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

Multiple 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 proteasome inhibitors (bortezomib, carfilzomib, and ixazomib), immunomodulatory drugs (lenalidomide and pomalidomide), and mAbs (daratumumab and elotuzumab; refs. 2, 3) have achieved remarkable clinical responses and improved patient outcome, relapse of disease is common, highlighting the need for novel treatment strategies (4, 5).

Ribonucleotide reductase (RR) is an enzyme that catalyzes the conversion of ribonucleotide diphosphate to deoxynucleotide diphosphate, which is further phosphorylated into deoxynucleotide triphosphate. Deoxynucleotide triphosphate is a direct substrate of DNA polymerases and therefore plays a central role in de novo DNA synthesis during cell replication, DNA repair, and cell growth (6, 7). The RR enzyme primarily exists as a heterodimeric tetramer of large and catalytic subunit RRM1, with small and regulatory 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 been 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 with ERCC1, and that higher RRM1 expression in non–small cell lung carcinoma is associated with better disease-free and overall survival (9, 10). In pancreatic cancer, there was no benefit of gemcitabine therapy after surgery in tumors highly expressing RRM1 group, and higher RRM1 expression was associated with shorter survival (11). In multiple myeloma, a genome-scale siRNA's lethality study in multiple myeloma identified RRM1 (12); however, the biological role of RR in multiple myeloma pathogenesis has not yet been further elucidated.

In this study, we characterize the biological significance of RR in multiple myeloma pathogenesis. We show that knockdown of RR, especially RRM1, leads to apoptotic cell death in multiple myeloma both in vitro and in vivo, even in the presence of BM microenvironment, associated with upregulation of DNA damage response and p53 pathway. Nonspecific RRM1 inhibitor clofarabine also triggers apoptotic multiple myeloma cell death, upregulates DNA damage response and p53 pathway, and triggers synergistic multiple myeloma cytotoxicity when combined with melphalan (MEL). Our data therefore provide the rationale for a novel treatment strategy inhibiting RRM1 to improve patient outcome in multiple myeloma.

Human multiple myeloma cell lines NCI-H929, MM.1S, RPMI8226, and U266 were purchased from the American Type Culture Collection (ATCC). KMS-11 cells were obtained from Japanese Collection of Research Bioresources Cell Bank. Cell lines have been tested and authenticated by STR DNA fingerprinting analysis (Molecular Diagnostic Laboratory, Dana-Farber Cancer Institute) and used within 3 months after thawing. MOLP-8 cells were recently obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures). OPM2 was provided from Dr. Edward Thompson (University of Texas Medical Branch, Galveston, TX). All multiple myeloma cell lines were cultured in RPMI1640 medium supplemented with 10% (v/v) heat-inactivated FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 μmol/L l-glutamine (Life Technologies). 293T cell lines were obtained from the ATCC and maintained in DMEM supplemented with 10% (v/v) FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. BM samples were obtained from multiple myeloma patients after informed consent and approval by the Institutional Review Board of the Dana-Farber Cancer Institute. Mononuclear cells were separated by Ficoll-Paque PLUS (GE Healthcare Life Sciences), and multiple myeloma cells were purified by CD138-positive selection with anti-CD138 magnetic-activated cell separation microbeads (Miltenyi Biotec). Long-term BM stromal cells (BMSC) were established by culturing CD138-negative BM mononuclear cells for 4 to 6 weeks in DMEM containing 15% (v/v) FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cell lines were tested to rule out mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza).

Clofarabine was purchased from Selleck Chemicals. MEL was purchased from Sigma-Aldrich. Primary antibodies for the immunoblot were as follows: anti-RRM1, -RRM2 (Abcam); anti-GAPDH, –caspase-8, –caspase-9, –caspase-3, –phosphorylated (p)-p53, -p21, -PUMA, –γ-H2A.X, –p-ATM, -ATM, –p-ATR, -ATR, –p-Chk1, -Chk1, –p-Chk2, -Chk2, -RAD51, -53BP1, -BRCA1 (Cell Signaling Technology); anti-p53 (DO-1; Santa Cruz Biotechnology); anti-Noxa (Millipore/Merck); and anti-BRCA2 (Bethyl Laboratories).

