Novel DNA Damage Checkpoints Mediating Cell Death Induced by the NEDD8-Activating Enzyme Inhibitor MLN4924

Published OnlineFirst October 24, 2012; DOI: 10.1158/0008-5472.CAN-12-1729

Cancer Molecular and Cellular Pathobiology Research

Jonathan L. Blank1, Xiaozhen J. Liu1, Katherine Cosmopoulos1, David C. Bouck1, Khristofer Garcia1, Hugues Bernard1, Olga Tayber1, Greg Hather2, Ray Liu2, Usha Narayanan1, Michael A. Milhollen1, and Eric S. Lightcap1

Abstract MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identiﬁed, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, andbaseexcisionrepair seemedtobeimportantforMLN4924lethality. Incontrast, geneswithin

the G2–M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by ﬂow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S–

G2 phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924. Cancer Res; 73(1); 1–10. 2012 AACR.

Introduction in the activation of the class of E3 ubiquitin ligases known as MLN4924 is an investigational small-molecule inhibitor of the cullin-RING ligases (CRL). These enzymes conjugate the NEDD8-activating enzyme (NAE; ref. 1). MLN4924 has proteins with Lys48-linked polyubiquitin chains (8, 9) there- shown antitumor activity in multiple xenograft models of by promoting the degradation of a relatively small subset human cancer at well-tolerated doses (1–3). MLN4924 is of proteasomal substrates (1). CRLs have been shown to currently being explored in phase I clinical trials, where some monoubiquitinate some proteins, especially histones (9). clinical activity has been observed in solid tumor and hema- Recently, the function of Cul1-Rbx1-Skp1 (CRL1) has been tologic malignancies (4). Therefore, a more complete under- expanded to include Lys63-linked polyubiquitin chain for- standing of the impact of NAE inhibition on cancer cell biology mation (10). fi is warranted. Initial studies to de ne the mechanism of action of NAE catalyzes the activation and transfer of NEDD8 onto MLN4924 have shown that blocking of CRL1-Skp2 and Cul4- Ubc12 and Nce2 in an ATP-dependent manner (5). Activated Rbx1-Ddb1 (CRL4)-Cdt2 function results in Cdt1 stabilization, NEDD8 is then transferred onto 1 of 8 cullins in a DCN1- which leads to DNA rereplication and accumulation of cells in dependent manner (6, 7). Neddylation of the cullins results S-phase, thereby promoting cell death in most cancer cell lines studied (1, 11, 12), although stabilization of IkB via inhibition of CRL1-bTRCP plays a role in some settings (2). To prevent genomic instability, initiation of DNA replication is tightly controlled by a well orchestrated process that Authors' Affiliations: 1Discovery and 2Medical Biostatistics, Millennium Pharmaceuticals, Inc., Cambridge, Massacchusetts involves the origin recognition complex (ORC), Cdc6, Cdt1, geminin, the Mcm2–7 complex, Mcm10, Cdc45, and the GINS Note: Supplementary data for this article are available at Cancer Research – Online (http://cancerres.aacrjournals.org/). complex (13 17). Following DNA replication initiated by the protein kinases Cdk2 and Dbf4-Cdc7, Cdt1 is degraded, Corresponding Author: Eric S. Lightcap, Discovery Oncology Biology, Millennium Pharmaceuticals, Inc., 40 Landsdowne Street, Cambridge, MA whereas Cdc6 is exported from the nucleus, thereby prevent- 02139. Phone: 617-551-3717; Fax: 617-551-8906; E-mail: ing refiring of the origin. Degradation of Cdt1 is controlled in [email protected] a Cdk2-dependent manner by CRL1-Skp2 and in a PCNA- doi: 10.1158/0008-5472.CAN-12-1729 dependent manner by CRL4-Cdt2 (18–21). Cdt2 is also known 2012 American Association for Cancer Research. as denticleless (DTL).

