, suggesting that WRN helicase inhibition interferes with later steps of homologous recombination at ICL-induced DSBs. Thus, when the Fanconi anemia pathway is defective, WRN helicase inhibition perturbs the normal ICL response, leading to NHEJ activation. Potential implication for treatment of Fanconi anemia–deﬁcient tumors by their sensitization to DNA cross-linking agents is discussed. Cancer Res; 73(17); 1–11. 2013 AACR.

Introduction Werner syndrome cells are sensitive to interstrand cross- – Werner syndrome, characterized by premature aging and link (ICL) inducing agents (8) and undergo apoptosis in S- cancer predisposition (1), is caused by autosomal recessive phase (9). Cellular studies suggest WRN helicase, in conjunc- mutations in WRN (2), encoding a RecQ DNA helicase-nuclease tion with BRCA1, is required to process DNA ICLs (10). WRN is – implicated in genomic stability (3, 4). WRN helicase can required for ataxia telangiectasia mutated (ATM) activation unwind key homologous recombination DNA intermediates and the intra-S-phase checkpoint in response to ICL-induced and interacts with proteins implicated in DNA replication, double-strand breaks (DSB; ref. 11), suggesting WRN and ATM recombinational repair, and telomere maintenance (5). Wer- collaborate in response to collapsed forks at ICL-induced DSBs. ner syndrome cells are sensitive to certain DNA-damaging Biochemical studies using a reconstituted system with a DNA agents, and display defects in resolution of recombination substrate harboring a psoralen ICL suggested that WRN heli- intermediates and a prolonged S-phase (6). WRN is required case is required for ICL processing (12). for normal replication fork progression after DNA damage or Patients with Fanconi anemia suffer from progressive bone fork arrest (7). marrow failure and cancer predisposition, and their cells (such as Werner syndrome cells) are sensitive to ICL agents, only to a greater extent (13). In fact, a chromosome breakage test using Fanconi anemia patient cells exposed to an ICL-inducing agent Authors' Afﬁliations: 1Laboratory of Molecular Gerontology, National Institute on Aging, NIH Biomedical Research Center, NIH, Baltimore; is used as a primary diagnostic tool for Fanconi anemia (14). 2Screening Technologies Branch, Developmental Therapeutics Program, The Fanconi anemia pathway is involved in initial recognition Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, NIH, Frederick, Maryland; and 3Section of Experimental and and unhooking of an ICL and subsequent repair of the ICL- Computational Carcinogenesis, Istituto Superiore di Sanita, Rome, Italy induced DSB in conjunction with homologous recombination or translesion synthesis pathways, but the detailed molecular Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). mechanism is an active area of investigation (15). One function of the Fanconi anemia pathway is to channel DSBs through Current address for M. Aggarwal: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, the homologous recombination pathway, thereby preventing Georgetown University, 3800 Reservoir Road, NW, Washington, DC 20007. inappropriate engagement of breaks by error-prone nonho- Corresponding Author: Robert M. Brosh Jr, National Institute on Aging, mologous end-joining (NHEJ; refs. 16, 17). However, the rela- NIH, 251 Bayview Blvd, Baltimore, MD 21224. Phone: 410-558-8578; Fax: tionship between DNA repair pathways is complex, as evi- 410-558-8162; E-mail: [email protected] denced by a recent mouse study showing that deletion of 53BP1 doi: 10.1158/0008-5472.CAN-12-2975 or Ku exacerbates genomic instability in FANCD2-deﬁcient 2013 American Association for Cancer Research. cells (18).

