RRM1) As a Novel Therapeutic Target in Multiple Myeloma Morihiko Sagawa1,2, Hiroto Ohguchi1, Takeshi Harada1, Mehmet K

Published OnlineFirst April 25, 2017; DOI: 10.1158/1078-0432.CCR-17-0263

Biology of Human Tumors Clinical Cancer Research Ribonucleotide Reductase Catalytic Subunit M1 (RRM1) as a Novel Therapeutic Target in Multiple Myeloma Morihiko Sagawa1,2, Hiroto Ohguchi1, Takeshi Harada1, Mehmet K. Samur3, Yu-Tzu Tai1, Nikhil C. Munshi1,4, Masahiro Kizaki2, Teru Hideshima1, and Kenneth C. Anderson1

Abstract

Purpose: To investigate the biological and clinical significance BRCA1, and BRCA2 were upregulated/activated. Moreover, of ribonucleotide reductase (RR) in multiple myeloma. immunoblots showed that p53, p21, Noxa, and Puma were acti- Experimental Design: We assessed the impact of RR expression vated in p53 wild-type multiple myeloma cells. Clofarabine, a on patient outcome in multiple myeloma. We then characterized purine nucleoside analogue that inhibits RRM1, induced growth the effect of genetic and pharmacologic inhibition of ribonucle- arrest and apoptosis in p53 wild-type cell lines. Although clofarabine otide reductase catalytic subunit M1 (RRM1) on multiple mye- did not induce cell death in p53-mutant cells, it did trigger synergistic loma growth and survival using siRNA and clofarabine, respec- toxicity in combination with DNA-damaging agent melphalan. tively, in both in vitro and in vivo mouse xenograft models. Finally, we demonstrated that tumor growth of RRM1-knockdown Results: Newly diagnosed multiple myeloma patients with multiple myeloma cells was significantly reduced in a murine higher RRM1 expression have shortened survival. Knockdown of human multiple myeloma cell xenograft model. RRM1 triggered significant growth inhibition and apoptosis in Conclusions: Our results therefore demonstrate that RRM1 is multiple myeloma cells, even in the context of the bone marrow a novel therapeutic target in multiple myeloma in the preclin- microenvironment. Gene expression profiling showed upregula- ical setting and provide the basis for clinical evaluation of tion of DNA damage response genes and p53-regulated genes after RRM1 inhibitor, alone or in combination with DNA-damaging RRM1 knockdown. Immunoblot and qRT-PCR analysis con- agents, to improve patient outcome in multiple myeloma. firmed that g-H2A.X, ATM, ATR, Chk1, Chk2, RAD51, 53BP1, Clin Cancer Res; 1–13. 2017 AACR.

Introduction Ribonucleotide reductase (RR) is an enzyme that catalyzes the conversion of ribonucleotide diphosphate to deoxynucleotide Multiple myeloma is a plasma cell disorder characterized by diphosphate, which is further phosphorylated into deoxynucleo- excess malignant plasma cells in the bone marrow (BM), tide triphosphate. Deoxynucleotide triphosphate is a direct sub- increased monoclonal gammaglobulin in blood and/or urine, strate of DNA polymerases and therefore plays a central role in and end organ damage in kidney and bone (1). Although protea- de novo DNA synthesis during cell replication, DNA repair, and cell some inhibitors (bortezomib, carfilzomib, and ixazomib), immu- growth (6, 7). The RR enzyme primarily exists as a heterodimeric nomodulatory drugs (lenalidomide and pomalidomide), and tetramer of large and catalytic subunit RRM1, with small and mAbs (daratumumab and elotuzumab; refs. 2, 3) have achieved regulatory subunit RRM2 (6). RRM1 expression is ubiquitous, remarkable clinical responses and improved patient outcome, whereas RRM2 expression is cell-cycle dependent (6). relapse of disease is common, highlighting the need for novel RR is expressed in different types of cancers and has been treatment strategies (4, 5). associated with drug resistance, cancer cell growth, and metastasis (8). However, other reports show that RRM1 suppresses metastasis through induction of PTEN, that RRM1 expression correlates – 1Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, with ERCC1, and that higher RRM1 expression in non small cell Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts. lung carcinoma is associated with better disease-free and overall 2Department of Hematology, Saitama Medical Center, Saitama Medical Univer- survival (9, 10). In pancreatic cancer, there was no benefitof sity, Kawagoe, Saitama, Japan. 3Department of Biostatistics and Computational gemcitabine therapy after surgery in tumors highly expressing Biology, Dana-Farber Cancer Institute and Harvard School of Public Health, RRM1 group, and higher RRM1 expression was associated with 4 Boston, Massachusetts. West Roxbury Division, VA Boston Healthcare System, shorter survival (11). In multiple myeloma, a genome-scale West Roxbury, Massachusetts. siRNA's lethality study in multiple myeloma identified RRM1 Note: Supplementary data for this article are available at Clinical Cancer (12); however, the biological role of RR in multiple myeloma Research Online (http://clincancerres.aacrjournals.org/). pathogenesis has not yet been further elucidated. Corresponding Author: Kenneth C. Anderson, Dana-Farber Cancer Institute, In this study, we characterize the biological significance of RR in 450 Brookline Avenue, Boston, MA 02215. Phone: 617-632-2144; Fax: 617-632- multiple myeloma pathogenesis. We show that knockdown of RR, 2140; E-mail: [email protected] especially RRM1, leads to apoptotic cell death in multiple mye- doi: 10.1158/1078-0432.CCR-17-0263 loma both in vitro and in vivo, even in the presence of BM 2017 American Association for Cancer Research. microenvironment, associated with upregulation of DNA damage