Gene Expression Omnibus data sets (GSE6477, GSE5900, GSE13591, GSE 39754, GSE2658, and GSE36133) were used for gene expression analyses (13–18). Both 201476_s_at and 201477_s_at are the probes for RRM1, and 201890_at is the probe for RRM2 transcript on Affymetrix Human Genome U133A Array or Human Genome U133 Plus 2.0 Array.

NCI-H929, MM.1S, RPMI8226, and KMS-11 cells were transiently transfected with scramble or targeted siRNA (GE Healthcare Dharmacon) against RRM1, RRM2, and p53. siRNA transfection was performed by electroporation using Nucleofector Kit V (Lonza), according to the manufacturer's instructions.

The human RRM2 cDNA was amplified using PCR and ligated into the HpaI and XhoI sites of pMSCV retroviral expression vector (Clontech).

On day 0, 293T packaging cells were plated at a density of 6 × 10⁵ cells per 6-well plates. On day 1, cells were transfected with 500 ng of pMSCVpuro plasmid, 500 ng of pMD-MLV, and 100 ng of VSV-G, using TransIT-LT1 Transfection Reagent (Mirus Bio), according to the manufacturer's instructions. On day 2, media were replaced and cells were cultured for an additional 24 hours to obtain viral supernatants. On day 3, media containing virus were harvested, passed through 0.45-μm cellulose acetate membrane filters, and used fresh for infection. Overall, 2 × 10⁶ cells per 1 mL of crude viral supernatants in the presence of 8 μg/mL polybrene (Sigma-Aldrich) were spinoculated at 800 × g for 30 minutes at room temperature, and then incubated in 5% CO₂ at 37°C for 5 hours. Media were then replaced. After 24 hours of viral infection, cells expressing cDNA were selected with puromycin dihydrochloride (Sigma-Aldrich) at 1 μg/mL for 2 days, and clones expressing cDNAs were subjected to rescue experiments. Puromycin concentrations were titrated to identify the minimum concentration of each drug that caused complete cell death of uninfected cells after 2 days.

The growth-inhibitory effect was assessed by measuring 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma-Aldrich) dye absorbance, as previously described (19). The synergistic effect was assessed by combination index using the CompuSyn software program (ComboSyn Inc.).

Cells were treated, harvested, washed with PBS, and lysed in RIPA buffer (Boston BioProducts) containing protease inhibitor cocktail (Roche). Protein concentration was measured with Bio-Rad Protein Assay (Bio-Rad Laboratories). Whole-cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose membrane (Bio-Rad Laboratories), immunoblotted with antibodies described above, and visualized using ECL Western Blotting Detection Reagents (GE Healthcare Life Sciences), as previously described (20).

Apoptotic cell death was assessed by the FITC Annexin-V Apoptosis Detection Kit (BD Biosciences), according to the manufacturer's instructions. Cells stained with Annexin V and propidium iodide were analyzed with BD FACS Canto II (BD Biosciences) using the FACS DIVA software (BD Biosciences), as previously described (21).

Cells were harvested and fixed with 70% ethanol for 20 minutes on ice. After washing with PBS twice, cells were incubated with 5 μg/mL RNase (Roche) in PBS for 20 minutes at room temperature, and then resuspended in PBS containing 10 μg/mL propidium iodide (Sigma-Aldrich). The stained cells were analyzed with BD FACS Canto II (BD Biosciences), and the percentage of cells in G₁, S, and G₂–M phases was determined using the ModFit LT software (Verity Software House).