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To understand the role of WRN in the ICL response, we plated to 50% to 60% confluence in 10-cm dishes 24 hours examined effects of a small-molecule WRN helicase inhibitor before transfection. siRNA (0.6 nmol) was mixed with 30 mLof in a Fanconi anemia–mutant genetic background. A more Lipofectamine 2000 in 3 mL of Opti-MEM (Invitrogen). The potent structural analog of the previously identified parent mixture was added to cells that were subsequently incubated compound NSC 19630 (19) was discovered that is blocked for 6 hours. After 24 hours, a second transfection was carried from potential thiol reactivity but retains the ability to mod- out similarly. Seventy-two hours after the initial transfection, ulate WRN function in vivo. The WRN helicase inhibitor NSC cells were harvested for preparing lysate or treated with small- 617145 acted synergistically with a very limited concentration molecule compounds or dimethyl sulfoxide (DMSO) at the of mitomycin C to induce DSB accumulation and chromosom- indicated concentrations and cell proliferation was measured al abnormalities, and activate the DNA damage response in using WST-1 reagent (Roche) as described for HeLa cells. For Fanconi anemia–mutant cells. NSC 617145 exposure resulted Fanconi anemia–mutant cells, siRNA transfection and treat- in enhanced accumulation of DNA-dependent protein kinase, ment with NSC 617145 was carried out as described previously catalytic subunit (DNA-PKcs) pS2056 foci and Rad51 foci in except that OD450 was measured after 48 hours of treatment mitomycin C–treated Fanconi anemia–deficient cells, suggest- with mitomycin C and/or NSC 617145. ing that WRN helicase inhibition prevents processing of Rad51- For lysate preparation, cells were washed twice with 1 PBS. mediated recombination products and activates NHEJ. WRN Radioimmunoprecipitation assay (RIPA) buffer [10 mmol/L helicase may be a suitable target for chemotherapy strategies sodium phosphate (pH 7.2), 300 mmol/L NaCl, 0.1% SDS, 1% in Fanconi anemia–deficient tumors. NP-40, 1% sodium deoxycholate, and 2 mmol/L EDTA] was added to the cells and the cells were incubated at 4C for 30 Materials and Methods minutes. Cells were scraped and the suspension was further Cell lines and culture incubated on ice for 30 minutes. Cell suspension was centri- HeLa (CCL-2), U2OS (HTB96), and HCT116 (CCL-247) cell fuged at 18,500 g for 10 minutes at 4C and supernatant was lines were obtained from American Type Culture Collection collected. Twenty microgram of the lysate was loaded on 8% to where they were tested and authenticated. HCT116 p53 double 16% SDS-PAGE. Protein was transferred onto a polyvinylidene knockout cells were obtained from the laboratory of Dr. B. difluoride (PVDF) membrane and blot was probed with anti- Vogelstein (Johns Hopkins University, Baltimore, MD) where WRN mouse monoclonal antibody (1:1,000; Spring Valley they were tested and authenticated (20). All these cell lines Laboratories). For secondary antibody, peroxidase-conjugated were grown in Dulbecco's Modified Eagle Medium supplemen- anti-mouse immunoglobulin G (IgG; 1:1,000; Vector Labora- ted with 10% FBS, 1% penicillin–streptomycin, and 1% L- tories) was used. Blot was developed using ECL Plus Western glutamine at 37 Cin5%CO2. PSNF5 [Bloom syndrome Blot Detection Kit as per the manufacturer's protocol (Amer- (BLM)–corrected] and PSNG13 (BLM-mutant) are isogenic sham). As a loading control, blot was stripped and then cell lines that were obtained from the laboratory of Dr. I. reprobed with anti-actin antibody (1:5,000; Sigma). Hickson (University of Copenhagen, Copenhagen, Denmark) where they were tested and authenticated (21, 22). PSNF5 is a Analysis of metaphase chromosomes stable BLM cell line expressing a FLAG epitope-tagged, wild- Cell harvest and metaphase slide preparation was carried type BLM protein from the cytomegalovirus (CMV) promoter out for metaphase analysis as described previously (23). in a pcDNA3-based construct. PSNG13 is a control BLM cell See Supplementary Data for additional information. transfectant containing the pcDNA3 vector only. The BLM- mutant and -corrected cell lines were grown in the same media Results as HeLa cells but supplemented with 350 mg/mL G418. The Inhibition of WRN helicase activity by small-molecule simian virus 40–transformed Fanconi anemia–mutant fibro- NSC 617145 blasts, PD20 (FA-D2), GM6914 (FA-A), and their respective To identify small molecules that inhibit WRN helicase corrected counterparts were provided by Fanconi Anemia Cell activity in a more potent manner than the previously identified Repository at Oregon Health & Science University (Portland, compound NSC 19630 (19), we selected four close structural OR) where they were tested and authenticated. Fanconi ane- analogs and three compounds whose pattern of activity in the mia–mutant and -corrected cells were grown in the same NCI60 screen matched NSC 19630 for analysis (Supplementary medium as HeLa cells but supplemented with 0.2 mg/mL Fig. S1). One of the latter compounds, NSC 617145, is also a puromycin. structural analog, but blocked with respect to thiol reactivity in the five-membered rings by Cl atoms. NSC 617145 inhibited Cell proliferation assays WRN helicase activity in a concentration-dependent manner in Proliferation was measured using WST-1 assay (Roche) as vitro (Supplementary Fig. S2A), yielding an IC50 value of 230 described previously (19). nmol/L. NSC 617145 inhibited WRN ATPase in a dose-dependent manner (Supplementary Fig. S2B). No detectable effect on siRNA transfection and Western blot analysis WRN exonuclease was observed (Supplementary Fig. S2C). WRN siRNA (WH: 50-UAGAGGGAAACUUGGCAAAUU-30 To examine specificity of WRN helicase inhibition, we tested 0 0 and/or CG: 5 -GUGUAUAGUUACGAUGCUAGUGAUU-3 ) and 5 mmol/L NSC 617145 (20-fold >IC50 value) on DNA unwind- control siRNA (19) were transfected using Lipofectamine 2000 ing catalyzed by various helicases. No significant inhibition of as per the manufacturer's protocol (Invitrogen). Cells were unwinding was observed with BLM, FANCJ, ChlR1, RecQ, and

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AB Day 0 120 120 DMSO Day 1 100 Day 2 100 1.5 µmol/L NSC 617145 80 Day 3 80 60 60 40 40 20 20 Proliferation (%) Proliferation (%) 0 0 321.510.750 3210 NSC 617145 (μmol/L) Days C 120 HeLa 100 WRN siRNA NS siRNA 80 60 40 20 Proliferation (%) 0 3210 Days

Figure 1. NSC 617145 inhibits cell proliferation in a WRN-specific manner. A, HeLa cells were treated with DMSO or indicated NSC 617145 concentration for 0 to 3 days. B, HeLa cells were exposed to 1.5 mmol/L NSC 617145 for 3 days, replenished with media lacking NSC 617145, and allowed to recover for 0 to 3 days. C, HeLa cells either untransfected (HeLa) or transfected with nonspecific siRNA (NS siRNA) or WRN-specific siRNA (WRN siRNA) were treated with DMSO or 1.5 mmol/L NSC 617145 for indicated number of days. Cell proliferation was then determined with WST-1 reagent. Percentage proliferation was calculated. Experiments were repeated three times, and error bars indicate SD. This applies to all figures.