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DNA rereplication results from the multiple refiring of the than 0.995. Cell lines were passaged for fewer than 8 weeks origins of replication during DNA synthesis, generally resulting following resuscitation. in partial polyploidy (16). Overexpression of CDT1 or CDC6 or depletion of GMNN, encoding an inhibitor of Cdt1, can induce Genome-wide screen and hit deconvolution DNA rereplication in some tumor cell lines, although check- A375 melanoma cells (300 cells/well) were reverse trans- point control is activated in normal cells and in other tumor fected in duplicate on 384-well poly-D-lysine (PDL)-coated cell lines (22, 23). black, clear bottom plates (CELLCOAT, Greiner) using 15 Several studies have shown that cullin-dependent ubiquiti- nmol/L siRNA oligos (siGENOME SMARTpool, Dharmacon) nation along with the COP9 signalosome, which is responsible and 28 nL/well DharmaFECT 4 reagent (DH4, Dharmacon) in for deneddylation of the cullins, have additional roles in DNA 64 mL Opti-MEM (Life Technologies). After 48 hours of knock- damage response pathways. These include global genome and down, cells were treated with 0, 250, or 650 nmol/L MLN4924, transcription-coupled nucleotide excision repair, histone mod- in the continued presence of siRNA oligos, and incubated for a ification, and regulation of checkpoints via degradation of p21, further 48 hours, after which viability was assessed with p27, p53, Cdc25A, Wee1, claspin, and Fanconi anemia, com- ATPlite reagent according to the manufacturer's instructions plementation group M (FANCM) (9, 24–27). Therefore, it is (Perkin-Elmer). Luminescence was measured using a LEAD- likely that inhibition of NAE by MLN4924 impacts multiple seeker imaging system (GE Healthcare). Viability interactions DNA damage response pathways beyond those involving Cdt1 of SMARTpools with MLN4924 were scored according to Bliss stabilization. independence (BI) assumptions that enable siRNAs to be Therefore, to ascertain the relative importance of these ranked based on magnitude of effect (31). BI and Rescue scores potentially diverse mechanisms, we have conducted a allow for the assignment of synthetic lethal (BI < 0), epistatic genome-wide siRNA screen to evaluate the genetics of sensi- (BI > 0, Rescue > 0), or suppressor (BI > 0, Rescue < 0) tivity to MLN4924 in the A375 human melanoma-derived cell phenotypes to the RNAi results. line. RNA interference (RNAi) screens provide an important SMARTpool hits were deconvoluted in sextuplicate under avenue for understanding the sensitivity of tumor cells to drugs identical conditions, except that 8 nmol/L individual oligo was and drug candidates (28–30). used. Calculation of false discovery rates (FDR) was achieved This screen identified 154 genes whose knockdown had using a random permutation of deconvoluted data, as outlined significant effects on MLN4924-induced cell death. Collective- in Supplementary Materials and Methods. ly, the data suggest that NAE inhibition by MLN4924 primarily For HCT-116 cell transfections, changes included the use of causes cell death by dysregulating components of the cell cycle, 450 cells per well, transfection with Lipofectamine RNAiMax apoptotic machinery, the ubiquitin system, and the DNA (Life Technologies) at 20 nL/well, and McCoy5A growth medi- damage response, including the p53 pathway, BRCA1/BRCA2 um (Life Technologies). After 48 hours of knockdown, HCT-116 complex, transcription-coupled, and base excision repair. In cells were treated with 0, 91, or 116 nmol/L MLN4924 for an particular, we show that activation of the p53 pathway is additional 48 hours before assaying cell viability by ATPlite. independent of Cdt1 stabilization and ATR activation, showing MLN4924 will be made available to qualified researchers once a that MLN4924 affects cell survival by the stabilization of standard Materials Transfer Agreement has been executed. proteins beyond Cdt1. Flow cytometry Materials and Methods A375 cells were reverse transfected on 96-well PDL-coated black, clear-bottom plates (CELLCOAT, Greiner) using 15 Additional experimental details are contained within the nmol/L siRNA oligos (siGENOME SMARTpool, Dharmacon) Supplementary Materials and Methods. and 84 nL/well DharmaFECT 4 reagent (DH4, Dharmacon) in Opti-MEM (Life Technologies). Forty-eight hours after trans- Genomic characterization and authentication of cell fection, cells were treated in triplicate with 500 nmol/L lines MLN4924 or vehicle [0.005% dimethyl sulfoxide (DMSO)] and A375 and HCT-116 were received from the American Type incubated for 22 hours. Cells were pulsed with 10 mmol/L 5- Culture Collection (ATCC) in July 2006 and June 2009, bromo-2-deoxyuridine (BrdUrd, Life Technologies) for 30 min- respectively, and passaged twice before freezing. Genomic utes, trypsinized, fixed, and denatured. Cells were stained with DNA was isolated from 2 million cells under vendor-spec- undiluted fluorescein isothiocynate (FITC)-conjugated anti- ified protocols. Five-hundred micrograms of DNA per sam- BrdUrd (BD Pharmingen), washed, and stained with 1 mg/mL ple was then amplified,labeled,andprocessedonthe propidium iodide (Life Technologies). Flow cytometry was Affymetrix SNP-6 whole genome array platform on August conducted using a Becton Dickinson FACSCanto II with High 22, 2011 and evaluated using Partek Genomics Suite for copy Throughput Sampler. Flow cytometry data were analyzed as number variation analysis. Spearman correlation was deter- outlined in Supplementary Materials and Methods. mined for each cell line against an ATCC-authenticated stock vial received at Millennium (A375 ATCC CRL-1619 cDNA synthesis and TaqMan Gene Expression assays for and HCT-116 ATCC CCL-247). Consistent regions of ampli- qPCR fication and deletions were found between the cell lines of The cDNA to be used for the quantitative PCR (qPCR) thesameoriginandcorrelationongenotypesweregreater reactions was generated using ABI TaqMan Gene Expression