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Reagents Translational Relevance Clofarabine was purchased from Selleck Chemicals. MEL was Ribonucleotide reductase, an enzyme required for DNA purchased from Sigma-Aldrich. Primary antibodies for the synthesis and repair, is overexpressed in many cancers and immunoblot were as follows: anti-RRM1, -RRM2 (Abcam); associated with poor prognosis. Here, we investigate the anti-GAPDH, –caspase-8, –caspase-9, –caspase-3, –phosphory- biological significance of ribonucleotide reductase subunit lated (p)-p53, -p21, -PUMA, –g-H2A.X, –p-ATM, -ATM, –p-ATR, M1 (RRM1) in multiple myeloma cells. We demonstrate that -ATR, –p-Chk1, -Chk1, –p-Chk2, -Chk2, -RAD51, -53BP1, RRM1 knockdown and an RRM1 inhibitor clofarabine, alone -BRCA1 (Cell Signaling Technology); anti-p53 (DO-1; Santa and especially when combined with melphalan, trigger sig- Cruz Biotechnology); anti-Noxa (Millipore/Merck); and anti- nificant multiple myeloma cell growth inhibition both in vitro BRCA2 (Bethyl Laboratories). and in vivo in a mouse human multiple myeloma xenograft model. Importantly, activation of both DNA damage response Gene expression analysis using publicly available data sets and p53 pathways mediates combination treatment-induced Gene Expression Omnibus data sets (GSE6477, GSE5900, anti–multiple myeloma activity. Our findings provide the GSE13591, GSE 39754, GSE2658, and GSE36133) were used for rationale for clinical investigation of RRM1 inhibitor in com- gene expression analyses (13–18). Both 201476_s_at and bination with DNA-damaging agents as a novel treatment 201477_s_at are the probes for RRM1, and 201890_at is the strategy in multiple myeloma. probe for RRM2 transcript on Affymetrix Human Genome U133A Array or Human Genome U133 Plus 2.0 Array.

siRNA transfection response and p53 pathway. Nonspecific RRM1 inhibitor clofar- NCI-H929, MM.1S, RPMI8226, and KMS-11 cells were abine also triggers apoptotic multiple myeloma cell death, upre- transiently transfected with scramble or targeted siRNA (GE gulates DNA damage response and p53 pathway, and triggers Healthcare Dharmacon) against RRM1, RRM2, and p53. synergistic multiple myeloma cytotoxicity when combined with siRNA transfection was performed by electroporation using melphalan (MEL). Our data therefore provide the rationale for a Nucleofector Kit V (Lonza), according to the manufacturer's novel treatment strategy inhibiting RRM1 to improve patient instructions. outcome in multiple myeloma. Expression plasmid The human RRM2 cDNA was amplified using PCR and ligated Materials and Methods into the HpaI and XhoI sites of pMSCV retroviral expression vector Cell culture (Clontech). Human multiple myeloma cell lines NCI-H929, MM.1S, RPMI8226, and U266 were purchased from the American Type Viral production and infection Culture Collection (ATCC). KMS-11 cells were obtained from On day 0, 293T packaging cells were plated at a density of Japanese Collection of Research Bioresources Cell Bank. Cell lines 6 105 cells per 6-well plates. On day 1, cells were transfected have been tested and authenticated by STR DNA fingerprinting with 500 ng of pMSCVpuro plasmid, 500 ng of pMD-MLV, and analysis (Molecular Diagnostic Laboratory, Dana-Farber Cancer 100 ng of VSV-G, using TransIT-LT1 Transfection Reagent Institute) and used within 3 months after thawing. MOLP-8 cells (Mirus Bio), according to the manufacturer's instructions. On were recently obtained from Deutsche Sammlung von Mikroor- day 2, media were replaced and cells were cultured for an ganismen und Zellkulturen GmbH (German Collection of additional 24 hours to obtain viral supernatants. On day Microorganisms and Cell Cultures). OPM2 was provided from 3, media containing virus were harvested, passed through Dr. Edward Thompson (University of Texas Medical Branch, 0.45-mm cellulose acetate membrane filters, and used fresh for Galveston, TX). All multiple myeloma cell lines were cultured infection. Overall, 2 106 cells per 1 mL of crude viral super- in RPMI1640 medium supplemented with 10% (v/v) heat- natants in the presence of 8 mg/mL polybrene (Sigma-Aldrich) inactivated FBS, 100 U/mL penicillin, 100 mg/mL streptomycin, were spinoculated at 800 g for 30 minutes at room temper- and 2 mmol/L L-glutamine (Life Technologies). 293T cell lines ature, and then incubated in 5% CO2 at 37 Cfor5hours. were obtained from the ATCC and maintained in DMEM Media were then replaced. After 24 hours of viral infection, cells supplemented with 10% (v/v) FBS, 100 U/mL penicillin, and expressing cDNA were selected with puromycin dihydrochlor- 100 mg/mL streptomycin. BM samples were obtained from ide (Sigma-Aldrich) at 1 mg/mL for 2 days, and clones expres- multiple myeloma patients after informed consent and approv- sing cDNAs were subjected to rescue experiments. Puromycin al by the Institutional Review Board of the Dana-Farber Cancer concentrations were titrated to identify the minimum concen- Institute. Mononuclear cells were separated by Ficoll-Paque tration of each drug that caused complete cell death of unin- PLUS (GE Healthcare Life Sciences), and multiple myeloma fected cells after 2 days. cells were purified by CD138-positive selection with anti- CD138 magnetic-activated cell separation microbeads (Milte- Growth inhibition assay nyi Biotec). Long-term BM stromal cells (BMSC) were estab- The growth-inhibitory effect was assessed by measuring 3- lished by culturing CD138-negative BM mononuclear cells for (4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro- 4 to 6 weeks in DMEM containing 15% (v/v) FBS, 100 U/mL mide (MTT, Sigma-Aldrich) dye absorbance, as previously penicillin, and 100 mg/mL streptomycin. Cell lines were tested described (19). The synergistic effect was assessed by combi- to rule out mycoplasma contamination using the MycoAlert nation index using the CompuSyn software program (Combo- Mycoplasma Detection Kit (Lonza). Syn Inc.).