UvrD, and only a very modest (7%) inhibition of RECQ1 endogenous WRN, and siRNA-resistant WRN [wild-type or (Supplementary Fig. S2D) was observed. helicase inactive K577M (24)] was expressed. The resulting To determine their potency in vivo, HeLa cells were exposed cells were tested for sensitivity to NSC 617145. It was observed to increasing concentrations of selected analogs or DMSO for 0 that the WRN-depleted cells rescued with wild-type WRN were to 3 days. Of the selected analogs, NSC 617145 showed maximal sensitive to the WRN helicase inhibitor compared with empty inhibition of proliferation (98%) at the lowest concentration of vector–transfected cells, supporting the finding that the inhi- NSC 617145 (1.5 mmol/L; Fig. 1A; Supplementary Fig. S3). To bition of cell proliferation by NSC 617145 is WRN-dependent address whether the effect was cytostatic or cytotoxic, HeLa (Supplementary Fig. S5). However, WRN-depleted cells expres- cells were exposed to NSC 617145 for 3 days, replenished sing the helicase-inactive WRN-K577M behaved similar to the with media lacking NSC 617145, and allowed to recover for WRN-depleted cells transfected with empty vector, suggesting 0 to 3 days (Fig. 1B). The lack of recovery from NSC 617145 that they were resistant to the negative effect of WRN helicase suggested a cytotoxic effect. To determine whether the effect inhibitor NSC 617145 on cell viability. of NSC 617145 was WRN-dependent, we compared its effect Because NSC 617145 inhibited proliferation of p53-inacti- on HeLa cells depleted of WRN (90%) with NS-siRNA– vated HeLa cells, we examined its effect on proliferation of transfected cells. WRN-depleted cells grown in the presence U2OS cells with wild-type p53. NSC 617145 (1.5 mmol/L) of 1.5 mmol/L NSC 617145 were resistant to its antiproli- inhibited U2OS proliferation by 80% after day 2 (Supple- ferative effects, whereas NS-siRNA HeLa cells were highly mentary Fig. S6A), suggesting that the effect of NSC 617145 sensitive to NSC 617145 (Fig. 1C). These results were con- was not dependent on p53 status. We also tested a pair of firmed by 2 different WRN-siRNA molecules that indepen- isogenic p53-negative and -positive HCT116 cells for sensi- dently conferred resistance to NSC 617145 (Supplementary tivity to NSC 617145 (1.5 mmol/L). Both cell lines were Fig. S4). Western blot analyses showed that WRN depletion sensitive to the compound; however, p53-deficient cells were by either WRN-siRNA or combination was more than 90% 2-fold more resistant than p53-proficient cells (Supplemen- (Supplementary Fig. S4). tary Fig. S6B). Because NSC 617145 exerted a WRN-depen- NSC 617145 may exert its effect through a mechanism in dent effect on proliferation, we evaluated whether cells which the NSC 617145–inhibited WRN helicase induces greater mutated for the sequence-related BLM helicase were sensi- damage by interfering with a compensatory mechanism(s). To tive. BLM-null and -corrected cells displayed similar sensi- determine whether the effect of NSC 617145 is dependent on a tivity to NSC 617145 (Supplementary Fig. S6C), suggesting helicase-active version of WRN, HeLa cells were depleted of BLM does not play a role.

