ribonucleotide reductase catalytic subunit m1 (rrm1) as a ...gene expression omnibus data sets...

Biology of Human Tumors

Ribonucleotide Reductase Catalytic Subunit M1(RRM1) as a Novel Therapeutic Target in MultipleMyelomaMorihiko Sagawa1,2, Hiroto Ohguchi1, Takeshi Harada1, Mehmet K. Samur3,Yu-Tzu Tai1, Nikhil C. Munshi1,4, Masahiro Kizaki2, Teru Hideshima1, andKenneth C. Anderson1

Abstract

Purpose: To investigate the biological and clinical significanceof ribonucleotide reductase (RR) in multiple myeloma.

ExperimentalDesign:Weassessed the impact of RR expressionon patient outcome in multiple myeloma. We then characterizedthe effect of genetic and pharmacologic inhibition of ribonucle-otide reductase catalytic subunit M1 (RRM1) on multiple mye-loma growth and survival using siRNA and clofarabine, respec-tively, in both in vitro and in vivo mouse xenograft models.

Results: Newly diagnosed multiple myeloma patients withhigher RRM1 expression have shortened survival. Knockdown ofRRM1 triggered significant growth inhibition and apoptosis inmultiple myeloma cells, even in the context of the bone marrowmicroenvironment. Gene expression profiling showed upregula-tionofDNAdamage response genes andp53-regulated genes afterRRM1 knockdown. Immunoblot and qRT-PCR analysis con-firmed that g-H2A.X, ATM, ATR, Chk1, Chk2, RAD51, 53BP1,

BRCA1, and BRCA2 were upregulated/activated. Moreover,immunoblots showed that p53, p21, Noxa, and Puma were acti-vated in p53 wild-type multiple myeloma cells. Clofarabine, apurine nucleoside analogue that inhibits RRM1, induced growtharrest andapoptosis inp53wild-type cell lines.Althoughclofarabinedidnot inducecelldeath inp53-mutant cells, itdid trigger synergistictoxicity in combination with DNA-damaging agent melphalan.Finally, we demonstrated that tumor growth of RRM1-knockdownmultiple myeloma cells was significantly reduced in a murinehuman multiple myeloma cell xenograft model.

Conclusions:Our results therefore demonstrate that RRM1 isa novel therapeutic target in multiple myeloma in the preclin-ical setting and provide the basis for clinical evaluation ofRRM1 inhibitor, alone or in combination with DNA-damagingagents, to improve patient outcome in multiple myeloma.Clin Cancer Res; 23(17); 5225–37. �2017 AACR.

IntroductionMultiple myeloma is a plasma cell disorder characterized by

excess malignant plasma cells in the bone marrow (BM),increased monoclonal gammaglobulin in blood and/or urine,and end organ damage in kidney and bone (1). Although protea-some inhibitors (bortezomib, carfilzomib, and ixazomib), immu-nomodulatory drugs (lenalidomide and pomalidomide), andmAbs (daratumumab and elotuzumab; refs. 2, 3) have achievedremarkable clinical responses and improved patient outcome,relapse of disease is common, highlighting the need for noveltreatment strategies (4, 5).

Ribonucleotide reductase (RR) is an enzyme that catalyzesthe conversionof ribonucleotide diphosphate todeoxynucleotidediphosphate, which is further phosphorylated into deoxynucleo-tide triphosphate. Deoxynucleotide triphosphate is a direct sub-strate of DNA polymerases and therefore plays a central role inde novoDNA synthesis during cell replication,DNA repair, and cellgrowth (6, 7). The RR enzyme primarily exists as a heterodimerictetramer of large and catalytic subunit RRM1, with small andregulatory subunit RRM2 (6). RRM1 expression is ubiquitous,whereas RRM2 expression is cell-cycle dependent (6).

RR is expressed in different types of cancers and has beenassociated with drug resistance, cancer cell growth, andmetastasis(8). However, other reports show that RRM1 suppresses metas-tasis through induction of PTEN, that RRM1 expression correlateswith ERCC1, and that higher RRM1 expression in non–small celllung carcinoma is associated with better disease-free and overallsurvival (9, 10). In pancreatic cancer, there was no benefit ofgemcitabine therapy after surgery in tumors highly expressingRRM1 group, and higher RRM1 expression was associated withshorter survival (11). In multiple myeloma, a genome-scalesiRNA's lethality study in multiple myeloma identified RRM1(12); however, the biological role of RR in multiple myelomapathogenesis has not yet been further elucidated.

In this study, we characterize the biological significance of RR inmultiplemyelomapathogenesis.We show that knockdownofRR,especially RRM1, leads to apoptotic cell death in multiple mye-loma both in vitro and in vivo, even in the presence of BMmicroenvironment, associatedwith upregulation ofDNAdamage

1Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology,Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts.2Department of Hematology, Saitama Medical Center, Saitama Medical Univer-sity, Kawagoe, Saitama, Japan. 3Department of Biostatistics and ComputationalBiology, Dana-Farber Cancer Institute and Harvard School of Public Health,Boston, Massachusetts. 4West Roxbury Division, VA Boston Healthcare System,West Roxbury, Massachusetts.

