inhibition of the nedd8 system sensitizes cells to dna...

DNA Damage and Cellular Stress Responses

Inhibition of the Nedd8 System Sensitizes Cells to DNAInterstrand Cross-linking Agents

Younghoon Kee1, Min Huang2, Sophia Chang1, Lisa A. Moreau2, Eunmi Park2,Peter G. Smith3, and Alan D. D'Andrea2

AbstractThe Fanconi anemia pathway is required for repair of DNA interstrand cross-links (ICL). Fanconi anemia

pathway–deficient cells are hypersensitive to DNA ICL–inducing drugs such as cisplatin. Conversely, hyperactiva-tion of the Fanconi anemia pathway is a mechanism that may underlie cellular resistance to DNA ICL agents.Modulating FANCD2 monoubiquitination, a key step in the Fanconi anemia pathway, may be an effectivetherapeutic approach to conferring cellular sensitivity to ICL agents. Here, we show that inhibition of the Nedd8conjugation system increases cellular sensitivity to DNA ICL–inducing agents. Mechanistically, the Nedd8inhibition, either by siRNA-mediated knockdown of Nedd8-conjugating enzymes or treatment with a Nedd8-activating enzyme inhibitor MLN4924, suppressed DNA damage–induced FANCD2 monoubiquitination andCHK1 phosphorylation. Our data indicate that inhibition of the Fanconi anemia pathway is largely responsible forthe heightened cellular sensitivity to DNA ICLs upon Nedd8 inhibition. These results suggest that a combinationof Nedd8 inhibition with ICL-inducing agents may be an effective strategy for sensitizing a subset of drug-resistantcancer cells. Mol Cancer Res; 10(3); 369–77. �2012 AACR.

IntroductionCisplatin-based drugs have been used as a primary treat-

ment formany types of cancers formore than 30 years. Thesedrugs cause DNA damage, primarily via formation of inter-strand DNA cross-linkages (ICL). ICLs are highly toxic torapidly dividing cells, and cells that are unable to properlyrepair the damaged DNA die of apoptosis. However, theeffectiveness of the therapy is often compromised largelybecause cancer cells develop resistance to the drugs (1).Elevated DNA repair pathways are observed in a subset ofdrug-resistant tumor cells (2, 3). Thus, understanding thecellular response mechanisms that regulate the activation ofDNA repair pathways may provide a strategy for sensitizingsome drug-resistant tumors. The DNA repair pathways thatresolve DNA ICLs, such as nucleotide excision repair andhomologous recombination, are coordinated by a DNA

damage response pathway termed the Fanconi anemia path-way (4).Patients with Fanconi anemia, who have a germ line

disruption of the Fanconi anemia pathway, exhibit congen-ital abnormalities, bone marrow failure, and genomic insta-bility leading to cancers (4, 5). Cells from patients withFanconi anemia display abnormally high sensitivity to DNAICL–inducing agents, such as cisplatin, mitomycin C(MMC), andmelphalan. Fifteen Fanconi anemia genes havebeen identified to date (FANC-A, B,C,D1,D2, E, F,G, I, J,L,M, N, O, and P). These act cooperatively in the Fanconianemia pathway to coordinate the repair of DNA ICLs(6–8). The central regulatory event in the pathway ismonoubiquitination of FANCD2, which requires S-phaseor DNA damage–induced activation of 8 Fanconi anemiaproteins (A, B, C, E, F, G, L, andM) that form a nuclear E3ubiquitin ligase core complex. The activation of this Fanconianemia core complex is preceded by a cascade of upstreamDNA damage–induced signaling events involving the ATRand CHK1 kinases (4, 9). Monoubiquitinated FANCD2 isrequired for multiple steps during ICL repair, including theactivation of the nucleotide excision repair and translesionsynthesis (TLS) steps (4), and the recruitment of homolo-gous recombination repair factors such as BRCA1, BRCA2,RAD51, and FAN1 (4).Defects in the Fanconi anemia pathway also occur in

somatic cells of non–Fanconi anemia individuals, causingdiverse types of cancers (5, 10–12). Human tumors withFanconi anemia gene mutations are particularly sensitive toICL-inducing agents, such as cisplatin and MMC. Con-versely, restoration of a functional Fanconi anemia pathway

Authors' Affiliations: 1Department of Cell Biology, Microbiology, andMolecular Biology, University of South Florida, Tampa, Florida; 2Divisionof Genomic Stability and DNA Repair, Department of Radiation Oncology,Dana-Farber Cancer Institute, Harvard Medical School, Boston; and 3Mil-lennium Pharmaceuticals, Inc., Cambridge, Massachusetts

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

Corresponding Authors: Younghoon Kee, 4202 E. Fowler Avenue,BSF216 Tampa, FL 33620. Phone: 813-974-5352; Fax: 813-974-1614;E-mail: [email protected]; andAlanD. D'Andrea, 450Brookline Avenue,Mayer640, Boston, MA 02215. Phone: 617-632-2112; Fax: 617-632-5757;E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-11-0497

�2012 American Association for Cancer Research.