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Immunoblot analysis described (22). Expression data can be found at http://www. Cells were treated, harvested, washed with PBS, and lysed in ncbi.nlm.nih.gov/geo/ under accession number GSE93425. RIPA buffer (Boston BioProducts) containing protease inhibitor cocktail (Roche). Protein concentration was measured with Bio- Subcutaneous xenograft model Rad Protein Assay (Bio-Rad Laboratories). Whole-cell lysates were Five-week-old male CB17 SCID mice (Charles River Laborato- subjected to SDS-PAGE, transferred to nitrocellulose membrane ries, Inc.) were used for this study. Note that 3 106 viable MM.1S (Bio-Rad Laboratories), immunoblotted with antibodies cells transduced with the corresponding siRNA (siRRM1 or scram- described above, and visualized using ECL Western Blotting ble) were suspended in 100 mL of PBS, and then inoculated Detection Reagents (GE Healthcare Life Sciences), as previously subcutaneously into the left flank of 200-cGy–irradiated mice. described (20). Tumor growth was monitored twice a week using an electronic caliper, and the tumor volume was calculated using the formula: Annexin V/propidium iodide staining (length width2) 2 1, where length is greater than width. Apoptotic cell death was assessed by the FITC Annexin-V Animal studies were performed under a protocol approved by the Apoptosis Detection Kit (BD Biosciences), according to the man- Dana-Farber Institutional Animal Care and Use Committee and ufacturer's instructions. Cells stained with Annexin V and propi- followed the ARRIVE guidelines (23). dium iodide were analyzed with BD FACS Canto II (BD Bios- ciences) using the FACS DIVA software (BD Biosciences), as Statistical analysis previously described (21). The Student t test or ANOVA followed by the Dunnett test was used to compare differences between the treated group and Cell-cycle analysis relevant control group. Correlation of RRM1 and RRM2 expres- fi Cells were harvested and xed with 70% ethanol for 20 minutes sion with overall survival was measured using the Kaplan–Meier on ice. After washing with PBS twice, cells were incubated with method, with Cox proportional hazard regression analysis for 5 mg/mL RNase (Roche) in PBS for 20 minutes at room temper- group comparison. A value of P < 0.05 was considered significant. ature, and then resuspended in PBS containing 10 mg/mL propidium iodide (Sigma-Aldrich). The stained cells were analyzed with BD FACS Canto II (BD Biosciences), and the percentage of Results cells in G1, S, and G2–M phases was determined using the ModFit RRM1 and RRM2 are highly expressed in multiple myeloma LT software (Verity Software House). cells We first investigated the expression of RRM1 and RRM2 in ELISA primary multiple myeloma cells. Our evaluation of RRM1 and To isolate nuclear and cytoplasmic proteins, cells were treated, RRM2 messenger RNA (mRNA) expression in three independent harvested, washed with PBS, and lysed in the Nuclear Extract Kit publicly available data sets (13–15) revealed that RRM1 transcript (Active Motif), according to the manufacturer's instructions. levels are significantly higher in multiple myeloma than in DNA-binding activity of p53 was quantified by ELISA using the healthy donor in all data sets, and in monoclonal gammopathy Trans-AM p53 Transcription Factor Assay Kit (Active Motif), of undetermined significance (MGUS) in two of three data sets according to the manufacturer's instructions. (Fig. 1A–C, top); and that RRM2 transcript levels are also significantly higher in two of three data sets (Fig. 1A–C, bottom). These RNA extraction and quantitative real-time PCR results are consistent with previous studies in other cancers Total RNA was extracted using the RNeasy Mini Kit (Qiagen). (Supplementary Fig. S1). We also evaluated another two publicly cDNA was synthesized from 1 mg of total RNA with oligo(dT) available data sets of 170 (16) and 350 (17) newly diagnosed primers using the SuperScript III First-Strand Synthesis System patients and found that patients with higher expressions of RRM1 (Thermo Fisher Scientific). Real-time PCR was performed in 96- and RRM2 had significantly shorter overall survival (Fig. 1D and well plates using the Applied Biosystems 7300 Real-Time PCR Supplementary Fig. S2). We also examined RRM1 and RRM2 System (Thermo Fisher Scientific). The PCR mixture contained protein expression in multiple myeloma cells. We found that both 10 ng of reverse-transcribed RNA, 100 nmol/L of forward and RRM1 and RRM2 were detected in six human multiple myeloma reverse primers, and SYBR Green PCR Master Mix (Thermo Fisher cell lines and three patient multiple myeloma cells (Fig. 1E). Scientific), in a final volume of 20 mL. The conditions were 95C for 10 minutes, followed by 40 cycles of 15 seconds at 95 C and 1 RRM1 is required for multiple myeloma cell survival minute at 60 C. The relative amount of each transcript was To evaluate the biological function of RRM1 and RRM2, we calculated using the relative standard curve method. GAPDH transduced multiple myeloma cells with siRNA targeting RRM1, mRNA was used as the invariant control, and values were nor- RRM2, or control (scramble) by electroporation. Transduction of malized by GAPDH expression. Specific primers for each gene RRM1- and RRM2-specific siRNA markedly reduced the respective transcript are shown in Supplementary Table 1. protein expression in 4 cell lines (p53 wild-type; NCI-H929 and MM.1S, p53 mutant; RPMI8226, p53 null; KMS11) examined Affymetrix gene expression analysis (Fig. 2A). Importantly, knockdown of RRM1 or RRM2 signifi- Total RNAs for microarray analysis were extracted from NCI- cantly inhibited multiple myeloma cell line growth (Fig. 2A). Of H929 cells transfected with siRNA targeting RRM1, RRM2, or note, RRM2 knockdown did not enhance cell growth inhibition scramble siRNA in biological duplicate using the RNeasy Mini Kit induced by RRM1 knockdown. Along with cell growth inhibition, (Qiagen). Total RNA (1 mg) was processed, and labeled cRNA was apoptotic cell death was significantly increased by RRM1 or RRM2 hybridized to Human Genome U133 plus 2.0 arrays (Affymetrix) knockdown in NCI-H929 multiple myeloma cells (Fig. 2B). according to the standard Affymetrix protocols, as previously Apoptotic cell death was further confirmed by immunoblots

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*** AC*** B ** ** *** NS ) ) NS * *** )

2 NS 2

2 10 *** 11 * 13 ** NS NS 10 9 12 9 8 11 8 7 10 7 6 RRM1 Expression (Log Expression RRM1 RRM1 Expression (Log 6 RRM1 Expression (Log 9 5 r r

MM PCL MGUS MGUS MGUS New MM rmal dono Normal donor Relapsed MM Normal dono No Smoldering MM Smoldering MM