To isolate nuclear and cytoplasmic proteins, cells were treated, harvested, washed with PBS, and lysed in the Nuclear Extract Kit (Active Motif), according to the manufacturer's instructions. DNA-binding activity of p53 was quantified by ELISA using the Trans-AM p53 Transcription Factor Assay Kit (Active Motif), according to the manufacturer's instructions.

Total RNA was extracted using the RNeasy Mini Kit (Qiagen). cDNA was synthesized from 1 μg 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 PCR System (Thermo Fisher Scientific). The PCR mixture contained 10 ng of reverse-transcribed RNA, 100 nmol/L of forward and reverse primers, and SYBR Green PCR Master Mix (Thermo Fisher Scientific), in a final volume of 20 μL. The conditions were 95°C for 10 minutes, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. The relative amount of each transcript was calculated using the relative standard curve method. GAPDH mRNA was used as the invariant control, and values were normalized by GAPDH expression. Specific primers for each gene transcript are shown in Supplementary Table 1.

Total RNAs for microarray analysis were extracted from NCI-H929 cells transfected with siRNA targeting RRM1, RRM2, or scramble siRNA in biological duplicate using the RNeasy Mini Kit (Qiagen). Total RNA (1 μg) was processed, and labeled cRNA was hybridized to Human Genome U133 plus 2.0 arrays (Affymetrix) according to the standard Affymetrix protocols, as previously described (22). Expression data can be found at http://www.ncbi.nlm.nih.gov/geo/ under accession number GSE93425.

Five-week-old male CB17 SCID mice (Charles River Laboratories, Inc.) were used for this study. Note that 3 × 10⁶ viable MM.1S cells transduced with the corresponding siRNA (siRRM1 or scramble) were suspended in 100 μL of PBS, and then inoculated subcutaneously into the left flank of 200-cGy–irradiated mice. Tumor growth was monitored twice a week using an electronic caliper, 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 the Dana-Farber Institutional Animal Care and Use Committee and followed the ARRIVE guidelines (23).

The Student t test or ANOVA followed by the Dunnett test was used to compare differences between the treated group and relevant control group. Correlation of RRM1 and RRM2 expression with overall survival was measured using the Kaplan–Meier method, with Cox proportional hazard regression analysis for group comparison. A value of P < 0.05 was considered significant.

We first investigated the expression of RRM1 and RRM2 in primary multiple myeloma cells. Our evaluation of RRM1 and RRM2 messenger RNA (mRNA) expression in three independent publicly available data sets (13–15) revealed that RRM1 transcript levels are significantly higher in multiple myeloma than in healthy donor in all data sets, and in monoclonal gammopathy of undetermined significance (MGUS) in two of three data sets (Fig. 1A–C, top); and that RRM2 transcript levels are also significantly higher in two of three data sets (Fig. 1A–C, bottom). These results are consistent with previous studies in other cancers (Supplementary Fig. S1). We also evaluated another two publicly available data sets of 170 (16) and 350 (17) newly diagnosed patients and found that patients with higher expressions of RRM1 and RRM2 had significantly shorter overall survival (Fig. 1D and Supplementary Fig. S2). We also examined RRM1 and RRM2 protein expression in multiple myeloma cells. We found that both RRM1 and RRM2 were detected in six human multiple myeloma cell lines and three patient multiple myeloma cells (Fig. 1E).