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Cells-to-CT kit (Applied Biosystems #AM1729) according to by MLN4924. These hits represent genes whose function manufacturer's protocols and was conducted on Biomek FX may be directly dysregulated by MLN4924. In interpreting Liquid Handling System. TaqMan Gene Expression Assays kits RNAi screens, it is important to remember key limitations of were purchased from Applied Biosystems and data were RNAi, including transient protein knockdown, incomplete generated with an ABI7900HT system. elimination of the protein, genetic redundancy, and off-target effects. Western blotting Consistent with NAE being the primary target of MLN4924, A375 cells were treated with 650 nmol/L MLN4924 or vehicle the 2 hits with the greatest degree of synthetic lethality (0.065% DMSO) for the times indicated. For UV irradiation, following deconvolution (Table 1) were UBA3 and NAE1, A375 cells were exposed to 30 J/m2 UV and then incubated for encoding the heterodimeric subunits of NAE. Depletion of – UBA3 NAE1 24 hours. To induce a G1 S-phase arrest, A375 cells were either or by RNAi in cells therefore seems to reduce treated with 1 mg/mL aphidicolin for 24 hours. For each the effective concentration of MLN4924 required to achieve a sample, a large batch of cells was lysed in radioimmunopre- critical level of inhibition of the NEDD8 pathway that results in cipitation assay (RIPA) buffer and prepared for SDS-PAGE and cell death. quantitative immunoblot analysis with antisera as specified in The hits with the greatest degree of suppression of the Supplementary Table S1A using tubulin as a normalization MLN4924-induced lethality included CDT1, CASP8,and control so that the coefficient of variance across samples was TEX10, a protein associated with the SLX4 Holliday junction less than 16%. This set of samples was then used to run multiple resolvase (32). Depletion of CDT1 wouldbeexpectedto Western blots. Western blot intensity was quantified using an reduce prereplication licensing, thereby preventing cell Odyssey Infrared Imager (LI-COR Biosciences). Full-length death, consistent with its scoring as a suppressor. Hits were blots are presented in Supplementary Fig. S1. Antibody details also evaluated using 2 or 4 out of 4 oligos (O2 and O4 criteria, are presented in Supplementary Table S1A. Supplementary Table S2A and S2B), although these criteria captured fewer hits. Results Genome-wide siRNA screen Behavior of genes in HCT-116 Gene knockdown in A375 cells was conducted with 15 nmol/L The deconvoluted siRNA oligos against 240 genes were also Dharmacon SMARTpool siRNA oligos for 48 hours to deplete evaluated in HCT-116 cells at 91 and 116 nmol/L MLN4924 cellular proteins, followed by 48-hour treatment with 2 concen- (LC50 and LC80, respectively, Supplementary Tables S1H and trations of MLN4924 [LC25 (250 nmol/L) and LC70 (650 nmol/L)] S1I), of which 24 (10%) scored as hits by O4 criteria following or a vehicle control. These concentrations of MLN4924 enabled permutation of results from 240 randomly selected genes the evaluation of either enhancement or suppression of (Supplementary Table S2C). Fewer hits were identified by MLN4924-induced cell death by the SMARTpools. A375 cells either O2 or O3 criteria (Supplementary Table S1I). With the are highly transfectable and display an average amount of exception of the TCR/BER gene set, the depletion of the DDR rereplication following MLN4924 treatment relative to other and cell-cycle genes that impacted A375 sensitivity of cell lines, as measured by the accumulation of cells with a more MLN4924 did not similarly impact HCT-116 sensitivity. than 4N DNA content (Supplementary Fig. S2). The genome- Because HCT-116 rereplicates its DNA following treatment wide screen was conducted with 21,062 SMARTpools in dupli- with MLN4924 even more substantially than does A375 (Sup- cate (Supplementary Table S1B and Supplementary Fig. S3A). plementary Fig. S2), we hypothesized that genetic differences Following reconfirmation of 2239 SMARTpool hits (Supplemen- in DDR mechanisms between A375 and HCT-116 may sub- tary Table S1C and Supplementary Fig. S3B–E), 1221 different stantially impact the response to MLN4924. Therefore, decon- genes were deconvoluted, with separate wells for each individual voluted siRNA oligos against 317 genes with known DNA oligo at 8 nmol/L siRNA (Supplementary Table S1D and S1E and damage response roles were evaluated in HCT-116 cells at Supplementary Fig. S4). 91 nmol/L MLN4924 (Supplementary Table S1H & S1I, Run 3), Using permutation analysis and confirmation of gene of which 9 scored as hits (Supplementary Table S2C, annotated expression (Supplementary Table S1F and S1G), 109 hits were with #). Notably, 6 of these have known roles within the G2–M identified with an FDR P < 0.05 based on 3 of the 4 oligos checkpoint, with knockdown of FBXO5 (encoding Emi1), CDK1, interacting with MLN4924 (O3 criteria, Table 1). An additional and PLK1 giving the most significant effects. 45 hits were identified with an FDR 0.05 < P < 0.10 based on 3 of 4 oligos (O3 criteria). These hits were classified according to Cell-cycle interactions of hits with MLN4924 their gene annotation (Fig. 1). MLN4924 induces DNA rereplication by stabilizing the Importantly, the screen captured key aspects of biology replication licensing factor Cdt1 (12). To quantify the effect previously shown to be important for MLN4924 mechanism of MLN4924 on the cell cycle and to better characterize how of action, including genes involved in the ubiquitin pathway, MLN4924 might affect previously studied rereplication check- DNA damage repair (DDR), cell cycle, TNF family receptors, points, A375 cells transfected with control siRNA (GL2) were and apoptosis. However, genes within the p53 pathway, treated with vehicle or 500 nM MLN4924 for 22 hours, pulse- BRCA1/BRCA2 complex, transcription-coupled, and base exci- labeled with BrdUrd, stained with propidium iodide, and then sion repair pathways were identified that had not previously subjected to flow cytometry (Fig. 2A). MLN4924 resulted in been implicated in the mechanism of cell death induced changes to most phases of the cell cycle (Fig. 2B), including