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is a mechanism for acquired cellular resistance to DNA ICLagents (10, 13, 14). Interestingly, overexpression of Fanconianemia genes accounts for drug resistance in melphalan-resistant multiple myeloma (14, 15). For these reasons, theFanconi anemia pathway may be an effective target forchemosensitization in cancer treatment. Small-moleculeinhibitors of the Fanconi anemia pathway have been iden-tified by high-throughput platforms (13, 16), and an inhib-itor of HSP90 has been shown to inhibit the Fanconi anemiapathway (17). Recently, the proteasome inhibitor bortezo-mib, which is used for treating certain types of hematologictumors, was shown to inhibit the Fanconi anemia pathway,providing a mechanism for its antitumor effect (14, 18).The ubiquitin–proteasome system regulates several essen-

tial cellular functions, including the cell-cycle and DNAdamage responses. Protein ubiquitination is achieved by acascade of E1 ubiquitin–activating enzymes, E2 ubiquitin–conjugating enzymes, and E3 ubiquitin ligases whereasreversal of ubiquitination is regulated by deubiquitinatingenzymes. In addition to the ubiquitin system, eukaryoticcells use "ubiquitin-like modifiers" or Ubls, such as SUMO,Nedd8, and ISG15, which provide additional layers ofregulation for protein degradation. Nedd8 shares approxi-mately 60% sequence identity with ubiquitin (19) and it iscovalently attached to Lys residues on protein substrates in amanner similar to that of the ubiquitin system. The Nedd8conjugation system consists of a single E1, a heterodimer ofUBA3 and NAE1, two E2s, UBE2M (also known asUBC12) and UBE2F (20). The E3 for Nedd8 is not wellcharacterized, and Nedd8 from the E2 can be directlytransferred to Nedd8 substrates, including the cullin sub-units of Cullin RING Ligase complexes (CRL; ref. 21). Inhumans, at least 6 cullin subunits (Cul1, 2, 3, 4A, 4B, and 5)have been identified. These cullins form distinct CRLcomplexes with different substrate specificity adaptors forprotein degradation. Neddylation (protein modification byNedd8) of the cullin subunits induces conformationalchanges within the CRL complexes, resulting in increasedcatalysis of substrate ubiquitination (20, 22). Therefore, theNedd8 system affects a wide range of cellular functions thatare regulated by the CRL complexes. Recently, a pharma-cologic inhibitor of the Nedd8 system, MLN4924, wasdeveloped. MLN4924 has potent antitumor activity in cellculture as well as in a xenograft model (23), suggesting thatthe Nedd8 system may be an effective target for treatingcancers.To better understand the upstream signaling events that

regulate FANCD2 monoubiquitination, we undertook acandidate-based siRNA screening that uses FANCD2West-ern blot analysis as readouts. We found that knockdown ofindividual components in the Nedd8 conjugation systemdecreased DNA damage–induced FANCD2 monoubiqui-tination and foci formation and CHK1 phosphorylation.The results are phenocopied by treatment of cells withMLN4924, the pharmacologic inhibitor of NAE1. Consis-tent with these results, treatment with MLN4924, orsiRNA-mediated depletion of the Nedd8 system, synergis-tically elevates cellular sensitivity to DNA ICL–inducing

agents in various cell lines. These results suggest that acombination of a Nedd8 inhibitor with DNA ICL agentscan be an effective therapeutic strategy against chemoresis-tant cancer cells.