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

Corresponding Author: Kenneth C. Anderson, Dana-Farber Cancer Institute,450 Brookline Avenue, Boston, MA 02215. Phone: 617-632-2144; Fax: 617-632-2140; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-0263

�2017 American Association for Cancer Research.

ClinicalCancerResearch

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response and p53 pathway. Nonspecific RRM1 inhibitor clofar-abine also triggers apoptotic multiple myeloma cell death, upre-gulates DNA damage response and p53 pathway, and triggerssynergistic multiple myeloma cytotoxicity when combined withmelphalan (MEL). Our data therefore provide the rationale for anovel treatment strategy inhibiting RRM1 to improve patientoutcome in multiple myeloma.

Materials and MethodsCell culture

Human multiple myeloma cell lines NCI-H929, MM.1S,RPMI8226, and U266 were purchased from the American TypeCulture Collection (ATCC). KMS-11 cells were obtained fromJapanese Collection of Research Bioresources Cell Bank. Cell lineshave been tested and authenticated by STR DNA fingerprintinganalysis (Molecular Diagnostic Laboratory, Dana-Farber CancerInstitute) and used within 3 months after thawing. MOLP-8 cellswere recently obtained from Deutsche Sammlung von Mikroor-ganismen und Zellkulturen GmbH (German Collection ofMicroorganisms and Cell Cultures). OPM2 was provided fromDr. Edward Thompson (University of Texas Medical Branch,Galveston, TX). All multiple myeloma cell lines were culturedin RPMI1640 medium supplemented with 10% (v/v) heat-inactivated FBS, 100 U/mL penicillin, 100 mg/mL streptomycin,and 2 mmol/L L-glutamine (Life Technologies). 293T cell lineswere obtained from the ATCC and maintained in DMEMsupplemented with 10% (v/v) FBS, 100 U/mL penicillin, and100 mg/mL streptomycin. BM samples were obtained frommultiple myeloma patients after informed consent and approv-al by the Institutional Review Board of the Dana-Farber CancerInstitute. Mononuclear cells were separated by Ficoll-PaquePLUS (GE Healthcare Life Sciences), and multiple myelomacells were purified by CD138-positive selection with anti-CD138 magnetic-activated cell separation microbeads (Milte-nyi Biotec). Long-term BM stromal cells (BMSC) were estab-lished by culturing CD138-negative BM mononuclear cells for4 to 6 weeks in DMEM containing 15% (v/v) FBS, 100 U/mLpenicillin, and 100 mg/mL streptomycin. Cell lines were testedto rule out mycoplasma contamination using the MycoAlertMycoplasma Detection Kit (Lonza).

ReagentsClofarabine was purchased from Selleck Chemicals. MEL was

purchased from Sigma-Aldrich. Primary antibodies for theimmunoblot were as follows: anti-RRM1, -RRM2 (Abcam);anti-GAPDH, –caspase-8, –caspase-9, –caspase-3, –phosphory-lated (p)-p53, -p21, -PUMA, –g-H2A.X, –p-ATM, -ATM, –p-ATR,-ATR, –p-Chk1, -Chk1, –p-Chk2, -Chk2, -RAD51, -53BP1,-BRCA1 (Cell Signaling Technology); anti-p53 (DO-1; SantaCruz Biotechnology); anti-Noxa (Millipore/Merck); and anti-BRCA2 (Bethyl Laboratories).

Gene expression analysis using publicly available data setsGene Expression Omnibus data sets (GSE6477, GSE5900,

GSE13591, GSE 39754, GSE2658, and GSE36133) were used forgene expression analyses (13–18). Both 201476_s_at and201477_s_at are the probes for RRM1, and 201890_at is theprobe for RRM2 transcript on Affymetrix Human GenomeU133A Array or Human Genome U133 Plus 2.0 Array.

siRNA transfectionNCI-H929, MM.1S, RPMI8226, and KMS-11 cells were

transiently transfected with scramble or targeted siRNA (GEHealthcare Dharmacon) against RRM1, RRM2, and p53.siRNA transfection was performed by electroporation usingNucleofector Kit V (Lonza), according to the manufacturer'sinstructions.

Expression plasmidThe human RRM2 cDNA was amplified using PCR and ligated

into theHpaI andXhoI sites of pMSCV retroviral expression vector(Clontech).

Viral production and infectionOn day 0, 293T packaging cells were plated at a density of

6 � 105 cells per 6-well plates. On day 1, cells were transfectedwith 500 ng of pMSCVpuro plasmid, 500 ng of pMD-MLV, and100 ng of VSV-G, using TransIT-LT1 Transfection Reagent(Mirus Bio), according to the manufacturer's instructions. Onday 2, media were replaced and cells were cultured for anadditional 24 hours to obtain viral supernatants. On day3, media containing virus were harvested, passed through0.45-mm cellulose acetate membrane filters, and used fresh forinfection. Overall, 2 � 106 cells per 1 mL of crude viral super-natants in the presence of 8 mg/mL polybrene (Sigma-Aldrich)were spinoculated at 800 � g for 30 minutes at room temper-ature, and then incubated in 5% CO2 at 37�C for 5 hours.Media were then replaced. After 24 hours of viral infection, cellsexpressing cDNA were selected with puromycin dihydrochlor-ide (Sigma-Aldrich) at 1 mg/mL for 2 days, and clones expres-sing cDNAs were subjected to rescue experiments. Puromycinconcentrations were titrated to identify the minimum concen-tration of each drug that caused complete cell death of unin-fected cells after 2 days.