*** ** Figure 1. ** NS

NS ) RRM1 and RRM2 expression in multiple 2 NS ) NS ) NS 2 14 *** 2 14 *** 14 myeloma (MM) cells. A–C, RRM1 (top) ** NS NS ** NS 12 and RRM2 (bottom) mRNA expression 12 12 in multiple myeloma patient samples. 10 Three independent data sets (A, 10 10 8 GSE6477; B, GSE5900; and C, GSE13591) were analyzed for RRM1 8 6 8 and RRM2 expression in normal RRM2 Expression (Log Expression RRM2 RRM2 Expression (Log Expression RRM2 RRM2 Expression (Log Expression RRM2 6 4 donors, MGUS, smoldering multiple myeloma, newly diagnosed multiple MM PCL MGUS MGUS MGUS myeloma, relapsed multiple myeloma, New MM Normal donor and plasma cell leukemia (PCL). , P < Normal donor Relapsed MM Normal donor Smoldering MM Smoldering MM 0.05; , P < 0.01; , P < 0.001; NS, not D signiﬁcant; ANOVA followed by the RRM1 (OS HR = 1.65 Cox P = 0.0042) RRM2 (OS HR = 1.41 Cox P = 0.0015) Dunnett test. D, Survival analysis in newly diagnosed multiple myeloma patients related to RRM1 and RRM2 expression (GSE39754). Red line indicates upper 1/3 of each gene expression, whereas blue line indicates lower 2/3 of each gene expression. E, Immunoblot analysis of Survival

Survival RRM1 and RRM2 in 6 multiple myeloma cell lines, 3 multiple myeloma patient samples (CD138-positive cells from bone marrow), and 3 normal donor

High 1/3 High 1/3 PBMC samples. Low 2/3 Low 2/3 P = 0.039 P

0.0 0.2 0.4 0.6 0.8 1.0 = 0.021 0.0 0.2 0.4 0.6 0.8 1.0 0 20406080 0 20406080

Months Months E

NCI-H929MM.1S RPMI8226U266 OPM2 KMS-11 Pt #1 Pt #2 Pt #3 MM.1S PBMC #1PBMC #2PBMC #3MM.1S RRM1

RRM2

GAPDH

showing cleavages of caspase-3, -8 and -9, and PARP in NCI-H929 As seen in Fig. 2A, RRM1 knockdown induced upregulation cells (Fig. 2C). Consistent with Annexin V–PI staining, apoptotic of RRM2, whereas RRM2 knockdown did not induce upregula- cell death triggered by RRM1 or RRM2 knockdown was modest in tion of RRM1. These results suggested that, although precise RPMI8226 cells (Fig. 2C). We also performed cell-cycle analysis molecular mechanism has not yet been elucidated, RRM2 could and found that cells in S-phase were increased when RRM1 and compensate RRM1 knockdown effect, although growth-inhib- RRM2 were knocked down. As previously reported (24), this itory assay showed RRM2 upregulation could not compensate result suggests RRM1- and RRM2 knockdown triggered S-phase the RRM1-knockdown effect. Therefore, we further induced arrest (Fig. 2D). RRM2 expression to NCI-H929 and RPMI8226 cells by using

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A NCI-H929 MM.1S 120% 120% 100% 100% 80% 80% ** ** **** 60% ** ** ScramblesiRRM1 siRRM2 siRRM1+siRRM2 60% ScramblesiRRM1 siRRM2 siRRM1+siRRM2 48 h ** ** 48 h 40% ** ** RRM1 40% RRM1 72 h ** ** 72 h MTT (% control) 96 h MTT (% control) 20% 96 h RRM2 20% RRM2 0% 0% Figure 2. GAPDH GAPDH RRM2 In vitro and in vivo effects of RRM1 and siRRM1 siRRM2 Scramble siRRM1 siRRM2 Scramble RRM2 knockdown in multiple myeloma cells. A, RRM1- and RRM2- siRRM1+siRRM2 siRRM1+si speciﬁc siRNAs were used to KMS-11 120% RPMI8226 120% knockdown respective genes in 100% 100% multiple myeloma cell lines. Growth 80% ** 80% ** ** ** ** ScramblesiRRM1 siRRM2 siRRM1+siRRM2 inhibition of the cells was measured ** ** ScramblesiRRM1 siRRM2 siRRM1+siRRM2 60% 60% ** ** by MTT assay. The growth of all ** RRM1 48 h RRM1 40% ** 40% 72 h 48 h

4 multiple myeloma cell lines was MTT (% control) MTT (% control) 96 h RRM2 20% 72 h RRM2 20% significantly reduced at 72 and/or 96 96 h 0% 0% hours, especially in siRRM1 cells. Blue GAPDH GAPDH bar: 48 hours; orange bar: 72 hours; siRRM1 siRRM2 siRRM1 siRRM2 and gray bar: 96 hours. , P < 0.01 Scramble Scramble compared with scramble (control) at siRRM1+siRRM2 siRRM1+siRRM2 the same time period; Student t test. B C Immunoblots confirmed RRM1 and Scramble siRRM1 siRRM2 NCI-H929 RPMI8226 RRM2 knockdown. Whole-cell lysates Scramble siRRM1 siRRM2 ScramblesiRRM1 siRRM2 13.4% 38.5% 19.3% were subjected to immunoblot 48 72 48 72 48 72 48 72 48 72 48 72 (h) analysis, and GAPDH served as the NCI-H929 RRM1 loading control for each membrane. 9.4% 18.8% 17.3% B, RRM1 and RRM2 were knocked RRM2 PI down in NCI-H929 and RPMI8226 cells 15.9% 16.8% 12.4% with RRM1- and RRM2-specific siRNA, Caspase-9 and the number of apoptotic cells RPMI8226 was examined at 72 hours. Although 4.4% 5.8% 5.9% Caspase-8 significant apoptosis was triggered by siRNA knockdown in NCI-H929 cells, Annexin V only mild apoptosis was observed in Caspase-3 RPMI8226 cells. , P < 0.01 compared 100% with scramble; Student t test. ** PARP 80% ** C, Immunoblot analysis of apoptosis- GAPDH related proteins in RRM1- and RRM2- 60% knockdown NCI-H929 and RPMI8226 40% cells. Whole-cell lysates were Early apoptosis 20% subjected to immunoblot analysis, Apoptosis (%) Late apoptosis and GAPDH served as the loading 0% control for each membrane. D, RRM1 siRRM1 siRRM1 siRRM2 and RRM2 were knocked down in siRRM2 Scramble Scramble NCI-H929 cells with RRM1- and RRM2- NCI-H929 RPMI8226 specific siRNA, and the cell-cycle analysis was performed at 48 hours. G S G -M Increase in the number of cells in S- D Scramble siRRM1 siRRM2 1 2 120% Dip G1 Dip G1 phase was seen in siRNA knockdown Dip G1 Dip G2 Dip G2 Dip G2 Dip S Dip S cells. (Continued on the following Dip S 100% page.) 80%