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Cellular exposure to NSC 617145 causes accumulation of mmol/L) for 6 hours was reduced compared with DMSO- DSBs, blocked replication forks, and apoptosis treated cells (Fig. 2B). Inclusion of proteasome inhibitor Because WRN-deficient cells are defective in resolution of MG132 restored WRN level to that of DMSO-treated cells (Fig. recombination intermediates (6), NSC 617145 inhibition of 2B). Thus, NSC 617145 causes WRN to become degraded by a WRN helicase activity might result in DSBs at blocked repli- proteasome-mediated pathway. Consistent with reduction of cation forks. Therefore, we analyzed the effect of NSC 617145 WRN protein in NSC 617145–treated cells, helicase activity on g-H2AX foci, a marker of DSBs. Exposure of HeLa cells to catalyzed by immunoprecipitated WRN from an equivalent 0.25 mmol/L NSC 617145 elevated g-H2AX foci approximately amount of total extract protein from HeLa cells treated with 18-fold compared with DMSO-treated cells (Supplementary NSC 617145 (0.75 mmol/L; 4 hours) was reduced 4-fold com- Fig. S7A and S7B). WRN-siRNA–treated HeLa cells showed a pared with helicase activity by immunoprecipitated WRN from similar low number of g-H2AX foci in NSC 617145- and DMSO- DMSO-treated cells (Fig. 2C). treated cells, suggesting that inhibition of WRN activity in vivo by NSC 617145 led to DSB accumulation in a WRN-dependent Synergistic effect of NSC 617145 and DNA cross-linking manner. H2AX phosphorylation can be induced by a wide agent mitomycin C on cell proliferation range of phenomenon including DSBs; therefore, we examined We sought to use the newly identified WRN helicase inhib- the effect of NSC 617145 on 53BP1 foci, an independent marker itor as a tool to explore whether WRN helicase activity helps of DNA damage (25). Cellular exposure to 0.25 mmol/L NSC cells cope with stress imposed by the DNA cross-linking agent 617145 elevated 53BP1 foci 3.5-fold compared with DMSO- mitomycin C. Treatment with either NSC 617145 (0.5 mmol/L) treated cells, confirming NSC 617145 induced DNA damage or mitomycin C (9.4 nmol/L) exerted no significant effect on (Supplementary Fig. S7C and S7D). proliferation. However, cotreatment with both NSC 617145 (0.5 HeLa cells exposed to 1.5 mmol/L NSC 617145 showed a 4- mmol/L) and mitomycin C (9.4 nmol/L) resulted in a 45% fold increase in apoptosis as compared with DMSO-treated reduction in proliferation (Fig. 3A). In contrast, treatment with cells (Supplementary Fig. S8). WRN-depleted HeLa cells NSC 617145 (0.5 mmol/L) and hydroxyurea (0–5 mmol/L) had a showed no difference in level of apoptosis in NSC 617145- similar effect on proliferation compared with hydroxyurea and DMSO-treated cells, showing WRN-dependent induc- alone (Supplementary Fig. S11A), suggesting that the apparent tion of apoptosis by NSC 617145. Failure to repair DNA synergism between NSC 617145 and mitomycin C is not a damage would affect replication fork progression; therefore, general effect imposed by other forms of replicative stress such we determined the effect of NSC 617145 on proliferating cell as hydroxyurea that depletes the nucleotide pool. nuclear antigen (PCNA) foci formation. HeLa cells exposed to 0.25 mmol/L NSC 617145 showed an elevated number of NSC 617145–treated Fanconi anemia–mutant cells are PCNA foci (22-fold)comparedwithDMSO-treatedcells hypersensitive to mitomycin C (Supplementary Fig. S9A and S9B). In contrast, WRN-deplet- On the basis of the synergistic effect of NSC 617145 and ed cells showed similar levels of PCNA staining for NSC mitomycin C, we hypothesized that WRN might participate 617145- or DMSO-treated cells, suggesting WRN-dependent in a pathway that is partially redundant with Fanconi accumulation of stalled replication foci. Consistent with the anemia–mediated ICL repair. For this, we examined sensi- PCNA induction, exposure of HeLa cells to NSC 617145 tivity of FA-D2–mutant and -corrected cells to mitomycin C (1 mmol/L) resulted in an increased population in S-phase and NSC 617145. Dose–response curves for mitomycin C and compared with DMSO-treated cells (Supplementary Fig. NSC 617145 sensitivity of FA-D2 cells indicated drug con- S10). We also observed from fluorescence-activated cell centrations at which only modest effects on proliferation – sorting (FACS) analysis a sub-G1 fraction of NSC 617145 were observed (Supplementary Fig. S11B and S11C). NSC treated HeLa cells (data not shown), consistent with the 617145 (0.125 mmol/L) or mitomycin C (9.4 nmol/L) exerted induction of apoptosis by the WRN helicase inhibitor. WRN- only a mild effect on cell proliferation; however, a significant depleted cells exposed to 1 mmol/L NSC 617145 showed a 45% reduction was observed for cells treated with both NSC þ þ similar percentage of cells in S-phase compared with DMSO- 617145 and mitomycin C (Fig. 3B). In contrast, FA-D2 / treated cells, suggesting that NSC 617145 induced prolonged cells showed only a 10% reduction of proliferation by com- S-phase in a WRN-dependent manner. bined treatment of NSC 617145 and mitomycin C (Fig. 3B). Sensitization of FA-D2 / cells to mitomycin C by WRN NSC 617145 induces WRN binding to chromatin and helicase inhibition is distinct from the effect imposed by a proteasomal degradation deficiency or inhibition of proteins in the NHEJ pathway We reasoned that NSC 617145 might target cellular WRN for (LIG-4 and Ku-70), which suppress ICL-sensitivity of Fanconi helicase inhibition and induce toxic DNA lesions mediated by anemia–deficient cells (16, 17). WRN's interaction with genomic DNA, prompting us to ask Combinatorial treatment of FA-A / cells with NSC 617145 whether poisoned WRN became enriched in the chromatin (0.125 mmol/L) and mitomycin C (9.4 nmol/L) resulted in 60% fraction. Western blot analysis of nuclear soluble and chro- reduction in proliferation compared with very modest effects matin-bound fractions prepared from HeLa cells exposed to exerted by either agent alone (Fig. 3C). Moreover, the effect was NSC 617145 showed a dose-dependent increase in WRN bound dependent on FANCA status as only 10% reduction in prolif- þ þ to chromatin (Fig. 2A). We also observed that the amount of eration was observed for FA-A / cells exposed to both WRN from extract of HeLa cells exposed to NSC 617145 (0.75 mitomycin C and NSC 617145. Thus, NSC 617145 acts in a

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A C NSC 617145 NSC 617145 NSC 617145 (0.75 μmol/L) α DMSO -WRN DMSO IgG ATP (2 mmol/L)