Growth inhibition assayThe growth-inhibitory effect was assessed by measuring 3-

(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-mide (MTT, Sigma-Aldrich) dye absorbance, as previouslydescribed (19). The synergistic effect was assessed by combi-nation index using the CompuSyn software program (Combo-Syn Inc.).

Translational Relevance

Ribonucleotide reductase, an enzyme required for DNAsynthesis and repair, is overexpressed in many cancers andassociated with poor prognosis. Here, we investigate thebiological significance of ribonucleotide reductase subunitM1 (RRM1) in multiple myeloma cells. We demonstrate thatRRM1 knockdown and an RRM1 inhibitor clofarabine, aloneand especially when combined with melphalan, trigger sig-nificant multiple myeloma cell growth inhibition both in vitroand in vivo in a mouse human multiple myeloma xenograftmodel. Importantly, activation of bothDNAdamage responseand p53 pathways mediates combination treatment-inducedanti–multiple myeloma activity. Our findings provide therationale for clinical investigation of RRM1 inhibitor in com-bination with DNA-damaging agents as a novel treatmentstrategy in multiple myeloma.

Sagawa et al.

Clin Cancer Res; 23(17) September 1, 2017 Clinical Cancer Research5226




Immunoblot analysisCells were treated, harvested, washed with PBS, and lysed in

RIPA buffer (Boston BioProducts) containing protease inhibi-tor cocktail (Roche). Protein concentration was measured withBio-Rad Protein Assay (Bio-Rad Laboratories). Whole-celllysates were subjected to SDS-PAGE, transferred to nitrocellu-lose membrane (Bio-Rad Laboratories), immunoblotted withantibodies described above, and visualized using ECL Western

Blotting Detection Reagents (GE Healthcare Life Sciences), aspreviously described (20).

Annexin V/propidium iodide stainingApoptotic cell death was assessed by the FITC Annexin-V

Apoptosis Detection Kit (BD Biosciences), according to the man-ufacturer's instructions. Cells stained with Annexin V and propi-dium iodide were analyzed with BD FACS Canto II (BD

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Figure 1.

RRM1 and RRM2 expression inmultiplemyeloma (MM) cells. A–C, RRM1 (top)and RRM2 (bottom)mRNA expressionin multiple myeloma patient samples.Three independent data sets (A,GSE6477; B, GSE5900; and C,GSE13591) were analyzed for RRM1and RRM2 expression in normaldonors, MGUS, smoldering multiplemyeloma, newly diagnosed multiplemyeloma, relapsed multiple myeloma,and plasma cell leukemia (PCL). � , P <0.05; ��,P <0.01; �� ,P<0.001; NS, notsignificant; ANOVA followed by theDunnett test. D, Survival analysis innewly diagnosed multiple myelomapatients related to RRM1 and RRM2expression (GSE39754). Red lineindicates upper 1/3 of each geneexpression, whereas blue lineindicates lower 2/3 of each geneexpression. E, Immunoblot analysis ofRRM1 andRRM2 in 6multiplemyelomacell lines, 3 multiple myeloma patientsamples (CD138-positive cells frombone marrow), and 3 normal donorPBMC samples.

Targeting RRM1 as a Novel Treatment for Multiple Myeloma

www.aacrjournals.org Clin Cancer Res; 23(17) September 1, 2017 5227




Biosciences) using the FACS DIVA software (BD Biosciences), aspreviously described (21).

Cell-cycle analysisCellswere harvested andfixedwith 70%ethanol for 20minutes

on ice. After washing with PBS twice, cells were incubated with

5 mg/mL RNase (Roche) in PBS for 20 minutes at room temper-ature, and then resuspended in PBS containing 10 mg/mL propi-dium iodide (Sigma-Aldrich). The stained cells were analyzedwith BD FACS Canto II (BD Biosciences), and the percentage ofcells in G1, S, and G2–Mphases was determined using the ModFitLT software (Verity Software House).

48 4848 7272 72Scramble siRRM2siRRM1

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Figure 2.