Materials and MethodsCell culture and chemicalsHeLa, HCT116, 293T, U2OS, andMCF7 cell lines were

cultured in Dulbecco's Modified Eagle's Media supplemen-ted with 15% FBS and L-glutamine. These cell lines wereobtained fromAmericanTypeCulture Collection and testedfor mycoplasma contamination using Plasmocin (Invivo-gen). We also tested these cell lines for morphology usinglight microscopy, under low density. Nontransformed LO2liver cells and WI-38 lung human cell lines were grown inRPMI-1640 media. FA-F cells (2008 cells) corrected withFANCF cDNA was described previously (10). Cisplatin(Sigma) was dissolved as 10 mmol/L stock solution in PBSand used as final concentration of 10 mmol/L. Hydroxyurea(Sigma) was dissolved as 2 mol/L stock solution in PBS andused as final concentration of 2 mmol/L.MMC (Sigma) wasdissolved in 70% ethanol as 3.3 mmol/L stock concentra-tion. MLN4924 was provided by Millennium Pharmaceu-ticals Inc. and dissolved in dimethyl sulfoxide (DMSO) as astock concentration of 10 mmol/L.

siRNAs and antibodiesThe siRNAs against UBA3 were siRNA#1 TGTTC-

TGGTAGCCTGGGCATAGATG and siRNA#2 CCGA-GCACTGAATCTCTCCAGTTCT; siRNAs againstUBE2M were siRNA#1 GGGCTTCTACAAGAGTGG-GAAGTTT and siRNA#2 (ACTCCATAATTTATGGC-CTGCAGTA); siRNA against NAE1 was AGCACAGTG-GTATAGTGAAACAAAT; siRNAs against UBE2F weresiRNA#1 CGGAGGGTTTCTGTGAGAGACAAAT andsiRNA#2 ACTTCCGGAATAAAGTGGATGACTA; andsiRNA against FANCM was AAGCTCATAAAGCTCT-CGGAA. Antibodies used in this study are as follows: anti-FANCD2, anti-PCNA, and anti-CDC25A antibodies(Santa Cruz Biotechnology); anti-phospho-CHK1 (S317),anti-phospho-SMC1 (S966), anti-claspin, and anti-phos-pho-CHK2 (T68; Cell Signaling Technology); anti-CDT1and anti-53BP1 (Bethyl Laboratories); anti-RPA2 (EMDBiosciences); anti-g-H2AX (Upstate); anti-g-tubulin (Sig-ma); anti-UBE2M, anti-BRCA1 (Abcam); and anti-phos-pho-NBS1 (S343; Oncogene) antibodies.

ImmunofluorescenceHeLa cells were pretreated with extraction buffer (0.5%

Triton X-100 in PBS buffer) on ice for 3 minutes prior tofixation with 4% paraformaldehyde. Anti-FANCD2, anti-53BP1, anti-RPA, and anti-g-H2AX antibodies were used asprimary antibodies. Alexa Fluor 488- and 594–conjugatedantibodies (Invitrogen) were used as secondary antibodies.For the FANCD2 foci in Fig. 3E, images were collected by aZeiss Axiovert microscope equipped with a Yokogawa CSU-22 spinning disk confocal head and a 100�/1.45NA oil

Kee et al.

Mol Cancer Res; 10(3) March 2012 Molecular Cancer Research370




objective using Slidebook software (Intelligent ImagingInnovations). Three-dimensional images were taken with0.3 mm step size and then displayed as a maximal Z-projection.

Clonogenic and growth sensitivity assaysFor clonogenic assays, HeLa cells were transfected with

control, FANCM, or UBE2M siRNA in 6-well plates.Twenty-four hours after transfection, cells were replated in6-well plates at 500 cells per well, followed by treatment withMMC at gradient concentration in the following day.HCT116 cells were treated with 20 nmol/L of MLN4924,and 24 hours later, 20 nmol/L ofMMCwas added. Colonieswere allowed to grow for 7 to 10 days, fixed with a solutioncontaining 10% methanol and 10% acetic acid at roomtemperature for 15 minutes, and then stained with 1%crystal violet dissolved in methanol. Colonies of more than50 cells were counted, and the surviving fraction wascalculated and normalized to untreated control. The viablecells were determined by staining nucleic acids with a dye(CyQUANT;Molecular Probes) and subsequently analyzedby a fluorescence microplate reader (FL � 800; Bio-TekInstruments) according to the manufacturer's protocol. Forgrowth sensitivity assays using 2008 and 2008 þ FANCFcells, cells growing in 96-well plate were treated with 20nmol/L of MLN2949 for 24 hours and then exposed tocisplatin for 4 more days. Twenty microliters of MTT(Sigma) at 5 mg/mL was added to each well and incubatedat 37�C for 4 hours. The metabolic product was dissolved ina solution containing 10% SDS, 5% isopropanol, and 0.01mol/L HCl and optical density at 565 nm was measuredusing an automatic microplate reader. The percentage of cellsurvival was determined by normalizing to solvent vehicle–treated cells.