WRN ATPγS (2 mmol/L) WRN control

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Figure 2. NSC 617145 induces WRN to bind chromatin and WRN degradation. A, HeLa cells were exposed to increasing concentrations (0.75, 1.0, 1.5, and 2.0 mmol/L) of NSC 617145 for 4 hours. Chromatin-bound and nuclear soluble fractions were prepared and 10 mg of protein was resolved by SDS-PAGE, followed by analysis for WRN protein by immunoblotting. A representative Western blot analysis from 1 of 3 independent experiments is shown. Histone H3 and TopoI served as markers for the chromatin and soluble nuclear fractions, respectively. B, whole cell extracts were prepared from HeLa cells exposed to NSC 617145 (0.75 mmol/L) or DMSO for 6 hours and proteins resolved by SDS-PAGE followed by Western blot analysis for WRN. As indicated, the proteasome inhibitor MG132 (10 mmol/L) was added to the cell culture at the same time as NSC 617145. b-Actin and RECQL1 served as a loading control. C, immunoprecipitated WRN obtained from an equivalent amount of protein derived from extracts of HeLa cells that had been exposed to 0.75 mmol/L NSC 617145 or DMSO for 4 hours was tested for helicase activity on a forked duplex DNA substrate as described in Materials and Methods. A representative helicase gel and quantitative data from at least 3 independent experiments are shown. Helicase activity from anti-WRN immunoprecipitate (white bar), IgG control (gray bar), or anti-WRN immunoprecipitate in the absence of ATP (black bar) is indicated in bar graph. synergistic manner with mitomycin C in either the FANCA- or ment with NSC 617145 and mitomycin C, as evidenced by FANCD2-mutant background. 40% reduction in proliferation (Fig. 3E). In contrast, no Treatment with both NSC 617145 (0.125 mmol/L) and difference in proliferation was observed with WRN-depleted þ þ hydroxyurea (0–1.25 mmol/L) resulted in a very similar effect or NS-siRNA–treated FA-D2 / cells cotreated with these on proliferation as compared with hydroxyurea alone in both NSC 617145 and mitomycin C concentrations (Fig. 3D and þ þ FA-D2 / and FA-D2 / cells (Supplementary Fig. S11D and E). Furthermore, WRN depletion in FA-D2 / cells did not S11E). Pretreatment of FA-D2 / cells with hydroxyurea for 24 affect mitomycin C sensitivity as compared with NS-siRNA– hours followed by exposure to 0.125 mmol/L NSC 617145 for 48 treated or untransfected cells (Supplementary Fig. S12), sug- hours also showed very similar inhibition of proliferation gesting WRN helicase inhibition by NSC 617145 interfered compared with cells only exposed to hydroxyurea (Supple- with the ability of FA-D2 / cells to cope with mitomycin C– mentary Fig. S11F), indicating NSC 617145 does not sensitize induced DNA damage. cells to hydroxyurea. NSC 617145 and mitomycin C act synergistically to NSC 617145 causes FA-D2–mutant cells to be induce DNA damage and activate ATM hypersensitive to mitomycin C in a WRN-dependent Failure to repair DNA ICLs in dividing cells would be manner expected to result in blocked replication forks and hence To determine whether enhanced mitomycin C sensitivity DSBs. Cotreatment of FA-D2 / cells with NSC 617145 of FA-D2 / cells was mediated through inhibition of WRN (0.125 mmol/L) and mitomycin C (9.4 nmol/L) resulted in a function by NSC 617145, WRN was depleted by RNA inter- substantial increase in percentage (80%) of cells with more þ þ þ þ ference in FA-D2 / or FA-D2 / cells by 90% or more than 15 g-H2AX foci (Fig. 4A). In contrast, only 30% FA-D2 / compared with NS-siRNA cells (data not shown). WRN-deplet- cells exposed to NSC 617145 and mitomycin C showed more ed FA-D2 / cells grown in the presence of 0.125 mmol/L NSC than 15 g-H2AX foci (Fig. 4B). The difference in sensitivity of þ þ 617145 and 9.4 nmol/L mitomycin C were resistant to the FA-D2 / versus FA-D2 / cells exposed to very low concen- antiproliferative effects of combined treatment (Fig. 3D), trations of either mitomycin C or WRN inhibitor was modest whereas NS-siRNA FA-D2 / cells were sensitive to cotreat- (NSC 617145) or hardly apparent (mitomycin C) compared

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A B C 120 MMC 120 MMC 140 MMC 100 NSC 617145 100 NSC 617145 120 NSC 617145 MMC + NSC 617145 MMC + NSC 617145 100 MMC + NSC 617145 80 80 80 60 60 60 40 40 40 20 20

Proliferation (%) 20 Proliferation (%) 0 (%) Proliferation 0 0 03 FA-D2+/+ FA-D2–/– FA-A+/+ FA-A–/– Days D E 140 140 MMC MMC 120 120 NSC 617145 NSC 617145 100 100 MMC + NSC 617145 MMC + NSC 617145 80 80 60 60 40 40 20 20 Proliferation (%) 0 Proliferation (%) 0 FA-D2+/+ FA-D2–/– FA-D2+/+ FA-D2–/– WRN siRNA WRN siRNA NS siRNA NS siRNA

Figure 3. NSC 617145 exposure enhances sensitivity of Fanconi anemia–mutant cells to mitomycin C in a WRN-dependent manner. A, HeLa cells were treated þ þ with NSC 617145 (0.5 mmol/L), mitomycin C (9.4 nmol/L), or both compounds for 3 days. B, FA-D2 / and FA-D2 / cells were treated with NSC 617145 þ þ (0.125 mmol/L), mitomycin C (9.4 nmol/L), or both compounds for 2 days. C, FA-A / and FA-A / cells were treated with NSC 617145 (0.75 mmol/L), mitomycin C (9.4 nmol/L), or both compounds for 2 days. D, FA-D2/ and FA-D2þ/þ cell lines transfected with WRN siRNA were treated with NSC 617145 þ þ (0.125 mmol/L), mitomycin C (9.4 nmol/L), or both compounds for 2 days. E, FA-D2 / and FA-D2 / cell lines transfected with NS siRNA were treated with NSC 617145 (0.125 mmol/L), mitomycin C (9.4 nmol/L), or both compounds for 2 days. Cell proliferation was determined. Percentage proliferation was calculated.