To evaluate the biological function of RRM1 and RRM2, we transduced multiple myeloma cells with siRNA targeting RRM1, RRM2, or control (scramble) by electroporation. Transduction of RRM1- and RRM2-specific siRNA markedly reduced the respective protein expression in 4 cell lines (p53 wild-type; NCI-H929 and MM.1S, p53 mutant; RPMI8226, p53 null; KMS11) examined (Fig. 2A). Importantly, knockdown of RRM1 or RRM2 significantly inhibited multiple myeloma cell line growth (Fig. 2A). Of note, RRM2 knockdown did not enhance cell growth inhibition induced by RRM1 knockdown. Along with cell growth inhibition, apoptotic cell death was significantly increased by RRM1 or RRM2 knockdown in NCI-H929 multiple myeloma cells (Fig. 2B). Apoptotic cell death was further confirmed by immunoblots showing cleavages of caspase-3, -8 and -9, and PARP in NCI-H929 cells (Fig. 2C). Consistent with Annexin V–PI staining, apoptotic cell death triggered by RRM1 or RRM2 knockdown was modest in RPMI8226 cells (Fig. 2C). We also performed cell-cycle analysis and found that cells in S-phase were increased when RRM1 and RRM2 were knocked down. As previously reported (24), this result suggests RRM1- and RRM2 knockdown triggered S-phase arrest (Fig. 2D).

As seen in Fig. 2A, RRM1 knockdown induced upregulation of RRM2, whereas RRM2 knockdown did not induce upregulation of RRM1. These results suggested that, although precise molecular mechanism has not yet been elucidated, RRM2 could compensate RRM1 knockdown effect, although growth-inhibitory assay showed RRM2 upregulation could not compensate the RRM1-knockdown effect. Therefore, we further induced RRM2 expression to NCI-H929 and RPMI8226 cells by using retroviral expression vector, and then performed RRM1 knockdown. As shown in Fig. 2E and Supplementary Fig. S3, RRM2 overexpression could not rescue the growth-inhibitory effect of RRM1 knockdown, suggesting that RRM1, but not RRM2, is a survival factor and potential therapeutic target in multiple myeloma.

The BM microenvironment plays a crucial role in multiple myeloma pathogenesis by promoting tumor cell proliferation, survival, and drug resistance (1). To examine whether the BM microenvironment protects against the effects of RRM1 or RRM2 knockdown, we next cocultured siRNA-transfected NCI-H929 and RPMI8226 cells in the presence or absence of BMSC. We observed that the effects of knockdown of both RRM1 and RRM2 were not attenuated even in the presence of BMSC (Fig. 2F). These data suggest that the BM microenvironment cannot overcome RRM1- or RRM2-knockdown–mediated multiple myeloma cell growth inhibition.

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

RR is involved in rate-limiting deoxynucleotide (dNTP) generation and functions to maintain centrosome integrity, as well as provide dNTPs during replication or DNA damage repair (24, 25). Therefore, RRM1 knockdown may affect DNA damage response and/or repair genes. Indeed, immunoblots showed that RRM1 knockdown triggered DNA damage response in multiple myeloma cells, including γ-H2A.X, phosphorylated (p)-ATM, and p-ATR, as well as their downstream effectors p-Chk1 and p-Chk2 (Fig. 3A). We next examined downstream target genes RAD51, 53BP1, BRCA1, and BRCA2. As shown in Fig. 3B, quantitative real-time PCR (qRT-PCR) analysis showed that RRM1 knockdown induced these genes in both NCI-H929 and RPMI8226 cells. Consistent with qRT-PCR, immunoblots showed that RRM1 knockdown also induced increased RAD51, 53BP1, BRCA1, and BRCA2 protein levels (Fig. 3C).

To identify novel downstream targets of RRM1 (and RRM2) which mediate multiple myeloma cell growth, we next performed gene expression profiling after RRM1 or RRM2 knockdown in NCI-H929 cells. RRM1-knockdown upregulated 665 genes, including p53 pathway genes CDKN1A (p21^WAF1), 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 knockdown than in cells with mutant p53 (Fig. 2A).

We next used ELISA and immunoblots to examine activation of p53 pathway by RRM1 or RRM2 knockdown in NCI-H929 cells. ELISA showed that p53 was activated by both RRM1 and RRM2 knockdown (Fig. 4C). Immunoblot also showed that p53 pathway is activated, evidenced by induction of p53 phosphorylation at Ser15, as well as upregulation of p21^WAF1, Noxa, and PUMA (Fig. 4D). Importantly, p53 knockdown partially abrogated the effect of RRM1 knockdown (Fig. 4E), further validating p53 as a key molecule in RRM1-knockdown–induced multiple myeloma cell growth inhibition.