Cytogenetic analysisEach cell line had one of its two plates treated with 20 ng/

mLMMC for 48 hours. Following treatment, the cells wereexposed to colcemid (final concentration of 100 ng/mL) for2 hours, treated with a hypotonic solution (0.075 mol/LKCL) for 20 minutes, and fixed with 3:1 methanol:aceticacid. Slides were stained with Wright's stain and, whenpossible, 50 metaphase spreads were scored for aberrations.Metaphase spreads were observed using a Zeiss Axio Imagermicroscope and captured using CytoVision software fromApplied Imaging.

ResultsDepletion of the Nedd8 conjugation system abrogatesFANCD2 monoubiquitinationTo identify upstream factors that affect the damage-

inducible FANCD2 monoubiquitination (FANCD2-ub),we undertook a candidate-based siRNA screen. VariousDNA-damaging agents (UV, hydroxyurea, cisplatin, IR)induce FANCD2 monoubiquitination, and there are fewknown upstream factors that regulate DNA damage–induced FANCD2 monoubiquitination including the

Fanconi anemia core complex members, ATR, and CDK1(9, 24). We hypothesized that there might be other ubiqui-tin-mediated signaling events upstream of the monoubiqui-tination of FANCD2. Depletion of UBA3, an E1 compo-nent of the Nedd8 conjugation system, strongly inhibiteddamage-inducible FANCD2 monoubiquitination (Fig. 1Aand B). To further test whether other components in theNedd8 conjugation system also affect FANCD2 monoubi-quitination, we tested knockdown of UBE2M, an E2-conjugating enzyme forNedd8. UBE2Mdepletion similarlyinhibited FANCD2 monoubiquitination after UV (Fig. 1Dand F; HCT116 and HeLa cells, respectively) or afterpsoralen þ UVA (PUVA) treatment (Fig. 1E and G;HCT116 and HeLa cells, respectively). The level of CHK1phosphorylation (p-CHK1) was also consistently lower,when the Nedd8 conjugation pathway was inhibited. Thelevel of monoubiquitinated PCNA (PCNA-ub) remainedsimilar upon depletion of UBE2M, suggesting that the effecton FANCD2 monoubiquitination is specific. Phosphoryla-tion of other DNA damage response proteins, such asH2AX, BRCA1, and SMC1, was elevated or remained ata similar level upon UBE2M knockdown, suggesting thatknockdown of UBE2M caused some level of DNA damageand did not disrupt the overall DNA damage response.Knockdown ofUBA3 orUBE2Mdid not significantly affectcellular proliferation, as measured by bromodeoxyuridinestaining (Fig. 1C). Consistent with a previous report (20),knockdown of UBE2Mmodestly increased the G2–M peakin the cell-cycle distribution analysis (SupplementaryFig. S1).The monoubiquitinated form of FANCD2 localizes to

chromatin and forms damage-inducible foci. Consistentwith the reduction of FANCD2 monoubiquitination, fociformation of FANCD2 was also reduced upon UBE2Mknockdown (Fig. 2). Interestingly, foci formation of otherDNA damage response proteins, such as g-H2AX, 53BP1,and RPA, were not affected, suggesting that the effect isspecific for FANCD2. Because other DNA damageresponse proteins are not affected, the effect on FANCD2is not simply due to a disruption in the cell cycle.Knockdown of NAE1, the catalytic subunit of the Nedd8E1–activating enzyme, also inhibited damage-inducibleFANCD2 monoubiquitination and CHK1 phosphoryla-tion, whereas knockdown of UBE2F, a Nedd8 E2 thatparticipates in neddylation of a smaller subset of cullins(20), did not affect the level of FANCD2 monoubiqui-tination (Supplementary Fig. S2). Depletion of USP1, adeubiquitinating enzyme targeting FANCD2, elevatesthe level of FANCD2 monoubiquitination (25). Surpris-ingly, depletion of UBE2M did not reduce the FANCD2monoubiquitination that is upregulated by USP1 deple-tion (Supplementary Fig. S3), suggesting that theNedd8 inhibition specifically abrogates FANCD2 mono-ubiquitination that is induced by exogenous DNA dam-age. Altogether, these results suggest that the Nedd8conjugation system is required for DNA damage–induc-ible FANCD2 monoubiquitination and CHK1phosphorylation.