with the cotreatment. Thus, accumulation of mitomycin C– accumulated DSBs in FA-D2 / cells, when WRN helicase induced DNA damage in FA-D2 / cells is increased by NSC activity is pharmacologically inhibited, might promote geno- 617145. mic instability when captured by error-prone pathways. To Exposure of FA-D2 / cells with a dose range (0.125–1.5 address this, we examined metaphase spreads from FA-D2 / þ þ mmol/L) of NSC 617145 led to activation of ATM as detected and FA-D2 / cells for chromosomal aberrations (Fig. 5B; ref. by pATM-Ser1981 at 0.5 mmol/L NSC 617145 (Supplemen- 27). FA-D2 / cells exposed to NSC 617145 (0.125 mmol/L) and tary Fig. S13); however, no significant activation of ATM mitomycin C (9.4 nmol/L) showed a 4-fold increase in abnor- þ þ was observed at lower doses. In FA-D2 / cells, no appreci- mal chromosome structures compared with either agent alone able ATM activation was detected except at the highest NSC (Fig. 5C). Enhanced chromosomal instability was dependent 617145 concentration tested, 1.5 mmol/L. Upon coexposure on FANCD2 status as evidenced by the relatively low level of þ þ of FA-D2 / cells with a very low dose of NSC 617145 (0.125 chromosomal breaks in FA-D2 / cells exposed to NSC 617145 mmol/L) and mitomycin C (9.4 nmol/L), there was a sig- and/or mitomycin C. nificant accumulation of pATM-Ser1981, whereas ATM DNA-dependent protein kinase complex is required for þ þ activation was not detected in FA-D2 / cells (Fig. 5A). NHEJ in conjunction with Ku70/80 and XRCC4/Ligase IV; FA-D2 / cells exposed to very low dose of NSC 617145 moreover, DNA-PKcs pS2056 is detected at DSBs (28). To (0.125 mmol/L) and mitomycin C (9.4 nmol/L) retained high substantiate the role of NHEJ in DSB processing when WRN levels of pATM-Ser1981 even after 24-hour exposure (data helicase is inhibited, FA-D2 / cells were examined for DNA- notshown),suggestingasignificant delay in damage repair PKcs pS2056 foci formation. Cotreatment of FA-D2 / cells when WRN helicase was inhibited. Thus, NSC 617145 and with limited concentrations of NSC 617145 (0.125 mmol/L) and mitomycin C act synergistically to induce DNA damage and mitomycin C (9.4 nmol/L) resulted in 70% of cells with more activate ATM. than 15 DNA-PKcs pS2056 foci (Fig. 6A and B). In contrast, only þ þ 15% of FA-D2 / cells exposed to NSC 617145 and mitomycin C NSC 617145 elevated mitomycin C–induced showed more than 15 DNA-PKcs pS2056 foci (Fig. 6A and B). þ þ chromosomal instability and induced accumulation of The difference in sensitivity between FA-D2 / and FA-D2 / DNA-PKcs pS2056 foci in FA-D2–mutant cells cells exposed to very low concentrations of either mitomycin C Recent studies suggest the Fanconi anemia pathway plays an or NSC 617145 was modest (mitomycin C) or hardly apparent important role in preventing aberrant DNA repair (16, 17). (NSC 617145) compared with cotreatment, suggesting that Because NHEJ factors have high affinity for DNA ends (26), the when WRN helicase activity is inhibited, FA-D2 / cells show

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A FA-D2+/+ FA-D2–/–

γ-H2AX DAPI Merge γ-H2AX DAPI Merge

DMSO

MMC

Figure 4. Increased accumulation of g-H2AX foci in FA-D2–mutant NSC 617145 cells cotreated with NSC 617145 and mitomycin C. A, FA-D2 / and FA-D2þ/þ cells were treated with NSC 617145 (0.125 mmol/L), NSC 617145 mitomycin C (9.4 nmol/L), or both + compounds for 2 days. Cells were MMC stained with anti-g-H2AX antibody or 40,6-diamidino-2-phenylindole (DAPI) and imaged. B, percentage B ≤ 15 γ-H2AX foci of cells with g-H2AX foci (15 or 100 >15, as indicated). > 15 γ-H2AX foci 80

-H2AX foci 60 γ

20 % Cells with 0 DMSO MMC NSC 617145 NSC 617145 DMSO MMC NSC 617145 NSC 617145 + + MMC MMC FA-D2+/+ FA-D2–/–

increased chromosomal instability due to more DSBs repaired -corrected cells. We did not observe any signiﬁcant difference þ þ by the error-prone pathway. Strikingly, FA-D2 / cells cot- in percentage of cells that displayed Rad51 foci between þ þ reated with mitomycin C (9.4 nmol/L) and NSC 617145 (0.125 FA-D2 / and FA-D2 / upon cellular exposure with mito- mmol/L) showed similar levels of DNA-PKcs pS2056 foci as mycin C (9.4 nmol/L), as reported previously at higher doses of individually treated or DMSO control cells (Fig. 6A and B), mitomycin C (29) or NSC 617145 (0.125 mmol/L; Fig. 7A and B). suggesting an important role for WRN helicase in processing of In both cell lines, approximately 50% showed Rad51 foci, mitomycin C–induced cross-links converted to DSBs when the similar to that observed for DMSO-treated cells. Cotreatment Fanconi anemia pathway is defective. of FA-D2 / cells with NSC 617145 and mitomycin C resulted in 90% of cells staining positive for Rad51 foci. In contrast, þ þ NSC 617145 exposure results in accumulation of Rad51 FA-D2 / cells exposed to both agents showed a percentage of foci in FA-D2–mutant cells cells with Rad51 foci very similar to DMSO-treated cells (Fig. 7A WRN plays a physiologic role in resolution of Rad51-depen- and B). Collectively, these results suggest that mitomycin dent homologous recombination products (6). Expression of C–induced DNA cross-links in FA-D2 / cells exposed to WRN dominant-negative Rad51 or bacterial resolvase protein helicase inhibitor are converted to DSBs and the homologous RusA could suppress homologous recombination or lead to recombination pathway is activated but homologous recom- the generation of recombinants, respectively, resulting in bination intermediates fail to be subsequently resolved. improved survival of WRN / cells. Because Rad51 foci are formed at sites of ICL lesions independently of FA-D2 status Discussion (29), we reasoned that WRN helicase inhibition in mitomycin In this study, we discovered a new inhibitor of WRN helicase C–treated FA-D2 / cells might result in accumulation of activity (NSC 617145) that negatively affects cell proliferation homologous recombination intermediates. Therefore, we and induces DNA damage in a WRN-dependent manner. investigated the effect of NSC 617145 and mitomycin C treat- WRN depletion negates biologic activity of NSC 617145, ment on Rad51 foci formation in Fanconi anemia–mutant and suggesting targeted WRN helicase inhibition interferes with