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Table 1. 154 Genes whose knockdown signiﬁcantly affects the induction of cell death by MLN4924

DNA Damage repair Cell cycle

p53 BRCA TCR/BER Histone Other S G2–M Other PPP1R10 CDR2L GPN1 HIST1H2BO HUS1 TK1 BRD4 LZTS2 RAB35 PIAS4 XRCC1 PAF1 ILF2 MYBL2 MPHOSPH9 C4orf7 KLF5 RAD50 THOC2 SSB RAD21 RPA1 SPIN1 IPMK CYFIP2 MDC1 XAB2 ZMYM3 PCNA INCENP BRAF CDKN1A ATM FEN1 RRM2 CENPI TSC22D2 WWOX CLSPN TTF1 REPIN1 EI24 FANCM SUPT6H MCM7 SESN2 SFR1 DTL HDAC4 BARD1 DHFR TNFRSF10B ABL2 PPAT DDX3X TEX10 CDT1

mRNA Protein TNFR and Processing processing apoptosis Ubiquitin Cell Adhesion GPCR Unassigned

MED6 WDR4 MCL1 UBA3 SPOCK1 PDE9A C11orf75 WWC3 C2orf29 GTPBP5 CD40 NAE1 LCE1E GNG8 SYNGR2 RAB22A MED21 SERP1 IL8 UBE2Q1 DOCK1 ARHGEF6 TMEM110 ATG4A SOX21 NOB1 TNFRSF11B FBXO17 AFAP1 XPR1 TMCC3 CLCN7 JUNB NUPL1 LASS1 UBE2L3 SCYL3 OR10A3 FETUB FKSG2 NFIX PRKAA2 BAX CUL2 PTK2 GLUD1 AS3MT PRCC EIF2B4 ARL6IP1 CUL1 PLSCR1 TMEM69 THUMPD2 RPL27A BCL2 CDC34 FAHD2A SH2D4A DDX41 EMG1 GULP1 CUL7 C17orf88 WDR19 ZFP82 TUFM DFFB PSMD7 STIM2 CNPY2 ZNF184 NUP93 FAIM PSMD14 AVPI1 RDH13 SFRS2B EIF3G IL1A PSMD2 KIAA1468 MED28 FAU CASP8 ATG7 RBM43 NUP205 NHEDC2 NIF3L1 TBCCD1 MSI2 C20orf3 ZNF280B ZNF229 SOX10 SF3A1

NOTE: Oligo 3 scored set (109 genes: FDR P < 0.05, 45 genes: 0.05 < FDR P < 0.10). Genes contained within each classiﬁcation of Fig. 1 ordered according to their BI score from most negative. Gray background genes are synthetic lethal hits, white background genes are epistasis hits, except for the red-lettered genes, which are suppressor hits. Bold letters indicate the 15 genes with the strongest BI scores. Human Genome Organization (HUGO) names are given for the genes.

increases in the number of cells with S-phase DNA content but cation licensing proteins results in defective replication lead- not incorporating BrdUrd (labeled as 2N-4N) and cells with ing to cell-cycle arrest at a G2–M checkpoint rather than in more than 4N DNA actively incorporating BrdUrd. Of note, sub- inhibition of origin ﬁring. Engagement of the G2–M checkpoint G1 cells were not captured in this analysis due to their loss in is consistent with the response to rereplication as previously plate washing. characterized genetically.(15, 16) Following treatment by We evaluated the effect of depleting 11 genes directly MLN4924, a loss of S-phase was again seen, although the associated with the prereplication complex or origin ﬁring, as pattern shifted, as depletion of key replication licensing pro- well as UBA3 (Fig. 2C), treated with either vehicle (DMSO) or teins resulted in an accumulation of cells with 2N-4N (arrested) MLN4924. Knockdown of 9 of these genes in vehicle-treated or more than 4N (rereplicating) DNA content, suggesting that cells resulted in an accumulation in G2–M, generally with loss the rereplicating cells were less able to progress to G2 in the of S-phase. These results suggest that depletion of key repli- presence of MLN4924.