The Nedd8 System Maintains DNA Damage Response

www.aacrjournals.org Mol Cancer Res; 10(3) March 2012 371




Nedd8 inhibitor MLN4924 inhibits DNA damage–induced activation of the Fanconi anemia pathwayRecently, a pharmacologic inhibitor of the Nedd8 system

was described (23). This investigational agent, MLN4924,targets the NAE1 component of the Nedd8 E1 enzyme,stabilizes substrates of CRL E3 ligases such as CDT1,

induces DNA rereplication and checkpoint activation,and triggers apoptosis (23, 26, 27). We tested whetherMLN4924 phenocopies the siRNA results described above.We pretreated cells with MLN4924 and then exposed themto UV or PUVA for variable times (Fig. 3A and B; HCT116and HeLa cells, respectively; and Supplementary Fig. S4 for

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Figure 1. Knockdown of Nedd8conjugation system disruptsFANCD2 monoubiquitination andCHK1 phosphorylation. A,representative Western blotanalysis for the candidate siRNAscreens. HeLa cells were treatedwith siRNAs against various E3ligase components. Forty-eight to60 hours later, cells were treatedwith UV (30 J/m2), then the cellswere harvested and lysed for theWestern blot analysis. B, HeLacells were treated with UBA3siRNA#1, followed by UVtreatment. Cells were harvested atindicated time points. C, HeLa cellswere transfected with the siRNAs,and 48 hours later, cells wereincubated with 10 mmol/Lbromodeoxyuridine (BrdUrd) for 40minutes. Integrated BrdUrd wasvisualized using fluoresceinisothiocyanate (FITC)-conjugatedBrdUrd antibody and quantified byfluorescence-activated cell-sorting analysis. D, HCT116 cellswere treated with 2 differentUBE2M siRNAs, followed by UV(30 J/m2) treatment before beingharvested for Western blotanalysis. E, HCT116 cells weretreated with UBE2M#1 siRNA,followed by PUVA treatment, thenharvested at indicated time pointsfor Western blot analysis. F and G,HeLa cells were treated withUBE2M siRNAs followed by UV(30 J/m2) and PUVA treatment,respectively, for Western blotanalysis. Cul1, cullin 1; PI,propidium iodide.

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HeLa treated with UV). Consistent with previous reports,phosphorylation of BRCA1, SMC1, CHK1, CHK2,g-H2AX, andmonoubiquitination of FANCD2 and PCNAwere induced by the compound treatment (compare 0time points between DMSO and MLN4924). When com-bined with DNA-damaging agents, the compound specif-ically inhibited the sustained activation of p-CHK1 andFANCD2-ub whereas activation of other DNA repair pro-teins remained unaffected. These effects were observed inother cell types, such as U2OS (Supplementary Fig. S5),MCF7 (Supplementary Fig. S6), and Fanconi anemia–deficient ovarian tumor line 2008 (Fig. 4C, right). Longertreatment ofMLN4924 was required to observe the reducedlevel of p-CHK1 and FANCD2-ub at the lower inhibitordosage (Supplementary Fig. S7). In a reverse experiment,when MLN4924 was added after exogenous DNA damage,p-CHK1 and FANCD2-ub levels were suppressed, whereasp-CHK2, PCNA-ub, and g-H2AX remained unaffected(Fig. 3C). The effect of MLN4924 was dose-dependent, asshown in Fig. 3D. One discrepancy between the siRNA andMLN4924 results is that the spontaneous activation ofDNAcheckpoints was not observed following Nedd8 knockdownby siRNA. Perhaps longer treatment of cells with the siRNAs(>60 hours) may elicit secondary effects that suppressactivation of DNA checkpoint completely.Consistent with the siRNA results, MLN4924 effectively

suppressed DNA damage–induced foci formation ofFANCD2, whereas foci formation of g-H2AX and 53BP1was not affected (Fig. 3E). Treatment with MLN4924without additional damage caused foci formation of theseDNA damage response proteins to varying degrees, suggest-ing that the drug itself triggers DNA damage. Altogether,MLN4924 phenocopied the siRNA results in which theinhibition of the Nedd8 conjugation system suppressesDNA damage–induced FANCD2 monoubiquitination andCHK1 phosphorylation. Therefore, Nedd8-targeting inhi-bitors, such as MLN4924, may sensitize cancer cells toDNA-damaging agents, particularly to agents that generateDNA ICLs, which require the functional Fanconi anemiapathway for repair.We previously reported that the FANCM subunit of the