60%

% of Cells 40%

20%

siRRM1 siRRM2 Scramble retroviral expression vector, and then performed RRM1 knock- The BM microenvironment plays a crucial role in multiple down. As shown in Fig. 2E and Supplementary Fig. S3, RRM2 myeloma pathogenesis by promoting tumor cell proliferation, overexpression could not rescue the growth-inhibitory effect of survival, and drug resistance (1). To examine whether the BM RRM1 knockdown, suggesting that RRM1, but not RRM2, is microenvironment protects against the effects of RRM1 or RRM2 a survival factor and potential therapeutic target in multiple knockdown, we next cocultured siRNA-transfected NCI-H929 myeloma. and RPMI8226 cells in the presence or absence of BMSC. We

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E NCI-H929 120% ** ** 100%

80% RRM1 60% RRM2 40%

20% GAPDH MTT (% control) 0% –– ++ RRM2 cDNA + – –+ Scramble –– ++ RRM2 cDNA + –– + siRRM1 + – –+ Scramble Figure 2. – + – + siRRM1 (Continued.) E, NCI-H929 cells were induced with either control or pMSCV- RRM2 plasmid, and then knocked down F NCI-H929 RPMI8226 with scramble or RRM1-targeted siRNA. Growth inhibition of the cells was measured by MTT assay. , P < 0.01 200% ** ** 200% ** ** ** ** compared with scramble (control) at ** ** the same time period; Student t test. 150% 150% Immunoblots conﬁrmed RRM1 knockdown and RRM2 overexpression. GAPDH served as the loading control. 100% 100% F, RRM1 and RRM2 were knocked down in NCI-H929 and RPMI8226 cells with 50% 50% RRM1- and RRM2-speciﬁc siRNA, and MTT (% control) MTT (% control) cocultured in the presence or absence of patients' BMSC for 72 hours. Data 0% 0% indicate that the bone marrow siRRM1 – + – – + – siRRM1 – + – – + – microenvironment could not abrogate siRRM2 – – + – – + siRRM2 – – + – – + the knockdown effect of RRM1 and RRM2. , P < 0.01 compared with BMSC BMSC scramble; Student t test. G, Multiple myeloma cells transduced with siRRM1 G or scramble (3 106 viable cells) were – (7 weeks) subcutaneously injected into 200-cGy irradiated SCID mice. Data represent mean SEM. N ¼ 5 mice per group. An 2,000 Scramble siRRM1 image of tumors in each group is shown (top). , P ¼ 0.0159; Student t test. Data are representative of at least two )

3 1,500 independent experiments except for Scramble xenograft experiment. siRRM1 1,000

500

Tumor volume (mm *

0 1 2 3 4 5 6 7 Weeks

observed that the effects of knockdown of both RRM1 and RRM2 DNA damage response and p53 pathways are required for were not attenuated even in the presence of BMSC (Fig. 2F). These RRM1-knockdown–induced multiple myeloma cell death data suggest that the BM microenvironment cannot overcome RR is involved in rate-limiting deoxynucleotide (dNTP) gen- RRM1- or RRM2-knockdown–mediated multiple myeloma cell eration and functions to maintain centrosome integrity, as well growth inhibition. as provide dNTPs during replication or DNA damage repair To demonstrate the in vivo efﬁcacy of RRM1 downregulation, (24, 25). Therefore, RRM1 knockdown may affect DNA damage RRM1-knockdown MM.1S cells were implanted in mice. As response and/or repair genes. Indeed, immunoblots showed that shown in Fig. 2G, cell growth was signiﬁcantly reduced in RRM1 knockdown triggered DNA damage response in multiple RRM1-knockdown cells compared with control cells. myeloma cells, including g-H2A.X, phosphorylated (p)-ATM, and

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Scramble siRRM1 A siRRM2 NCI-H929 RPMI8226 B 1MRR 2MRR 2 2 ** Scramble siRRM1 siRRM2 Scramble siRRM1 siRRM2 RRM1 1.5 1.5

RRM2 1 1

γ-H2A.X 0.5 0.5 Relative mRNA level Relative mRNA ** ** level Relative mRNA ** ** 0 0 phospho-ATM (Ser1981) H929 RPMI8226 H929 RPMI8226

ATM RAD51 53BP1 2 ** 2 phospho-ATR (Ser428) 1.6 ** ** ** 1.5 ** ATR 1.2 * 1 0.8 phospho-Chk1 (Ser317) 0.4 0.5 Chk1 Relative mRNA level Relative mRNA Relative mRNA level Relative mRNA 0 0 H929 RPMI8226 H929 RPMI8226 phospho-Chk2 (Thr68)

BRCA1 BRCA2 Chk2 2.5 ** 5 ** ** GAPDH 2 4 ** ** 1.5 3 **

1 2

0.5 1 Relative mRNA level Relative mRNA level C 0 0 NCI-H929 RPMI8226 H929 RPMI8226 H929 RPMI8226

Scramble siRRM1 siRRM2 ScramblesiRRM1 siRRM2

RAD51

53BP1

BRCA1

BRCA2

GAPDH

Figure 3. DNA damage response pathway plays essential role in RRM1-knockdown multiple myeloma cells. A, Immunoblot analysis of DNA damage response pathway genes in RRM1- and RRM2-knockdown NCI-H929 and RPMI8226 cells. GAPDH served as the loading control for each membrane. B, qRT-PCR analysis of RRM1, RRM2, RAD51, 53BP1, BRCA1,andBRCA2 in NCI-H929 and RPMI8226 cells transduced with siRNA targeting RRM1, RRM2, or scramble (control). Shown are relative signal intensity (scramble ¼ 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 each membrane, and data are representative of at least two independent experiments.

p-ATR, as well as their downstream effectors p-Chk1 and p-Chk2 Consistent with qRT-PCR, immunoblots showed that RRM1 (Fig. 3A). We next examined downstream target genes RAD51, knockdown also induced increased RAD51, 53BP1, BRCA1, and 53BP1, BRCA1, and BRCA2. As shown in Fig. 3B, quantitative real- BRCA2 protein levels (Fig. 3C). time PCR (qRT-PCR) analysis showed that RRM1 knockdown To identify novel downstream targets of RRM1 (and RRM2) induced these genes in both NCI-H929 and RPMI8226 cells. which mediate multiple myeloma cell growth, we next