In vitro and in vivo effects of RRM1 andRRM2 knockdown in multiplemyeloma cells. A, RRM1- and RRM2-specific siRNAs were used toknockdown respective genes inmultiple myeloma cell lines. Growthinhibition of the cells was measuredby MTT assay. The growth of all4 multiple myeloma cell lines wassignificantly reduced at 72 and/or 96hours, especially in siRRM1 cells. Bluebar: 48 hours; orange bar: 72 hours;and gray bar: 96 hours. ��, P < 0.01compared with scramble (control) atthe same time period; Student t test.Immunoblots confirmed RRM1 andRRM2 knockdown. Whole-cell lysateswere subjected to immunoblotanalysis, and GAPDH served as theloading control for each membrane.B, RRM1 and RRM2 were knockeddown inNCI-H929 andRPMI8226 cellswith RRM1- and RRM2-specific siRNA,and the number of apoptotic cellswas examined at 72 hours. Althoughsignificant apoptosiswas triggered bysiRNA knockdown in NCI-H929 cells,only mild apoptosis was observed inRPMI8226 cells. �� , P < 0.01 comparedwith scramble; Student t test.C, Immunoblot analysis of apoptosis-related proteins in RRM1- and RRM2-knockdown NCI-H929 and RPMI8226cells. Whole-cell lysates weresubjected to immunoblot analysis,and GAPDH served as the loadingcontrol for each membrane. D, RRM1and RRM2 were knocked down inNCI-H929 cells with RRM1- andRRM2-specific siRNA, and the cell-cycleanalysis was performed at 48 hours.Increase in the number of cells in S-phase was seen in siRNA knockdowncells. (Continued on the followingpage.)

Sagawa et al.





ELISATo isolate nuclear and cytoplasmic proteins, cells were

treated, harvested, washed with PBS, and lysed in the NuclearExtract Kit (Active Motif), according to the manufacturer'sinstructions. DNA-binding activity of p53 was quantifiedby ELISA using the Trans-AM p53 Transcription FactorAssay Kit (Active Motif), according to the manufacturer'sinstructions.

RNA extraction and quantitative real-time PCRTotal RNA was extracted using the RNeasy Mini Kit (Qiagen).

cDNA was synthesized from 1 mg of total RNA with oligo(dT)primers using the SuperScript III First-Strand Synthesis System(Thermo Fisher Scientific). Real-time PCR was performed in 96-well plates using the Applied Biosystems 7300 Real-Time PCRSystem (Thermo Fisher Scientific). The PCR mixture contained10 ng of reverse-transcribed RNA, 100 nmol/L of forward and

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Figure 2.

(Continued. ) E, NCI-H929 cells wereinduced with either control or pMSCV-RRM2 plasmid, and then knocked downwith scramble or RRM1-targeted siRNA.Growth inhibition of the cells wasmeasured by MTT assay. �� , P < 0.01compared with scramble (control) atthe same time period; Student t test.Immunoblots confirmed RRM1knockdown and RRM2 overexpression.GAPDH served as the loading control.F, RRM1 and RRM2 were knocked downin NCI-H929 and RPMI8226 cells withRRM1- and RRM2-specific siRNA, andcocultured in the presence or absenceof patients' BMSC for 72 hours. Dataindicate that the bone marrowmicroenvironment could not abrogatethe knockdown effect of RRM1 andRRM2. �� , P < 0.01 compared withscramble; Student t test. G, Multiplemyeloma cells transduced with siRRM1or scramble (3 � 106 viable cells) weresubcutaneously injected into 200-cGy–irradiated SCID mice. Data representmean� SEM. N¼ 5 mice per group. Animage of tumors in each group is shown(top). �, P¼ 0.0159; Student t test. Dataare representative of at least twoindependent experiments except forxenograft experiment.






reverse primers, and SYBR Green PCRMaster Mix (Thermo FisherScientific), in a final volume of 20 mL. The conditions were 95�Cfor 10minutes, followed by 40 cycles of 15 seconds at 95�C and 1minute at 60�C. The relative amount of each transcript wascalculated using the relative standard curve method. GAPDHmRNA was used as the invariant control, and values were nor-malized by GAPDH expression. Specific primers for each genetranscript are shown in Supplementary Table 1.

Affymetrix gene expression analysisTotal RNAs for microarray analysis were extracted from NCI-

H929 cells transfected with siRNA targeting RRM1, RRM2, orscramble siRNA in biological duplicate using the RNeasyMini Kit(Qiagen). Total RNA (1 mg) was processed, and labeled cRNAwashybridized to Human GenomeU133 plus 2.0 arrays (Affymetrix)according to the standard Affymetrix protocols, as previouslydescribed (22). Expression data can be found at http://www.ncbi.nlm.nih.gov/geo/ under accession number GSE93425.

Subcutaneous xenograft modelFive-week-old male CB17 SCID mice (Charles River Laborato-

ries, Inc.) were used for this study.Note that 3� 106 viableMM.1Scells transducedwith the corresponding siRNA (siRRM1or scram-ble) were suspended in 100 mL of PBS, and then inoculatedsubcutaneously into the left flank of 200-cGy–irradiated mice.Tumor growth was monitored twice a week using an electroniccaliper, and the tumor volume was calculated using the formula:(length � width2) � 2�1, where length is greater than width.Animal studies were performed under a protocol approved by theDana-Farber Institutional Animal Care and Use Committee andfollowed the ARRIVE guidelines (23).

Statistical analysisThe Student t test or ANOVA followed by the Dunnett test was

used to compare differences between the treated group andrelevant control group. Correlation of RRM1 and RRM2 expres-sion with overall survival was measured using the Kaplan–Meiermethod, with Cox proportional hazard regression analysis forgroup comparison. A value of P < 0.05 was considered significant.