Fanconi anemia core complex is ubiquitinated and degradedduring mitosis, mediated by the CRLb-TRCP E3 ubiquitinligase. FANCMdegradation appear to be onemechanism for

cell-cycle–specific activation of the Fanconi anemia pathway(28). The failure of the mitosis-specific degradation ofFANCM leads to improper chromatin loading of the Fan-coni anemia core complex and heightened cellular sensitivityto MMC. Because the degradation is mediated by a CRLcomplex, we reasoned that inhibition of Nedd8 conjugationmay disrupt the timed degradation of FANCM duringmitosis. FANCM was phosphorylated and degraded whencells were released into themitotic phase of the cell cycle (Fig.3F, lanes 1–4), and treatment (3–4 hours) of proteasomeinhibitor MG132 inhibited the degradation of FANCM(lanes 5–8). Treatment with MLN4924 similarly stabilizedFANCM (lanes 9–12), suggesting that the CRL complexactivity is inhibited by the Nedd8 inhibitor. From theseresults, we suggest that the perturbed degradation ofFANCM during mitosis by inhibition of Nedd8 is onemechanism reflected by heightened cellular sensitivity tothe Nedd8 inhibition.

Inhibition of the Nedd8 conjugation systemhypersensitizes cancer cells to DNA ICL agentsWe next tested whether inhibition of the Nedd8 system

causes hypersensitization of cells to DNA ICL agents.Knockdown of UBE2M in HeLa cells significantly reducedcell growth after MMC treatment, as shown by clonogenicassay (Fig. 4A). Knockdown of FANCM, a Fanconi anemiagene shown to be required for resistance to MMC (29), wastested as a positive control. Treatment of HCT116 coloncancer cell lines with MLN4924 significantly reduced cellsurvival in combination with the cisplatin (Fig. 4B; 2independent experiments). These results show that theNedd8 system is required for cellular resistance to the DNAcross-linking agents. The degree of sensitization appears tobe preferential to the transformed cell lines, as 2 normalhuman diploid cells, LO2 and WI-38 cells, were not sig-nificantly sensitized to cisplatin by the Nedd8 inhibition(Supplementary Fig. S8). The MLN4924-mediated sensi-tization of cells toDNA ICL agents resulted, at least partially,from inhibition of the Fanconi anemia pathway, as FANCF-proficient and ICL-resistant ovarian tumor cells (2008þ F)showed enhanced sensitivity to MLN4924, compared withFANCF-deficient and ICL-sensitive counterpart 2008 cells(Fig. 4C). The slight sensitization of the Fanconi anemia–deficient tumor cells 2008 by MLN4924 to cisplatin

Figure 2. Knockdown of UBE2Mabrogates damage-inducibleFANCD2 foci formation. HeLa cellswere treated with control orUBE2M#1 siRNA for 48 hours,followed by hydroxyurea (2 mmol/L)treatment for 12 hours for analyzingdamage-inducible foci formation.DAPI, 40,6-diamidino-2-phenylindole; HU, hydroxyurea.

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suggests that MLN4924 disrupt targets other than theFanconi anemia pathway, including the proteins of theATR/CHK1 signaling pathway. To directly show thereduced DNA repair capacity upon Nedd8 inhibition, we

conducted cytogenetic analysis of 293T cells. We observedsynergistic effects of depletingUBE2M (Fig. 4D), or treatingMLN4924 (Fig. 4E), with MMC treatment in inducingchromosomal aberrations, suggesting that Nedd8 inhibition

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- (DMSO)

Cisplatin CisplatinMLN4924

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Figure 3. MLN4924 suppresses damage-inducible CHK1 phosphorylation and FANCD2 monoubiquitination. A, HeLa cells were treated with 1 mmol/L ofMLN4924 or DMSO vehicle for 20 hours, followed by PUVA treatment, and cells were harvested at indicated time points for Western blot analysis. B, HCT116cells were treated with 3 mmol/L of MLN4924 or DMSO vehicle for 18 hours, followed by UV (30 J/m2) treatment, and cells were harvested at indicatedtime points for Western blot analysis. C, HeLa cells were treated with cisplatin (10 mmol/L) for 16 hours, followed by treatment with MLN4924 (2 mmol/L) orDMSO for indicated times before being harvested for Western blot analysis. D, HeLa cells were treated with indicated concentrations of MLN4924 for12 hours followed by 12-hour treatment of 10 mmol/L cisplatin. E, HeLa cells were treated with MLN4924 (1 mmol/L) or DMSO for 16 hours, followed byhydroxyurea (2 mmol/L) treatment for 20 hours before fixation for the immunofluoresence assay. Experiments were carried out in triplicate, and the averagenumbers of foci were plotted as graphs. F, HeLa cells were synchronized in S-phase using double thymidine block, then the cells were released into freshmediumcontaining nocodazole. During the last 3 hours of incubation, cellswere treatedwithDMSO,MG132, orMLN4924before being harvested. The lysateswere analyzed by Western blot analysis. Protein band intensities were measured and plotted as graphs. DAPI, 40,6-diamidino-2-phenylindole; HU,hydroxyurea.