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A B DDB2 CDKN1A 14 TP53I3 MDM2 siRRM1 BBC3 SESN1 siRRM2 Scramble MDM2 DDIT3 PMAIP1 10 SAT1 TP53I3 BRCA1 DRAM1 IER3 PLK2 ANKRA2 ATF3 6 GADD45A NCI-H929_siRRM1 RAF2B XPC TAX1BP3 FUCA1 2 DRAM1 2 6 10 14 BBC3 NCI-H929_Scramble FAS PIDD1 RNF19B ZMAT3 14 CDKN1A SESN1 FAS SESN1 DDB2 DDB2 RNF19B PMAIP1 TNFSF9 S100A4 10 MDM2 DRAM1 FDXR FLXNB2 BRCA1 PHLDA3 CDKN1A Figure 4. 6 LRMP NCI-H929_siRRM2 PMAIP1 Transcriptional activity of TP53 pathway is crucial in TP53 wild-type multiple myeloma cells. A, Scatter plots depicting the relative gene expression in NCI-H929 cells treated with

2 siRRM1, siRRM2, or scramble. Genes related to TP53 and 2 6 10 14 –2 0 2 BRCA1 (plotted in red) were eluted together with >1.5 fold NCI-H929_Scramble change. B, Heatmap showed induction of TP53-related C D genes in RRM1- and RRM2-knockdown cells compared with NCI-H929 scramble. Yellow denoted higher expression, whereas blue ** 2 ** denoted lower expression. C, Transcription activity levels of TP53 in TP53 wild-type NCI-H929 cells. Fold changes ScramblesiRRM1siRRM2 1.5 relative to scramble are shown. D, Immunoblot analysis of RRM1 TP53 and its related proteins in whole-cell lysates from 1 RRM1- and RRM2-knockdown NCI-H929 cells. E, NCI-H929 RRM2 cells were treated with siRRM1, sip53, or both; left plot 0.5 shows survival of cells 72 hours after knockdown. Right plot phospho-p53 (Ser15) shows conﬁrmation of knockdown, and GAPDH served as

Expression (fold change) 0 the loading control for each membrane. Data are Scramble siRRM1 siRRM2 p53 representative of at least two independent experiments in NCI-H929 C–E. , P < 0.01; Student t test. p21 E 120% ** Noxa

100% PUMA 80% GAPDH 60%

40%

20% MTT assay (% control) 0% – + – + siRRM1 – – + + sip53 NCI-H929

RRM1

p53

GAPDH

– + – + siRRM1 – – + + sip53 NCI-H929

performed gene expression proﬁling after RRM1 or RRM2 (p21WAF1), PMAIP1 (Noxa), BBC3 (Puma), SESN1, DDB2, knockdown in NCI-H929 cells. RRM1-knockdown upregu- and DRAM1 as long as BRCA1 (Fig. 4A and B). Of note, lated 665 genes, including p53 pathway genes CDKN1A multiple myeloma cells with wild-type p53 showed more

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signiﬁcant growth inhibition by RRM1 knockdown than in by p53 pathway, whereas in p53-mutant/null cells, alternative cells with mutant p53 (Fig. 2A). pathway, such as BRCA1/2 pathway, might be critical. We next used ELISA and immunoblots to examine activation of p53 pathway by RRM1 or RRM2 knockdown in NCI-H929 cells. RRM1 inhibitor triggers growth inhibition in p53 wild-type ELISA showed that p53 was activated by both RRM1 and RRM2 multiple myeloma cells knockdown (Fig. 4C). Immunoblot also showed that p53 path- To assess the potential clinical relevance of RRM1 inhibition in way is activated, evidenced by induction of p53 phosphorylation multiple myeloma, we next examined the effect of the purine WAF1 at Ser15, as well as upregulation of p21 , Noxa, and PUMA nucleoside antimetabolite clofarabine, an RRM1 inhibitor that (Fig. 4D). Importantly, p53 knockdown partially abrogated the is approved for the treatment of acute lymphocytic and myeloid effect of RRM1 knockdown (Fig. 4E), further validating p53 as a leukemia (26–30), on multiple myeloma cell lines (NCI-H929, key molecule in RRM1-knockdown–induced multiple myeloma MM.1S, MOLP-8, RPMI8226, OPM2, U266, and KMS-11). TP53 cell growth inhibition. wild-type cells (NCI-H929, MM.1S, and MOLP-8) were more Therefore, we speculated that in p53 wild-type cells, RRM1- sensitive to clofarabine treatment compared with TP53-mutant knockdown effect derived upon DNA damage response followed (RPMI8226, OPM2, and U266) or TP53-null (KMS-11) cells

A 120%

100% H929 80% MM1S MOLP8 60% RPMI8226 40% KMS11 OPM2

MTT (% of control) 20% U266

0% (mmol/L) B C NCI-H929 NCI-H929 Control CLO 5 mmol/L Control CLO 5 mmol/L 3 24 48 3 24 48 (h) 24

Caspase-9 RRM1 Figure 5. RRM1 inhibitor induces apoptosis in RRM2 multiple myeloma cells. A, Seven Caspase-8 multiple myeloma cell lines (NCI-H929, phospho-p53 (ser15) MM.1S, MOLP8, RPMI8226, OPM2, Caspase-3 U266, and KMS-11 cells) were treated p53 with clofarabine (CLO; 0–30 mmol/L) for 48 hours, and growth was then p21 PARP measured by MTT assay. B–D, Immunoblot analysis of cell lysates of Noxa NCI-H929 cells treated with GAPDH clofarabine (5 mmol/L, 3–48 hours). Puma GAPDH served as the loading control for each membrane, and data are D GAPDH representative of at least two NCI-H929 independent experiments. Control CLO 5 mmol/L 3 8 24 48 3 8 24 48 (h) g-H2A.X

phospho-ATM (Ser1981)

ATM

phospho-ATR (Ser428)

ATR

phospho-Chk1 (Ser317)

Chk1

phospho-Chk2 (Thr68)