ResultsRRM1 and RRM2 are highly expressed in multiple myelomacells

We first investigated the expression of RRM1 and RRM2 inprimary multiple myeloma cells. Our evaluation of RRM1 andRRM2 messenger RNA (mRNA) expression in three indepen-dent publicly available data sets (13–15) revealed that RRM1transcript levels are significantly higher in multiple myelomathan in healthy donor in all data sets, and in monoclonalgammopathy of undetermined significance (MGUS) in two ofthree data sets (Fig. 1A–C, top); and that RRM2 transcript levelsare also significantly higher in two of three data sets (Fig. 1A–C,bottom). These results are consistent with previous studies inother cancers (Supplementary Fig. S1). We also evaluatedanother two publicly available data sets of 170 (16) and 350(17) newly diagnosed patients and found that patients withhigher expressions of RRM1 and RRM2 had significantly shorteroverall survival (Fig. 1D and Supplementary Fig. S2). We alsoexamined RRM1 and RRM2 protein expression in multiplemyeloma cells. We found that both RRM1 and RRM2 were

detected in six human multiple myeloma cell lines and threepatient multiple myeloma cells (Fig. 1E).

RRM1 is required for multiple myeloma cell survivalTo evaluate the biological function of RRM1 and RRM2, we

transduced multiple myeloma cells with siRNA targeting RRM1,RRM2, or control (scramble) by electroporation. Transduction ofRRM1- and RRM2-specific siRNA markedly reduced the respectiveprotein expression in 4 cell lines (p53 wild-type; NCI-H929 andMM.1S, p53 mutant; RPMI8226, p53 null; KMS11) examined(Fig. 2A). Importantly, knockdownof RRM1 or RRM2 significantlyinhibited multiple myeloma cell line growth (Fig. 2A). Of note,RRM2 knockdown did not enhance cell growth inhibition inducedbyRRM1knockdown.Alongwith cell growth inhibition, apoptoticcell death was significantly increased by RRM1 or RRM2 knock-down in NCI-H929 multiple myeloma cells (Fig. 2B). Apoptoticcell death was further confirmed by immunoblots showing clea-vages of caspase-3, -8 and -9, and PARP in NCI-H929 cells (Fig.2C). Consistent with Annexin V–PI staining, apoptotic cell deathtriggered by RRM1or RRM2 knockdownwasmodest in RPMI8226cells (Fig. 2C). We also performed cell-cycle analysis and foundthat cells in S-phase were increased when RRM1 and RRM2 wereknocked down. As previously reported (24), this result suggestsRRM1- and RRM2 knockdown triggered S-phase arrest (Fig. 2D).

As seen in Fig. 2A, RRM1 knockdown induced upregulation ofRRM2, whereas RRM2 knockdown did not induce upregulation ofRRM1. These results suggested that, although precise molecularmechanism has not yet been elucidated, RRM2 could compen-sate RRM1 knockdown effect, although growth-inhibitory assayshowed RRM2 upregulation could not compensate the RRM1-knockdown effect. Therefore, we further induced RRM2 expres-sion to NCI-H929 and RPMI8226 cells by using retroviral expres-sion vector, and then performed RRM1 knockdown. As shownin Fig. 2E and Supplementary Fig. S3, RRM2 overexpressioncould not rescue the growth-inhibitory effect of RRM1 knock-down, suggesting that RRM1, but not RRM2, is a survival factorand potential therapeutic target in multiple myeloma.

The BM microenvironment plays a crucial role in multiplemyeloma pathogenesis by promoting tumor cell proliferation,survival, and drug resistance (1). To examine whether the BMmicroenvironment protects against the effects of RRM1 or RRM2knockdown, we next cocultured siRNA-transfected NCI-H929and RPMI8226 cells in the presence or absence of BMSC. Weobserved that the effects of knockdown of both RRM1 and RRM2were not attenuated even in the presence of BMSC (Fig. 2F). Thesedata suggest that the BM microenvironment cannot overcomeRRM1- or RRM2-knockdown–mediated multiple myeloma cellgrowth inhibition.

To demonstrate the in vivo efficacy of RRM1 downregulation,RRM1-knockdown MM.1S cells were implanted in mice. Asshown in Fig. 2G, cell growth was significantly reduced inRRM1-knockdown cells compared with control cells.

DNA damage response and p53 pathways are required forRRM1-knockdown–induced multiple myeloma cell death

RR is involved in rate-limiting deoxynucleotide (dNTP) gen-eration and functions to maintain centrosome integrity, as wellas provide dNTPs during replication or DNA damage repair(24, 25). Therefore, RRM1 knockdown may affect DNA damageresponse and/or repair genes. Indeed, immunoblots showed thatRRM1 knockdown triggered DNA damage response in multiple

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http://www.ncbi.nlm.nih.gov/geo/

http://www.ncbi.nlm.nih.gov/geo/


myeloma cells, including g-H2A.X, phosphorylated (p)-ATM, andp-ATR, as well as their downstream effectors p-Chk1 and p-Chk2(Fig. 3A). We next examined downstream target genes RAD51,53BP1, BRCA1, andBRCA2. As shown in Fig. 3B, quantitative real-time PCR (qRT-PCR) analysis showed that RRM1 knockdowninduced these genes in both NCI-H929 and RPMI8226 cells.