Kee et al.





compromised cellular DNA repair capacity upon DNAICLs. Altogether, these results show that inhibition ofNedd8 conjugation system disrupts cellular resistance toDNA ICLs.

DiscussionWe have shown that inhibition of the Nedd8 conjugation

system sensitizes various cancer cells to DNA ICL–inducingagents. Mechanistically, Nedd8 inhibition abrogated DNAdamage–inducible activation of CHK1phosphorylation andFANCD2 monoubiquitination. Previous reports haveshown that treatment with MLN4924 activates DNA dam-age responses, such as CHK1 phosphorylation (23, 26).While our study is consistent with this result, it further showsthat damage-induced activation of CHK1 and FANCD2 issuppressed upon Nedd8 inhibition. Thus, the Nedd8 con-jugation system is required for sustained activation of CHK1and FANCD2.

The suppression of CHK1 phosphorylation andFANCD2 monoubiquitination was not due to alterationin cell-cycle progression. Other DNA damage responses thatoccur during S–G2 phases, such as proliferating cell nuclearantigen (PCNA) monoubiquitination, remain unchangedupon Nedd8 inhibition. Furthermore, phosphorylation ofATM substrates, such as CHK2, 53BP1, NBS1, BRCA1,and g-H2AX, remained unaffected or even elevated underthe same conditions. We therefore conclude that a Nedd8-mediated protein modification event is required for damage-inducible ATR/CHK1 activation and for subsequentFANCD2 monoubiquitination (Fig. 5).How Nedd8 inhibition suppresses CHK1 phosphoryla-

tion and FANCD2 activation requires further investigation.Increasing evidence supports a strong interaction betweenATR/CHK1 signaling and the Fanconi anemia pathway.ATR and CHK1 function is required for induction ofFANCD2 monoubiquitination (9, 30), and CHK1-medi-ated phosphorylation of FANCE is required for cellular

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Figure 4. Inhibition of the Nedd8 conjugation system sensitizes cells to DNA cross-linking agents and PARP1 inhibitor. A, HeLa cells were treated with siRNAsagainst control, FANCM, or UBE2M for 48 hours, followed by MMC treatment, and the cells were incubated for 7 to 10 days before fixation (see Methods fordetail). B, HCT116 cells were treated with 20 nmol/L of MLN4924 or DMSO control, and next day, MMC was added for further incubation of 7 days beforefixation. C, top, 2008 ovarian cancer cells harboring vector (2008) or FANCF cDNA (2008þ F) were treated with 20 nmol/L ofMLN4924, followed by treatmentwith indicated amount of cisplatin. After 5 days, viability of cells was measured as described in Methods. Bottom, the 2008 and 2008 þ FANCF cells weretreated with 2 mmol/L of MLN4924 for 20 hours, followed by UV (30 J/m2) irradiation, and the cells were harvested at indicated time points. D and E,chromosomal aberrations were measured in 293T cells that were treated with 100 nmol/L of MLN4924 (D) or transfected with siRNAs (E), followed by MMCtreatment for 3 days.






resistance to MMC (31). Consistent with reports thatCHK1 may function upstream of FANCD2, siRNA-medi-ated depletion of CHK1 partially suppressed damage-induc-ible FANCD2 monoubiquitination (data not shown). Theobservation that CHK1 phosphorylation is still suppressedby Nedd8 inhibition, regardless of the status in FANCD2monoubiquitination in FANCF-deficient 2008 cell lines(Fig. 4C), further supports the hypothesis. These dataaltogether suggest that the abrogation of FANCD2 mono-ubiquitination upon Nedd8 inhibition may be due, at leastin part, to the failure in CHK1 activation.The primary role of Nedd8 is to activate CRL complex