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We also evaluated the effect of knockdown of a further 86 genes that function more broadly in cell cycle and DNA damage responses for changes to the cell-cycle effects of MLN4924. In total, depletion of 69 genes significantly impacted the cell-cycle distribution following MLN4924 treatment (Fig. 3, Supplementary Fig. S5 and Supplementary Table S1J). Broadly, depletion of these genes can be categorized as affecting MLN4924-induced cell-cycle changes by their most significant impact on the cell cycle, namely increase in G1 content (BRIP1-XRCC3), decrease in S-phase content (ATR- UBE2L3 Figure 1. Classification of 154 genes whose knockdown significantly ), accumulation of cells with 2N-4N (arrested) DNA affects the induction of cell death by MLN4924. Classification of genes content (CHEK2-RPA1), accumulation of cells with more than with at least 3 of 4 oligos having significant interactions with MLN4924 4N (rereplicating) DNA content (ATM-XRCC2), or reduction in (FDR, P < 0.10). DDR and cell-cycle genes constitute 36% of all hits, the number of cells with 2N-4N (arrested) DNA (DDX3X- whereas TNFR/apoptosis and ubiquitin genes constitute 16% of all TNFRSF10B hits. ). Notably, knockdown of 6 of the 12 genes from Fig. 2 caused a similar impact following treatment with

A GL2 siRNA, Vehicle, 22 h C 1

P3: >4N DNA, 1.0% ± 0.1%

GL2 siRNA, 500 nmol/L MLN4924, 22 h –M 2

P3: >4N DNA, %G 10% ± 2%

B %>4N %2N–4N %S %G % Cells 1 S G –M 2 >4N

G Gene targeted by siRNA 2N–4N

Figure 2. Effect of MLN4924 on cell-cycle distribution of A375 melanoma cells. A, the impact of MLN4924 (500 nmol/L for 22 hours) on cell-cycle distribution in A375 cells transfected with control (GL2) siRNA was evaluated by flow cytometry in a 96-well plate format. B, quantitation of flow cytometry analysis was based on 108 separate samples. Cells with 2N-4N that are not incorporating BrdUrd DNA content was captured by the P1 gate; cells with more than 4N DNA content were captured by the P3 gate. Error bars represent the SEM. MLN4924 induced significant changes (P < 0.001) to cell-cycle content for all gates except S-phase (for which P ¼ 0.10), as measured by Student t test. C, the effect of gene knockdown by SMARTpool RNAi on MLN4924-induced cell- cycle changes was evaluated by flow cytometry. The solid line represents the control RNAi (GL2) value from Fig. 2B. Values were averaged from 2 to 4 separate triplicate runs; error bars for each gene represent SEM. Black bars indicate vehicle-treated samples, whereas red bars indicate MLN4924-treated samples. Solid and stippled bars represent data significantly different from the GL2 control with an FDR P < 0.001 and FDR P < 0.05, respectively. Hits from the viability screen are labeled parenthetically with the associated phenotype. L, synthetic lethal; E, epistatic; and S, suppressor (Table 1, Supplementary Table S2A and S2B, and Table III in Ref. 33).

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Set 1 Set 2 Set 3 Set 4 Set 5

Gene targeted by siRNA

Figure 3. Effect of gene knockdown on cell cycle distribution of A375 melanoma cells in combination with MLN4924. Same experiment as Fig. 2C, with a focus on 57 genes with broader cell cycle and DNA damage response roles. The genes have been grouped into 5 sets based on their most signiﬁcant impact on the cell cycle. All genes had an FDR P < 0.01, except ATR whose minimum FDR P ¼ 0.017 was for its S-phase reduction.

MLN4924 to those oligos in the third set (CHEK2-RPA1). Many suggest that the function of these genes in the response to of the genes whose depletion shifts cells between the arrested MLN4924 may not be fully realized by their effects on the cell population and the rereplicating population are likely to be cycle. involved in activation of an S-phase checkpoint in response to rereplication. Upregulation of p53 and p21 is independent of Cdt1 or ATR Extent of rereplication does not significantly correlate To understand better the sequence of events for induction of with cell death checkpoints, we evaluated the effect of depleting CDT1, TP53, Surprisingly, these cell-cycle changes (Fig. 2C, 3 and ATM, and ATR by Western blot analysis on key proteins (Fig. 4). Supplementary Fig. S5) did not significantly correlate with These data showed efficient knockdown of CDT1, TP53, and the viability effects of genes with significant MLN4924 ATM. As expected, knockdown of CDT1 or ATR following interactions. Genes whose knockdown resulted in the sig- treatment of cells with MLN4924 significantly reduced the fi ni cant accumulation of cells in G1 for vehicle-treated cells phosphorylation of ATR substrates, including Rpa32 and Chk1, were marginally negatively associated with a synthetic lethal as well as the stabilization of claspin and the phosphorylation phenotype (5 of 22, P ¼ 0.18). Genes whose knockdown led to of Rb. Surprisingly, knockdown of CDT1 or ATR had little increased >4N DNA content following MLN4924 treatment impact on increases in p53 or p21, whereas TP53 depletion were marginally negatively associated with a suppressor was very efficient in blocking these effects. A spliceform of phenotype (2 of 8, P ¼ 0.14). Thus, S-phase content before Puma was also unaffected by CDT1 depletion but reduced by treatment seems to be important for MLN4924 sensitivity TP53 depletion. Therefore, unexpectedly, the upregulation of and increased >4N DNA following treatment increased cell p53 and p21 seems to be independent of DNA damage induced death, although both effects were modest. These results by Cdt1 stabilization, particularly as detected by ATR signaling.