Chk2

GAPDH

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(Fig. 5A). To elucidate the molecular mechanism of multiple RRM1 inhibitor with MEL induces synergistic multiple myeloma cell death triggered by clofarabine, we carried out myeloma cytotoxicity immunoblots and observed time-dependent cleavage of cas- Because clofarabine enhanced DNA damage response pathway, pase-3, -8, -9 and PARP (Fig. 5B). Similar to RRM1 knockdown, we next combined clofarabine with DNA-damaging agent MEL to clofarabine treatment upregulated p53 and its downstream target assess for enhanced anti–multiple myeloma activity. Clofarabine proteins in NCI-H929 cells, without signiﬁcant alteration of in combination with MEL triggered synergistic cytotoxicity not RRM1 or RRM2 protein expression (Fig. 5C). DNA damage only in NCI-H929 and but also in RPMI8226 cells (Fig. 6A). response pathway proteins, including g-H2A.X, p-ATM, and effec- Consistent with cytotoxicity, clofarabine with MEL also markedly tors p-Chk1 and p-Chk2, were also upregulated by clofarabine upregulated Annexin V–positive cells and cleavage of caspase-3, treatment in a time-dependent fashion (Fig. 5D). -8, -9, and PARP in both cells (Fig. 6B and C), suggesting that the

A 120% NCI-H929 MEL 100% 0 mmol/L 5 mmol/L 80% 10 mmol/L 20 mmol/L 2 60% 1.5 40% 1 CI MTT (% control) 20% 0.5

0% 0 0 mmol/L 5 mmol/L 10 mmol/L 20 mmol/L CLO 0 0.2 0.4 0.6 0.8 1 Fa 120% RPMI8226

100%

80% 2

60% 1.5 Figure 6. 1 RRM1 inhibition combined with CI 40% DNA-damaging agent clofarabine has

MTT (% control) 0.5 20% synergistic effect on multiple 0 0 0.2 0.4 0.6 0.8 1 myeloma cells. A, (left) NCI-H929 and 0% Fa 0 mmol/L 5 mmol/L 10 mmol/L 20 mmol/L CLO RPMI8226 cells were treated with the combination of CLO and MEL for B Control CLO MEL CLO+MEL 48 hours at the indicated doses, and tumor growth reduction was 0.7% 1.5% 1.1% 14.4% measured by MTT assay. Right, Combination index (CI) was calculated NCI-H929 in each combination therapy. CI under 1 is recognized as synergy. B, NCI- H929 and RPMI8226 cells were treated 1.1% 1.0% 0.8% 3.9% with CLO (NCI-H929, 3 mmol/L; PI RPMI8226, 10 mmol/L) and MEL 1.5% 1.3% 1.0% 6.0% (20 mmol/L) for 48 hours, and the number of apoptotic cells was RPMI8226 examined. Combination treatment indicates higher percentage of apoptotic cells. C and D, Immunoblot 0.9% 1.5% 0.9% 4.5% analysis of cell lysates after combination treatment with CLO Annexin V (NCI-H929, 3 mmol/L; RPMI8226, 10 mmol/L) and MEL (20 mmol/L) for 48 C D NCI-H929 RPMI8226 hours. (Continued on the following NCI-H929 RPMI8226 CLO – + – + – + – + page.) CLO – + – + – + – + MEL – – + + – – + + MEL – – + + – – + + g-H2A.X RRM1 phospho-ATM (Ser1981) Caspase-9 ATM

phospho-ATR (Ser428) Caspase-8 ATR

phospho-Chk1 (Ser317) Caspase-3 Chk1

PARP phospho-Chk2 (Thr68) Chk2 GAPDH GAPDH

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NCI-H929 E F NCI-H929 RPMI8226 ** **** Scramble siRRM1 Scramble siRRM1 120% NS** ** MEL 0 10 20 0 10 20 0 10 20 0 10 20 (mmol/L) Figure 6. ** MEL 100% RRM1 (Continued.) E, NCI-H929 and 0 mmol/L 80% 10 mmol/L RPMI8226 cells were treated with γ-H2A.X combination of siRNA treatment 60% 20 mmol/L (siRRM1 or scramble) and MEL for 72 40% phospho-ATM (Ser1981) hours at the indicated doses, and MTT (% control) 20% ATM tumor growth was measured by MTT 0% phospho-ATR (Ser428) assay. , P < 0.01. NS, not signiﬁcant; Scramble siRRM1 Student t test. F, Immunoblot analysis ATR of cell lysates after combination siRNA RPMI8226 phospho-Chk1 (Ser317) treatment (siRRM1 or scramble) and ** Chk1 MEL at the indicated doses and time **** 120% ** ** (same condition as E). GAPDH served NS ** phospho-Chk2 (Thr68) 100% as the loading control for each 80% Chk2 membrane, and data are 60% representative of at least two GAPDH independent experiments. 40%