(21). Accumulation of CDT1, a CRL substrate and alicensing factor for the pre-replication (pre-RC) complex,is a key cytotoxic effect of MLN4924 (23, 26, 27). Accu-mulation of CDT1 induces DNA rereplication and triggersDNA damage and CHK1 activation (32, 33). CDT1 deg-radation by the CRL complex ensures that DNA replicationorigins fire only once per cell cycle (34, 35). Even thoughNedd8 inhibition caused an accumulation ofCDT1, it failedto activate CHK1 (Fig. 3). This result suggests that damage-inducible CHK1 activation requires additional events thatare mediated by the Nedd8 conjugation system.Besides the cullin subunits of the CRL complexes, p53 is

also modified by neddylation, resulting in inhibition of itsactivity (36). The effect of Nedd8 inhibition in suppressingtheDNAdamage response is unlikely to bemediated via p53modification, as our results were observed in several cell types

regardless of p53 status (Fig. 3 and Supplementary Figs. S5and S6). We previously reported that a CRL complexcontaining b-TRCP mediates mitotic degradation ofFANCM (28). The perturbation of timed degradation leadsto disruption of the Fanconi anemia core complex recruit-ment cycle and increase in the cellular sensitization toMMC.On the basis of our evidence that MLN4924 disrupts themitotic degradation of FANCM (Fig. 3F), we propose thatNedd8 inhibition disrupts Fanconi anemia pathway, at leastin part, through perturbation of mitotic FANCM degrada-tion. However, we cannot rule out the possibility that theremay be additional CRL complexes and substrates thatregulate the Fanconi anemia pathway.Because various cellular signaling pathways affect cellular

sensitivity to DNA ICL agents, a pharmacologic inhibitor ofsuch pathways may improve current chemotherapeutic reg-imen. Previous reports have shown that proteasome inhibitorssensitize cells to DNA-damaging agents, suggesting that theseagents may be synergistic in combination therapy (37, 38).Proteasome function is required for activation of DNAdamage responses including the Fanconi anemia pathway(14, 18), providing a basis for the proteasome-mediatedsensitization of cells to DNA-damaging agents. TheNedd8–CRL complexes account for approximately 20% ofproteasome-mediated protein degradation in cells (23). Fur-thermore, inhibition of proteasome, but not of the Nedd8conjugation, causes overaccumulation of overall ubiquitinconjugates in cells (not shown), suggesting more selectivityof the Nedd8 inhibition. Thus, inhibition of the Nedd8system, in combination with DNA-damaging agents, andmore specifically ICL agentsmight offer an alternative strategywhich could provide more specificity in targeting the DNAdamage response. Furthermore, our work suggests that theproteasome-mediated suppression of the Fanconi anemiapathway could be largely due to the suppression of theNedd8 and CRL-mediated protein degradation. Furtherinvestigation of the mechanism involved in this pathway willprovide better insight into DNA damage signaling and mayprovide a new avenue for the design of more specific anti-cancer drugs.

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

AcknowledgmentsThe authors thank Dr. Geoff Shapiro and members of the D'Andrea and the Kee

Laboratories for helpful discussions and suggestions; Millennium Pharmaceuticals Inc.for providing the Nedd8 inhibitor MLN4924; and Mijung Kwon for assistance withthe confocal fluorescence microscope.

Grant SupportThis work was supported byNIH grants 5R01DK43889 and 5R37HL52725 to A.

D. D'Andrea and departmental startup funds to Y. Kee. Y. Kee was a recipient of aLeukemia and Lymphoma Society Career Development Fellowship.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received October 13, 2011; revised December 12, 2011; accepted December 27,2011; published OnlineFirst January 4, 2012.

CHK2

γ-H2AX

SMC1

53BP1

NBS1

ATM

PCNA

ATR

CHK1

FANCD2

Resistance to cisplatin

Neddylation of protein X

Cullin-mediated destruction

of protein X

DNA damage

Figure 5. A protein neddylation event is required upstream of the Fanconianemia pathway. A schematic model for the Nedd8-mediated activationof the DNA damage–inducible FANCD2 monoubiquitination. Whileinterruption of the Nedd8 conjugation system suppresses the ATR-mediated signaling pathways, particularly CHK1 phosphorylation andFANCD2 monoubiquitination, ATM-mediated pathways or PCNAubiquitination remain unaffected. We hypothesize that specific proteinneddylation events, most likely the activation of specific CRL E3 ligases,is required for damage-inducible CHK1 phosphorylation and FANCD2monoubiquitination. We suggest that disruption in the CRL-mediateddestruction of FANCM is onemechanism leading to the abrogation of theFanconi anemia pathway.

Kee et al.





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2012;10:369-377. Published OnlineFirst January 4, 2012.Mol Cancer Res Younghoon Kee, Min Huang, Sophia Chang, et al. Cross-linking AgentsInhibition of the Nedd8 System Sensitizes Cells to DNA Interstrand

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