Consistent with qRT-PCR, immunoblots showed that RRM1knockdown also induced increased RAD51, 53BP1, BRCA1, andBRCA2 protein levels (Fig. 3C).

To identify novel downstream targets of RRM1 (and RRM2)which mediate multiple myeloma cell growth, we next performedgene expression profiling after RRM1 or RRM2 knockdown in

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DNA damage response pathway plays essential role in RRM1-knockdown multiple myeloma cells. A, Immunoblot analysis of DNA damage response pathwaygenes in RRM1- and RRM2-knockdownNCI-H929 and RPMI8226 cells. GAPDH served as the loading control for eachmembrane.B, qRT-PCR analysis of RRM1, RRM2,RAD51, 53BP1, BRCA1, and BRCA2 in NCI-H929 and RPMI8226 cells transduced with siRNA targeting RRM1, RRM2, or scramble (control). Shown arerelative signal intensity (scramble¼ 1) normalized by GAPDH. �� , P < 0.01 compared with scramble; Student t test. C, Immunoblot analysis of RAD51, 53BP1, BRCA1,and BRCA2 in NCI-H929 and RPMI8226 cells transduced with siRNA targeting RRM1, RRM2, or scramble. GAPDH served as the loading control for eachmembrane, and data are representative of at least two independent experiments.






NCI-H929 cells. RRM1-knockdownupregulated 665 genes, includ-ing p53 pathway genes CDKN1A (p21WAF1), PMAIP1 (Noxa),BBC3 (Puma), SESN1, DDB2, and DRAM1 as long as BRCA1(Fig. 4A and B). Of note, multiple myeloma cells with wild-type

p53 showed more significant growth inhibition by RRM1 knock-down than in cells with mutant p53 (Fig. 2A).

We next used ELISA and immunoblots to examine acti-vation of p53 pathway by RRM1 or RRM2 knockdown in

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Transcriptional activity of TP53 pathway is crucial in TP53wild-typemultiplemyeloma cells.A, Scatter plots depictingthe relative gene expression in NCI-H929 cells treated withsiRRM1, siRRM2, or scramble. Genes related to TP53 andBRCA1 (plotted in red) were eluted together with >1.5 foldchange. B, Heatmap showed induction of TP53-relatedgenes in RRM1- and RRM2-knockdown cells compared withscramble. Yellow denoted higher expression, whereas bluedenoted lower expression.C, Transcription activity levels ofTP53 in TP53 wild-type NCI-H929 cells. Fold changesrelative to scramble are shown. D, Immunoblot analysis ofTP53 and its related proteins in whole-cell lysates fromRRM1- and RRM2-knockdown NCI-H929 cells. E, NCI-H929cells were treated with siRRM1, sip53, or both; left plotshows survival of cells 72 hours after knockdown. Right plotshows confirmation of knockdown, and GAPDH served asthe loading control for each membrane. Data arerepresentative of at least two independent experiments inC–E. �� , P < 0.01; Student t test.

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NCI-H929 cells. ELISA showed that p53 was activated byboth RRM1 and RRM2 knockdown (Fig. 4C). Immunoblotalso showed that p53 pathway is activated, evidenced byinduction of p53 phosphorylation at Ser15, as well as upre-gulation of p21WAF1, Noxa, and PUMA (Fig. 4D). Importantly,p53 knockdown partially abrogated the effect of RRM1 knock-down (Fig. 4E), further validating p53 as a key molecule inRRM1-knockdown–induced multiple myeloma cell growthinhibition.

Therefore, we speculated that in p53 wild-type cells, RRM1-knockdown effect derived upon DNA damage response followedby p53 pathway, whereas in p53-mutant/null cells, alternativepathway, such as BRCA1/2 pathway, might be critical.

RRM1 inhibitor triggers growth inhibition in p53 wild-typemultiple myeloma cells

To assess the potential clinical relevance of RRM1 inhibitionin multiple myeloma, we next examined the effect of thepurine nucleoside antimetabolite clofarabine, an RRM1 inhib-itor that is approved for the treatment of acute lymphocyticand myeloid leukemia (26–30), on multiple myeloma celllines (NCI-H929, MM.1S, MOLP-8, RPMI8226, OPM2, U266,and KMS-11). TP53 wild-type cells (NCI-H929, MM.1S,and MOLP-8) were more sensitive to clofarabine treatmentcompared with TP53-mutant (RPMI8226, OPM2, and U266)or TP53-null (KMS-11) cells (Fig. 5A). To elucidate the molec-ular mechanism of multiple myeloma cell death triggered

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RRM1 inhibitor induces apoptosis inmultiple myeloma cells. A, Sevenmultiplemyeloma cell lines (NCI-H929,MM.1S, MOLP8, RPMI8226, OPM2,U266, and KMS-11 cells) were treatedwith clofarabine (CLO; 0–30 mmol/L)for 48 hours, and growth was thenmeasured by MTT assay. B–D,Immunoblot analysis of cell lysates ofNCI-H929 cells treated withclofarabine (5 mmol/L, 3–48 hours).GAPDH served as the loading controlfor each membrane, and data arerepresentative of at least twoindependent experiments.






by clofarabine, we carried out immunoblots and observedtime-dependent cleavage of caspase-3, -8, -9 and PARP (Fig.5B). Similar to RRM1 knockdown, clofarabine treatment upre-gulated p53 and its downstream target proteins in NCI-H929cells, without significant alteration of RRM1 or RRM2 proteinexpression (Fig. 5C). DNA damage response pathway proteins,including g-H2A.X, p-ATM, and effectors p-Chk1 and p-Chk2,were also upregulated by clofarabine treatment in a time-dependent fashion (Fig. 5D).