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Discussion Vehicle, 24 h MLN4924, 24 h Our original interest in developing an NAE inhibitor was Cdt1 based on an emerging knowledge of the cullin-dependent degradation of many cancer relevant proteins, the dysregula- Rpa32 (pT21) tion of which could provide a therapeutic benefit. Overall, the Chk1 (pS317) data obtained in our MLN4924 synthetic lethal screen align Claspin well with prior genetic studies on rereplication and in that many of the genes affecting MLN4924 sensitivity are directly CDT1-Dep H2Ax (pS139) involved in the prereplication complex and DNA replication, Rb (pS807/pS811) including PCNA, MCM7, DTL, CDT1, TK1, RPA1, RRM2, and DHFR. Importantly, DDR mechanisms were also well repre- p53 sented in these viability screen results. p53 (pS15) These DDR mechanisms included p53, BRCA1/2, transcription-coupled repair, base excision repair, and histones. Some p21 aspects of these results could have been anticipated. For TP53-Dep Puma example, CRL4 has clear roles in transcription-coupled repair (36) and histone monoubiquitination (9). However, while ATM multiple CRLs have been proposed to regulate p53 (37), the Tubulin precise CRL for p53 relevant in this setting is not clear. Generally, characterization of the role of p53 and p21 in siRNA GL2 GL2 MLN4924 and rereplication biology has included intra-S-phase ATR ATR ATM ATM TP53 TP53 CDT1 CDT1 and G2–M checkpoint control and the induction of senescence (11, 38, 39). Our data suggest that the intra-S-phase checkpoint Figure 4. MLN4924 affects the p53 pathway independently of Cdt1 or ATR is the most important in this setting. Finally, the roles of CRLs signaling. The ability of MLN4924 to regulate key proteins was evaluated in regulating BRCA1/BRCA2 and base excision repair have not following knockdown of the indicated genes. Knockdown of either CDT1 yet been characterized. The role of BRCA1 in rereplication is or ATR affected the regulation of one group of proteins (CDT1-Dep), but – had much less impact on another group of proteins (TP53-Dep). In understood to be promoting the G2 M checkpoint (40), there- contrast, knockdown of TP53 affected only the regulation of the TP53- by protecting against cell death. Contrary to these expecta- Dep group of proteins. tions, depletion of most of the BRCA1/BRCA2-associated genes, especially BARD1, resulted in suppression, suggesting a role in Activation of ATR by MLN4924 in A375 cells promoting cell death. Three BRCA-associated genes, BARD1, MLN4924 has been previously shown to induce the phos- BRCA1, and BRIP1, significantly reduced S-phase content (Fig. phorylation of ATR substrates including Chk1, Rad17, and 3), suggesting that the role of this complex in DNA replication Nbs1 in HCT-116 cells (12). However, depletion of ATR and and S-phase progression may account for this discordance (41). its substrates only moderately influenced the effect of Strikingly, early steps in BRCA1 complex formation are syn- MLN4924 on cell viability and the cell cycle (Table 1 thetic lethal, whereas genes in later steps are suppressors, and Fig. 3). Knockdown of ATR does efficiently block phos- suggesting that MLN4924 may affect BRCA1 function at the phorylation of Rpa32 (Fig. 4). Thus, activation of ATR or a transition between these 2 sets of genes, similar to the inter- critical downstream pathway may be blocked. Figure 5A action between bortezomib and genes within the Myc-poly- compares MLN4924 with 2 control treatments known to amine pathway (31). activate ATR, aphidicolin, and UV (34). Phosphorylation of the ATR targets Chk1, Mcm2, Nbs1, Rad17, and Rpa32 was Discordance with HCT-116 similar between all three treatments, whereas Cdc25A, a We were initially surprised by the lack of overlap in the substrate of Chk1 that is targeted for degradation by CRL1- results between HCT-116 and A375 (Supplementary Table bTRCP (35), was preferentially stabilized by MLN4924. S2C). However, evaluation of the 317 DNA damage response Among the other proteins previously shown to be CRL4-Cdt2 genes suggested that HCT-116 fails to engage the intra-S-phase – substrates (Cdt1, p21, Set8) or otherwise associated with checkpoint but proceeds to a G2 M checkpoint instead. Sta- MLN4924 activity (p-H2Ax and p53), Cdt1 and p21 stabilization bilization of Emi1 (encoded by FBXO5) through the inhibition most differentiated MLN4924 from the other 2 treatments. of CRL1-bTRCP by MLN4924 may account for the activation of Phosphorylation of ATR substrates following MLN4924 this checkpoint. Flow cytometry data suggest significant dif- treatment occurs in a single wave at approximately 10 hours ferences in the effect of MLN4924 on the cell-cycle profile of to half-maximal phosphorylation, consistent with these being HCT-116 and A375 consistent with activation of different substrates of a single kinase (Fig. 5B). Therefore, MLN4924 checkpoints (Supplementary Fig. S2). Garner and Eastman clearly activates ATR, as previously described (12). If MLN4924 found that of the 59 cell lines in the NCI-60 panel, only 4 directly interferes with the establishment of the ATR-depen- arrested in G2 rather than S-phase in response to the topo- dent checkpoint, it seems to be through the stabilization isomerase I inhibitor SN-38, including HCT-116 (42). Over- of Cdc25A or events further downstream from ATR expression of MRE11A in HCT-116 cells rescued the S-phase phosphorylation. arrest defect. Importantly, RAD50 siRNA was synthetic lethal