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A C DMSO 5 FA-D2–/– FA-D2+/+ MMC NSC 617145 4 MMC + NSC 617145

3 MMC + Cmpd MMC MMC DMSO DMSO Cmpd MMC + Cmpd Cmpd 2 pATM S1981 1 Breaks/Metaphase ATM 0 FA-D2+/+ FA-D2–/– B DMSO MMC NSC 617145 NSC 617145 + MMC

FA-D2+/+

FA-D2–/–

Figure 5. FA-D2–mutant cells cotreated with NSC 617145 and mitomycin C display ATM activation and increased chromosomal instability. A, FA-D2 / and þ þ FA-D2 / cells were cotreated with 0.125 mmol/L NSC 617145 and 9.4 nmol/L mitomycin C for 3 hours. Cell lysates were prepared and analyzed by immunoblotting by using anti-pATM Ser1981 antibody. As a control, blot was reprobed with anti-ATM antibody. Arrow indicates phosphorylated ATM. B, FA-D2/ and FA-D2þ/þ cells were treated with NSC 617145 (0.125 mmol/L), mitomycin C (9.4 nmol/L), or both compounds for 2 days. Chromosome spreads were analyzed. Arrow indicates chromatid break, chromatid loss, or radial. Each radial is counted as equivalent to two chromatid breaks. C, chromatid breaks per metaphase.

normal cellular DNA metabolism. The synergistic effect of The synergistic effect of NSC 617145 and mitomycin C in the NSC 617145 and mitomycin C was not observed for hydroxy- FA-D2–mutant was apparent by robust g-H2AX staining, ATM urea, suggesting that impairment of WRN helicase activity activation, and increased chromosomal abnormalities. More- exacerbates the effect of mitomycin C–induced DNA damage over, accumulation of DNA-PKcs pS2056 foci suggests that during replication but not an effect exerted by an agent FA-D2 cells attempted to deal with mitomycin C–induced (hydroxyurea) that primarily induces replication stress. We DSBs by error-prone NHEJ when WRN helicase was pharma- used the WRN helicase inhibitor to sensitize a Fanconi cologically inhibited. This finding builds on previous evidence anemia–mutant because mounting evidence points toward that the Fanconi anemia pathway plays an important role in a pivotal role of the Fanconi anemia pathway to coordinate a preventing aberrant NHEJ-mediated repair (16, 17), and impli- robust ICL response when WRN is also likely to act. WRN cates a role of WRN helicase in repair pathway choice. In helicase inhibition by NSC 617145 makes FA-A or FA-D2 cells addition to its helicase-dependent role in recombinational highly sensitive to very low mitomycin C concentrations that repair, WRN may have a structural role to enable repair of would otherwise be only marginally active in normal cells. mitomycin C–induced DNA damage. This would be akin to the Furthermore, the effect of combined mitomycin C/NSC observation that a WRN helicase/exonuclease double-mutant 617145 treatment is a consequence of a Fanconi anemia complemented the NHEJ defect of WRN / cells (30). Although pathway deficiency rather than a defect in a specificFanconi experimental data suggest that WRN participates in an alter- anemia gene. native NHEJ repair pathway of DSBs induced by increased

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Role of WRN Helicase in Fanconi Anemia–Deﬁcient Cells

A FA-D2+/+ FA-D2–/–

DNA-PKcsDAPI Merge DNA-PKcs DAPI Merge

DMSO

MMC

Figure 6. NSC 617145 exposure elicits NHEJ pathway in Fanconi anemia–deﬁcient cells upon NSC 617145 cotreatment with mitomycin C. A, þ þ FA-D2 / and FA-D2 / cells were treated with NSC 617145 (0.125 NSC 617145 + mmol/L), mitomycin C (9.4 nmol/L), MMC or both compounds for 2 days. Cells were stained with anti-DNA- PKcs-pS2056 antibody or 40,6- B ≤ diamidino-2-phenylindole (DAPI). 15 DNA-PKcs pS2056 foci 100 > B, percentage of cells with DNA- 15 DNA-PKcs pS2056 foci PKcs-pS2056 foci (15 or >15, as 80 indicated) is shown. 60

40 pS2056 foci 20 % Cells with DNA-PKcs 0 DMSO MMC NSC 617145 NSC 617145 DMSO MMC NSC 617145 NSC 617145 + + MMC MMC FA-D2+/+ FA-D2–/–