MTT (% Control) 20% 0% Scramble siRRM1 enhanced combination treatment-induced cytotoxicity was due plementary Fig. S4), indicating an alternative mechanism of to apoptotic cell death. Furthermore, g-H2A.X, biomarker of DNA action triggered by RRM1 inhibition. double-strand break and DNA damage (31), was activated upon Interestingly, we showed that BRCA1 and BRCA2 were also combination treatment (Fig. 6D). Because clofarabine may have upregulated in multiple myeloma cells by RRM1 knockdown off-target effects, we carried out combination treatment of MEL irrespective of p53 status. Harkins and colleagues reported that with RRM1 knockdown and confirmed that MEL enhanced inducible expression of BRCA1 leads to apoptotic cell death in RRM1-knockdown–induced cytotoxicity (Fig. 6E), associated osteosarcoma and breast cancer cells (35). Conversely, Rao and with enhanced activation of DNA damage response pathway (Fig. colleagues reported that selective reduction of BRCA1 mRNA 6F). These data indicate that RRM1 inhibition by either knock- levels using antisense RNA induces more rapid cell growth, down or clofarabine in combination with MEL triggers synergistic decreased susceptibility to apoptosis, and cell transformation in multiple myeloma cytotoxicity. NIH3T3 fibroblasts (36). Taken together, our results suggest that upregulation of BRCA1 mRNA and protein level may account, at least in part, for RRM1-knockdown/inhibition–induced apoptotic multiple myeloma cell death, putative alternative mechanisms. Discussion To assess clinical relevance of RRM1 inhibition in multiple As in many other cancers, RR is highly expressed in multiple myeloma, we showed that the purine analog clofarabine, known myeloma cells. More specifically, we here show that both RRM1 to inhibit RRM1 (26, 27), also induces multiple myeloma cell (large subunit) and RRM2 (small subunit) are highly expressed in growth inhibition. Similar to RRM1 knockdown, clofarabine multiple myeloma cells, but not in normal cells. Importantly, we treatment also induced DNA damage response proteins, g-H2A. demonstrate that RRM1 knockdown triggers significant multiple X, phosphorylated (p)-ATM, and p-ATR, followed by its down- myeloma cell growth inhibition and apoptosis, whereas RRM2 stream effectors, p-Chk1 and p-Chk2. Interestingly, clofarabine- knockdown shows modest growth-inhibitory effects. These data induced apoptosis is more potent in multiple myeloma cells with suggest that RRM1, but not RRM2, is a survival factor and wild-type TP53 compared with cells with mutant-p53 or null-p53. potential therapeutic target in multiple myeloma. We also showed that RRM1-induced apoptotic multiple myeloma Maintenance of genomic stability depends on an appropriate cell death was more evident in p53 wild-type cells than p53- response to DNA damage, and the protein kinases ATM and ATR mutant cells. Similar results were reported by Valdez and collea- are the master controllers of such DNA damage pathway gues (37). Upon DNA damage, p53 is stabilized, upregulated, responses (32, 33). We have previously reported that pervasive and phosphorylated at Ser15, cell-cycle arrest, leading to its constitutive and ongoing DNA damage is present in hematologic antiproliferative activity, and apoptosis (38). Our results further malignancies including multiple myeloma (34), and others have demonstrated that both RRM1 knockdown and clofarabine treat- reported that RRM1 maintains centrosomal integrity during rep- ment in NCI-H929 cells with p53 wild-type upregulate/activate lication stress (24). Importantly, in this study, our gene expression p53 pathway proteins including activation of p-p53 (Ser 15), data and qRT-PCR results showed that RRM1 knockdown upre- stabilization of p53, and upregulation of p21, Noxa, and Puma. gulated DNA damage response genes including RAD51 and These results suggest that p53 pathways play a critical role medi- 53BP1. Therefore, downregulation of RRM1 could inhibit the ating RRM1-induced multiple myeloma cell death. The preva- ability of multiple myeloma cells to survive in ongoing DNA lence of p53 mutation in newly diagnosed multiple myeloma is damage, leading to apoptotic cell death. We have previously quite low (ranging from 0%–20%) and is an independent poor reported that YAP1 knockdown can trigger p73-mediated apo- prognostic factor (39), whereas higher percentage of patients with ptosis in a subset of multiple myeloma with ongoing DNA p53 abnormalities (p53 mutation and p53 deletion) are noted in damage; however, RRM1 knockdown did not alter YAP1 (Sup- more advanced disease including relapsed refractory multiple

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myeloma (RRMM) and plasma cell leukemia (40). Therefore, pathway. Our studies provide the preclinical rationale for target- RRM1 knockdown/clofarabine treatment, as a single therapeutic ing RRM1 to enhance sensitivity of tumor cells to MEL and thereby strategy, might be difficult to utilize in RRMM patients, and improve patient outcome in multiple myeloma. combination treatment strategy is warranted. Finally, MEL is a member of the nitrogen mustard class of Disclosure of Potential Conflicts of Interest chemotherapeutic agents which alkylates DNA. It triggers forma- No potential conflicts of interest were disclosed. tion of DNA adducts and forms crosslinks. The formation of crosslinks between the two strands of DNA, interstrand cross- Authors' Contributions linking, is a critical event that correlates with in vitro cytotoxicity Conception and design: M. Sagawa, T. Hideshima, K.C. Anderson (41). A previous in vitro report has combined clofarabine with Development of methodology: M. Sagawa, H. Ohguchi, K.C. Anderson MEL and described synergistic effects (37), without elucidating its Acquisition of data (provided animals, acquired and managed patients, mechanism. Importantly, we here found that the synergistic provided facilities, etc.): T. Harada, Y.-T. Tai effects triggered by combining clofarabine with MEL are evident Analysis and interpretation of data (e.g., statistical analysis, biostatistics, not only in wild-type p53 cells, but also in mutant p53 cells, and, computational analysis): M. Sagawa, H. Ohguchi, M.K. Samur, K.C. Anderson Writing, review, and/or revision of the manuscript: M. Sagawa, M.K. Samur, importantly, are associated with induction of g-H2A.X. Further- N.C. Munshi, M. Kizaki, T. Hideshima, K.C. Anderson more, we found that BRCA1 and BRCA2 were upregulated upon Administrative, technical, or material support (i.e., reporting or organizing RRM1 knockdown in p53 wild-type cells as well as p53-mutant data, constructing databases): H. Ohguchi, T. Harada, Y.-T. Tai, K.C. Anderson (and null) cells. These results suggest that MEL can enhance anti– Study supervision: M. Kizaki, T. Hideshima, K.C. Anderson multiple myeloma activity of RRM1 inhibition–induced multiple myeloma cytotoxicity regardless of p53 status, and BRCA1/2 Grant Support pathway could be the possible alternative pathway for the This research was supported by NIH grants SPORE P50-100707 enhancement of this combination treatment. Because clofarabine (K.C. Anderson), R01-CA 050947 (K.C. Anderson), and R01-CA178264 is being used as a tool compound in preclinical setting because of (T. Hideshima and K.C. Anderson). K.C. Anderson is an American Cancer its unfavorable toxicities, combination treatment of clofarabine Society Clinical Research Professor. The costs of publication of this article were defrayed in part by the payment of with MEL may not be suitable for clinical settings. Therefore, page charges. This article must therefore be hereby marked advertisement in development of novel RRM1 inhibitor with less myelotoxicity is accordance with 18 U.S.C. Section 1734 solely to indicate this fact. needed. In conclusion, we have here elucidated a novel role of RRM1 in Received January 27, 2017; revised March 17, 2017; accepted April 18, 2017; multiple myeloma regulating DNA damage response and p53 published OnlineFirst April 25, 2017.

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Ribonucleotide Reductase Catalytic Subunit M1 (RRM1) as a Novel Therapeutic Target in Multiple Myeloma

Morihiko Sagawa, Hiroto Ohguchi, Takeshi Harada, et al.

Clin Cancer Res Published OnlineFirst April 25, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-17-0263

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