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A Vehicle MLN4924 B 31.5 h 4 12 24 48 4 12 24 48 h Aphidicolin Vehicle MLN4924 UV V M 8 18 36 8 18 36 Cdc25A

Chk1 (pS317)

Mcm2 (pS108)

Nbs1 (pS343)

Rad17 (pS656)

Rpa32 (pT21)

ATR

Cdt1

p21

Set8

H2Ax (pS139)

p53

p53 (pS15)

Tubulin

Figure 5. Phosphorylation of ATR substrates does not show dysregulation by MLN4924, although Cdc25A is rapidly stabilized. A, MLN4924 induces the phosphorylation of multiple ATR substrates, similar to other inducers of ATR activity, including aphidicolin and UV. Only the stabilization of Cdc25A, a Chk1- regulated event, differentiates MLN4924 from the other treatments. The kinetics of these phosphorylations are compared with the changes of other proteins previously shown to be substrates of CRL4-Cdt2 (Cdt1, p21, and Set8) or regulated by MLN4924. V, vehicle; M, MLN4924. B, the data were quantiﬁed by Odyssey Infrared Imager and normalized so that the 4-hour vehicle time point was 0 and the maximally regulated time point was 1. Maximum fold effect is indicated parenthetically beside each protein name. Cdt1 and Cdc25A achieved half-maximal stabilization within 3 hours. Phosphorylation of Chk1, Mcm2, Nbs1, Rad17, and Rpa32 achieved half-maximal levels in approximately 10 hours. Stabilization of p53 and p21 as well as phosphorylation of p53 and H2Ax achieved half-maximal levels in 15 to 23 hours. As we have not shown the linearity of response with our antibodies, kinetics of protein effects are only approximate.

with MLN4924 in A375 cells but not HCT-116 cells. Mismatch Intra S-phase checkpoint activation repair–deficient cells, including HCT-116, also fail to arrest in Of the 32 genes whose depletion resulted in a significant S-phase following ionizing radation treatment, in contrast to increase in extensive rereplication as indicated by the accu- mismatch repair–proficient cells (43, 44). mulation of more than 4N DNA content (Fig. 3), 11 were The evaluation of gene knockdown using orthogonal tech- associated with p53, including TP53, ATM, MDM2, CDKN1A, niques generally gives a much greater sense of the biologic SFN, RRM2B (45), FKBP3 (46), KLF5 (47), PPP1R10 (48), RAB35 impact of those genes. Notably, depletion of 69 genes gave (49), and RBM38 (50). Knockdown of most of these genes also significant cell-cycle interactions with MLN4924 (Fig. 2C and 3). resulted in the reduction of the cells with 2N-4N DNA content. The combined viability and cell-cycle data inform our under- These results suggest that p53 activation by MLN4924 limits standing of the phenotypic consequences of NAE inhibition as the extent of rereplication, consistent with genetic studies on discussed below and summarized in Fig. 6. rereplication (23). Surprisingly, depletion of ATR, CHEK1 and RAD1 did not Regulation of replication origin firing result in a significant loss of cells with 2N-4N DNA, although – CDC25A HUS1 The canonical response to rereplication stress is G2 M depletion of and did. Therefore, the intra-S- phase accumulation (Fig. 6, blue arrows; refs. 15, 16), consistent phase checkpoint is not clearly ATR-dependent, but is clearly with the results obtained for knockdown of replication genes p53-dependent, at least with continuous treatment by (Fig. 2C). Following treatment with MLN4924, the cell-cycle MLN4924. Washout experiments suggest that MLN4924 impact of depletion of these genes was more divergent (Fig. 6, requires more than 8 hours to induce a stable p53-dependent red arrows). intra-S-phase checkpoint, (11) consistent with the relatively