reactive oxygen species in chronic myeloid leukemia cells (31), other helicases may be useful for development of anticancer it is unclear whether this pathway is relevant to a role of WRN strategies that rely on synthetic lethality for targeting tumors helicase to confer mitomycin C resistance in Fanconi anemia– with preexisting DNA repair deficiencies (32). deficient fibroblasts. Further studies will be necessary to Demonstration that very low concentrations of the che- evaluate whether an alternate end-joining pathway or homol- motherapy drug mitomycin C are cytotoxic for Fanconi ogous recombination is important for repair of mitomycin anemia–mutant cells exposed to the helicase inhibitor may C–induced lesions in the WRN helicase-inhibited condition. have implications for strategies that target Fanconi anemia We also observed Rad51 foci accumulation in FA-D2 / DNA repair pathway–deficient tumors. Certain sporadic cells cotreated with mitomycin C and NSC 617145, suggesting head and neck, lung, ovarian, cervical, and hematologic that homologous recombination was activated and at least cancers are characterized by epigenetic silencing of wild- some DSBs were channeled into homology-mediated repair. type Fanconi anemia gene expression (33). It is estimated However, as WRN plays a role in resolution of homologous that 15% of all cancers harbor defects in the Fanconi anemia recombination intermediates (6), NSC 617145 inhibition of pathway (34). A significant number of these tumors may WRN helicase activity might lead to accumulation of homol- becomereliantonWRNorotherhelicasestodealwithDNA ogous recombination structures. We observed phosphoryla- damage such as strand breaks. WRN may be a suitable target tion of DNA-PKcs only under conditions of WRN helicase for pharmacologic inhibition to sensitize Fanconi anemia– inhibition in FA-D2 / cells, suggesting that NHEJ was elicited; deficient tumors to chemotherapy drugs such as DNA cross- however, the mechanism of DNA-PKcs recruitment is still linkers. unclear. In addition to epigenetic silencing, LOH from an additional Development of helicase inhibitors such as NSC 617145 that mutation in an Fanconi anemia gene in heterozygous carriers possess chemical stability and lack of nonspecific reactivity to may lead to increased cancer risk later in life (33, 35). Fanconi thiol and other functional groups may prove useful for study of anemia pathway–deficient fibroblasts were found to be highly compensatory DNA repair pathways dependent on DNA sensitive to silencing of ATM kinase (33). FANCG- and FANCC- unwinding by WRN or related helicases. In addition to their deficient pancreatic tumor lines were sensitive to a pharma- use as research tools, small-molecular inhibitors of WRN and cologic inhibitor of ATM, raising the possibility for an

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A FA-D2+/+ FA-D2–/–

Rad51 DAPI MergeRad51 DAPI Merge

DMSO

MMC

Figure 7. Increased accumulation of Rad51 foci in FA-D2–mutant NSC 617145 cells cotreated with NSC 617145 and mitomycin C. FA-D2 / and FA-D2þ/þ cells were treated with NSC 617145 (0.125 mmol/L), NSC 617145 mitomycin C (9.4 nmol/L), or both + compounds for 2 days. A, cells MMC were stained with anti-Rad51 antibody or 40,6-diamidino-2- phenylindole (DAPI). B, percentage B 100 of cells with Rad51 foci is shown. FA-D2+/+ FA-D2–/–

foci 80

20 % Nuclei with Rad51 % Nuclei 0 DMSO MMC NSC 617145 NSC 617145 + MMC

anticancer treatment. More recently, Fanconi anemia–defi- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Aggarwal, T. Banerjee, J.A. Sommers, C. Iannas- cient cell lines were shown to be hypersensitive to inhibition of coli, P. Pichierri, R.H. Shoemaker, R.M. Brosh, Jr. CHK1 kinase either by siRNA or a pharmacologic inhibitor of Writing, review, and/or revision of the manuscript: M. Aggarwal, T. Bane- rjee, J.A. Sommers, P. Pichierri, R.H. Shoemaker, R.M. Brosh, Jr. CHK1 kinase activity (36). Unlike the hypersensitivity of Fan- Administrative, technical, or material support (i.e., reporting or organiz- coni anemia–deficient cells to CHK1 silencing or kinase inhi- ing data, constructing databases): M. Aggarwal, J.A. Sommers, R.M. Brosh, Jr. bition, WRN depletion did not increase sensitivity of FA-D2 / Study supervision: R.M. Brosh, Jr. cells to mitomycin C. This suggests that NSC 617145 exerted its effect through a dominant-negative mechanism in which NSC Acknowledgments 617145–inhibited WRN helicase induced greater damage by The authors thank Fanconi Anemia Research Fund for FA-A and FA-D2 cell lines, and Dr. Ian Hickson (University of Copenhagen) for recombinant BLM þ þ blocking compensatory mechanism(s). Inhibition of WRN or a protein and BLM / (PSNG13) and BLM / (PSNF5) cells. The authors also related helicase by a small molecule may provide an alternative thank the Flow Cytometry and Cell Sorting Shared Resource at Georgetown – fi University, Lombardi Comprehensive Cancer Center, which is partially sup- strategy for targeting Fanconi anemia de cient tumors that is ported by NIH/NCI grant P30-CA051008. unique from approaches such as targeting the CHK1 or ATM kinase response. Grant Support This work was supported by Intramural Research Program of NIH, National Disclosure of Potential Conflicts of Interest Institute on Aging, and National Cancer Institute, and Fanconi Anemia Research No potential conflicts of interest were disclosed. Fund (R.M. Brosh, Jr.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Authors' Contributions advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this Conception and design: M. Aggarwal, P. Pichierri, R.H. Shoemaker, R.M. Brosh, fact. Jr. Development of methodology: M. Aggarwal, J.A. Sommers, R.M. Brosh, Jr. Acquisition of data (provided animals, acquired and managed patients, Received July 27, 2012; revised May 10, 2013; accepted June 7, 2013; provided facilities, etc.): M. Aggarwal, C. Iannascoli published OnlineFirst July 18, 2013.

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Werner Syndrome Helicase Has a Critical Role in DNA Damage Responses in the Absence of a Functional Fanconi Anemia Pathway

Monika Aggarwal, Taraswi Banerjee, Joshua A. Sommers, et al.

Cancer Res Published OnlineFirst July 18, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-12-2975

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