RRM1 inhibitor with MEL induces synergistic multiplemyeloma cytotoxicity

Because clofarabine enhancedDNAdamage response pathway,we next combined clofarabine with DNA-damaging agent MEL toassess for enhanced anti–multiple myeloma activity. Clofarabinein combination with MEL triggered synergistic cytotoxicity notonly in NCI-H929 and but also in RPMI8226 cells (Fig. 6A).Consistent with cytotoxicity, clofarabine withMEL also markedlyupregulated Annexin V–positive cells and cleavage of caspase-3,

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RRM1 inhibition combined withDNA-damaging agent clofarabine hassynergistic effect on multiplemyeloma cells. A, (left) NCI-H929 andRPMI8226 cells were treated with thecombination of CLO and MEL for48 hours at the indicated doses, andtumor growth reduction wasmeasured by MTT assay. Right,Combination index (CI)was calculatedin each combination therapy. CI under1 is recognized as synergy. B, NCI-H929 andRPMI8226 cellswere treatedwith CLO (NCI-H929, 3 mmol/L;RPMI8226, 10 mmol/L) and MEL(20 mmol/L) for 48 hours, and thenumber of apoptotic cells wasexamined. Combination treatmentindicates higher percentage ofapoptotic cells. C and D, Immunoblotanalysis of cell lysates aftercombination treatment with CLO(NCI-H929, 3 mmol/L; RPMI8226, 10mmol/L) and MEL (20 mmol/L) for 48hours. (Continued on the followingpage.)

Sagawa et al.





-8, -9, and PARP in both cells (Fig. 6B and C), suggesting that theenhanced combination treatment-induced cytotoxicity was dueto apoptotic cell death. Furthermore, g-H2A.X, biomarker ofDNAdouble-strand break and DNA damage (31), was activated uponcombination treatment (Fig. 6D). Because clofarabine may haveoff-target effects, we carried out combination treatment of MELwith RRM1 knockdown and confirmed that MEL enhancedRRM1-knockdown–induced cytotoxicity (Fig. 6E), associatedwith enhanced activation of DNA damage response pathway (Fig.6F). These data indicate that RRM1 inhibition by either knock-down or clofarabine in combination withMEL triggers synergisticmultiple myeloma cytotoxicity.

DiscussionAs in many other cancers, RR is highly expressed in multiple

myeloma cells. More specifically, we here show that both RRM1(large subunit) and RRM2 (small subunit) are highly expressed inmultiple myeloma cells, but not in normal cells. Importantly, wedemonstrate that RRM1 knockdown triggers significant multiplemyeloma cell growth inhibition and apoptosis, whereas RRM2knockdown shows modest growth-inhibitory effects. These datasuggest that RRM1, but not RRM2, is a survival factor andpotential therapeutic target in multiple myeloma.

Maintenance of genomic stability depends on an appropriateresponse to DNA damage, and the protein kinases ATM and ATRare the master controllers of such DNA damage pathwayresponses (32, 33). We have previously reported that pervasiveconstitutive and ongoing DNA damage is present in hematologicmalignancies including multiple myeloma (34), and others havereported that RRM1 maintains centrosomal integrity during rep-lication stress (24). Importantly, in this study, our gene expressiondata and qRT-PCR results showed that RRM1 knockdown upre-gulated DNA damage response genes including RAD51 and53BP1. Therefore, downregulation of RRM1 could inhibit theability of multiple myeloma cells to survive in ongoing DNAdamage, leading to apoptotic cell death. We have previouslyreported that YAP1 knockdown can trigger p73-mediated apo-ptosis in a subset of multiple myeloma with ongoing DNAdamage; however, RRM1 knockdown did not alter YAP1 (Sup-

plementary Fig. S4), indicating an alternative mechanism ofaction triggered by RRM1 inhibition.

Interestingly, we showed that BRCA1 and BRCA2 were alsoupregulated in multiple myeloma cells by RRM1 knockdownirrespective of p53 status. Harkins and colleagues reported thatinducible expression of BRCA1 leads to apoptotic cell death inosteosarcoma and breast cancer cells (35). Conversely, Rao andcolleagues reported that selective reduction of BRCA1 mRNAlevels using antisense RNA induces more rapid cell growth,decreased susceptibility to apoptosis, and cell transformation inNIH3T3 fibroblasts (36). Taken together, our results suggest thatupregulation of BRCA1 mRNA and protein level may account, atleast in part, for RRM1-knockdown/inhibition–induced apopto-ticmultiplemyeloma cell death, putative alternativemechanisms.