OF8 Cancer Res; 73(1) January 1, 2013 Cancer Research

MLN4924 Genome-Wide RNAi Screen

inhibition by MLN4924. Importantly, the ability of MLN4924 to CDC6 Origin Set 1 induce the p21-dependent intra-S-phase checkpoint seems to (CDT1) licensing be independent of Cdt1 stabilization or signaling through ATR G1 (Fig. 4). In addition, MLN4924 stabilizes Cdc25A (Fig. 5A), Set 2 S which may account for the direct interference of MLN4924 in (GMNN) the activation of the ATR-dependent intra-S-phase checkpoint MLN4924 Origin (53). The impact of MLN4924 beyond Cdt1 stabilization shows firing that MLN4924 will not simply mimic CDT1 overexpression. Set 3 UBA3 A genome-wide siRNA viability screen provides an unbiased MCM2/10, PCNA, way of surveying possible drug mechanisms. We have used CDC7, (MCM5/7, CDT1 ORC1) such a screen to characterize the genetics of sensitivity of the CRL4-DTL melanoma cell line A375 to MLN4924. Although it remains to be (CRL1-SKP2) p21-dependent determined precisely how NAE inhibition impacts these mul- Origin Intra S-phase Rereplication tiple pathways, these results together with those investigating misfiring checkpoint changes in protein expression following MLN4924 treatment Set 5 Set 4 (33, Supplementary Table S1K) will facilitate a better mecha- GMNN, nistic understanding of the cullin-dependent regulatory and MCM5/7, ORC1 repair processes that compromise the viability of cancer cells. ATR-dependent MLN4924 checkpoint Disclosure of Potential Conflicts of Interest S G. Hather is employed by Millennium Pharmaceuticals as a Principal Bio- statistician. E.S. Lightcap is employed by Millennium Pharmaceuticals as a fi G2 Scienti c Fellow and has ownership interest (including patents) in Millennium Pharmaceuticals. All authors were employed by Millennium Pharmaceuticals at p53-Dependent the time of their contribution to this work. No potential conflicts of interest were G2 Checkpoint disclosed by the other authors.

ﬂ Authors' Contributions Figure 6. Schematic of the in uence of gene depletion on rereplication Conception and design: J. Blank, M.A. Milhollen, E.S. Lightcap stress response. The canonical response of cells to rereplication stress is Development of methodology: J. Blank, X.J. Liu, K. Cosmopoulos, M.A. indicated in this schematic by the blue arrows. Apparent changes to this Milhollen response pathway induced by MLN4924 are indicated by red arrows. The Acquisition of data (provided animals, acquired and managed patients, role of replication origin licensing genes in these effects is inferred from provided facilities, etc.): J. Blank, X.J. Liu, K. Cosmopoulos, D.C. Bouck, K. the cell-cycle effects given in Fig. 2C. Roles dominant only for vehicle- Garcia, H. Bernard treated samples are indicated parenthetically. Roles dominant only for Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Cosmopoulos, D.C. Bouck, G. Hather, R. Liu, M.A. MLN4924-treated samples are indicated with red letters. A role for CRL1- Milhollen, E.S. Lightcap Skp2 in the degradation of Cdt1 has been previously characterized but is Writing, review, and/or revision of the manuscript: J. Blank, D.C. Bouck, G. not evaluated here. The likely step impacted by different gene sets Hather, E.S. Lightcap from Fig. 3 is also shown. Human Genome Organization (HUGO) names Administrative, technical, or material support (i.e., reporting or orga- for the genes are used. nizing data, constructing databases): O. Tayber, U. Narayanan Study supervision: E.S. Lightcap slow induction of p53 and p21 (Fig. 5B, 19 hours to half- Acknowledgments The authors thank Jean Courtemanche, John Donovan, Jesse Gray, Kara Hoar, maximal levels). In addition, depletion of ATR pathway genes Andrew Xiao, and Andrew Xu for technical help with the experiments. The did not affect sensitivity of HCT-116. authors also thank Margaret Quinlan and Petter Veiby for help with the These results highlight one key difference between our data manuscript and experimental design. and the genetic interference studies on the regulation of replication origin ﬁring, namely that very few genes within Grant Support the ATR-dependent checkpoints were found to interact with This work was supported by the funding provided by Millennium Pharma- ceuticals, Inc. MLN4924 in the A375 viability screen. Other researchers have The costs of publication of this article were defrayed in part by the payment of suggested that MLN4924 may interfere with the function of the page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ATR-dependent checkpoints (12, 51). Our data suggests that the p21-dependent intra-S-phase checkpoint (52) may play a Received May 7, 2012; revised September 24, 2012; accepted October 9, 2012; more dominant role in the response of A375 cells to NAE published OnlineFirst October 24, 2012.

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OF10 Cancer Res; 73(1) January 1, 2013 Cancer Research

Novel DNA Damage Checkpoints Mediating Cell Death Induced by the NEDD8-Activating Enzyme Inhibitor MLN4924

Jonathan L. Blank, Xiaozhen J. Liu, Katherine Cosmopoulos, et al.

Cancer Res Published OnlineFirst October 24, 2012.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-12-1729

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