To assess clinical relevance of RRM1 inhibition in multiplemyeloma, we showed that the purine analog clofarabine, knownto inhibit RRM1 (26, 27), also induces multiple myeloma cellgrowth inhibition. Similar to RRM1 knockdown, clofarabinetreatment also induced DNA damage response proteins, g-H2A.X, phosphorylated (p)-ATM, and p-ATR, followed by its down-stream effectors, p-Chk1 and p-Chk2. Interestingly, clofarabine-induced apoptosis is more potent inmultiple myeloma cells withwild-type TP53 comparedwith cellswithmutant-p53or null-p53.We also showed that RRM1-induced apoptoticmultiplemyelomacell death was more evident in p53 wild-type cells than p53-mutant cells. Similar results were reported by Valdez and collea-gues (37). Upon DNA damage, p53 is stabilized, upregulated,and phosphorylated at Ser15, cell-cycle arrest, leading to itsantiproliferative activity, and apoptosis (38). Our results furtherdemonstrated that both RRM1 knockdown and clofarabine treat-ment in NCI-H929 cells with p53 wild-type upregulate/activatep53 pathway proteins including activation of p-p53 (Ser 15),stabilization of p53, and upregulation of p21, Noxa, and Puma.These results suggest that p53 pathways play a critical role medi-ating RRM1-induced multiple myeloma cell death. The preva-lence of p53 mutation in newly diagnosed multiple myeloma isquite low (ranging from 0%–20%) and is an independent poorprognostic factor (39), whereas higher percentage of patients withp53 abnormalities (p53mutation and p53 deletion) are noted inmore advanced disease including relapsed refractory multiple

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(Continued. ) E, NCI-H929 andRPMI8226 cells were treated withcombination of siRNA treatment(siRRM1 or scramble) and MEL for 72hours at the indicated doses, andtumor growth was measured by MTTassay. �� , P < 0.01. NS, not significant;Student t test. F, Immunoblot analysisof cell lysates after combination siRNAtreatment (siRRM1 or scramble) andMEL at the indicated doses and time(same condition as E). GAPDH servedas the loading control for eachmembrane, and data arerepresentative of at least twoindependent experiments.






myeloma (RRMM) and plasma cell leukemia (40). Therefore,RRM1 knockdown/clofarabine treatment, as a single therapeuticstrategy, might be difficult to utilize in RRMM patients, andcombination treatment strategy is warranted.

Finally, MEL is a member of the nitrogen mustard class ofchemotherapeutic agents which alkylates DNA. It triggers forma-tion of DNA adducts and forms crosslinks. The formation ofcrosslinks between the two strands of DNA, interstrand cross-linking, is a critical event that correlates with in vitro cytotoxicity(41). A previous in vitro report has combined clofarabine withMEL and described synergistic effects (37), without elucidating itsmechanism. Importantly, we here found that the synergisticeffects triggered by combining clofarabine with MEL are evidentnot only in wild-type p53 cells, but also in mutant p53 cells, and,importantly, are associated with induction of g-H2A.X. Further-more, we found that BRCA1 and BRCA2 were upregulated uponRRM1 knockdown in p53 wild-type cells as well as p53-mutant(and null) cells. These results suggest that MEL can enhance anti–multiplemyeloma activity of RRM1 inhibition–inducedmultiplemyeloma cytotoxicity regardless of p53 status, and BRCA1/2pathway could be the possible alternative pathway for theenhancement of this combination treatment. Because clofarabineis being used as a tool compound in preclinical setting because ofits unfavorable toxicities, combination treatment of clofarabinewith MEL may not be suitable for clinical settings. Therefore,development of novel RRM1 inhibitor with less myelotoxicity isneeded.

In conclusion, we have here elucidated a novel role of RRM1 inmultiple myeloma regulating DNA damage response and p53

pathway. Our studies provide the preclinical rationale for target-ingRRM1 to enhance sensitivity of tumor cells toMELand therebyimprove patient outcome in multiple myeloma.

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

Authors' ContributionsConception and design: M. Sagawa, T. Hideshima, K.C. AndersonDevelopment of methodology: M. Sagawa, H. Ohguchi, K.C. AndersonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T. Harada, Y.-T. TaiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):M. Sagawa, H. Ohguchi, M.K. Samur, K.C. AndersonWriting, review, and/or revision of the manuscript: M. Sagawa, M.K. Samur,N.C. Munshi, M. Kizaki, T. Hideshima, K.C. AndersonAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):H. Ohguchi, T. Harada, Y.-T. Tai, K.C. AndersonStudy supervision: M. Kizaki, T. Hideshima, K.C. Anderson

Grant SupportThis research was supported by NIH grants SPORE P50-100707

(K.C. Anderson), R01-CA 050947 (K.C. Anderson), and R01-CA178264(T. Hideshima and K.C. Anderson). K.C. Anderson is an American CancerSociety Clinical Research Professor.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 27, 2017; revised March 17, 2017; accepted April 18, 2017;published OnlineFirst April 25, 2017.

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2017;23:5225-5237. Published OnlineFirst April 25, 2017.Clin Cancer Res Morihiko Sagawa, Hiroto Ohguchi, Takeshi Harada, et al. Therapeutic Target in Multiple MyelomaRibonucleotide Reductase Catalytic Subunit M1 (RRM1) as a Novel

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