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

To assess the potential clinical relevance of RRM1 inhibition in multiple myeloma, we next examined the effect of the purine nucleoside antimetabolite clofarabine, an RRM1 inhibitor that is approved for the treatment of acute lymphocytic and myeloid leukemia (26–30), on multiple myeloma cell lines (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 treatment compared with TP53-mutant (RPMI8226, OPM2, and U266) or TP53-null (KMS-11) cells (Fig. 5A). To elucidate the molecular mechanism of multiple myeloma cell death triggered by clofarabine, we carried out immunoblots and observed time-dependent cleavage of caspase-3, -8, -9 and PARP (Fig. 5B). Similar to RRM1 knockdown, clofarabine treatment upregulated p53 and its downstream target proteins in NCI-H929 cells, without significant alteration of RRM1 or RRM2 protein expression (Fig. 5C). DNA damage response pathway proteins, including γ-H2A.X, p-ATM, and effectors p-Chk1 and p-Chk2, were also upregulated by clofarabine treatment in a time-dependent fashion (Fig. 5D).

Because clofarabine enhanced DNA damage response pathway, we next combined clofarabine with DNA-damaging agent MEL to assess for enhanced anti–multiple myeloma activity. Clofarabine in combination with MEL triggered synergistic cytotoxicity not only in NCI-H929 and but also in RPMI8226 cells (Fig. 6A). Consistent with cytotoxicity, clofarabine with MEL also markedly upregulated Annexin V–positive cells and cleavage of caspase-3, -8, -9, and PARP in both cells (Fig. 6B and C), suggesting that the enhanced combination treatment-induced cytotoxicity was due to apoptotic cell death. Furthermore, γ-H2A.X, biomarker of DNA double-strand break and DNA damage (31), was activated upon combination treatment (Fig. 6D). Because clofarabine may have off-target effects, we carried out combination treatment of MEL with RRM1 knockdown and confirmed that MEL enhanced RRM1-knockdown–induced cytotoxicity (Fig. 6E), associated with enhanced activation of DNA damage response pathway (Fig. 6F). These data indicate that RRM1 inhibition by either knockdown or clofarabine in combination with MEL triggers synergistic multiple myeloma cytotoxicity.

As 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 in multiple myeloma cells, but not in normal cells. Importantly, we demonstrate that RRM1 knockdown triggers significant multiple myeloma cell growth inhibition and apoptosis, whereas RRM2 knockdown shows modest growth-inhibitory effects. These data suggest that RRM1, but not RRM2, is a survival factor and potential therapeutic target in multiple myeloma.

Maintenance of genomic stability depends on an appropriate response to DNA damage, and the protein kinases ATM and ATR are the master controllers of such DNA damage pathway responses (32, 33). We have previously reported that pervasive constitutive and ongoing DNA damage is present in hematologic malignancies including multiple myeloma (34), and others have reported that RRM1 maintains centrosomal integrity during replication stress (24). Importantly, in this study, our gene expression data and qRT-PCR results showed that RRM1 knockdown upregulated DNA damage response genes including RAD51 and 53BP1. Therefore, downregulation of RRM1 could inhibit the ability of multiple myeloma cells to survive in ongoing DNA damage, leading to apoptotic cell death. We have previously reported that YAP1 knockdown can trigger p73-mediated apoptosis in a subset of multiple myeloma with ongoing DNA damage; however, RRM1 knockdown did not alter YAP1 (Supplementary Fig. S4), indicating an alternative mechanism of action triggered by RRM1 inhibition.

Interestingly, we showed that BRCA1 and BRCA2 were also upregulated in multiple myeloma cells by RRM1 knockdown irrespective of p53 status. Harkins and colleagues reported that inducible expression of BRCA1 leads to apoptotic cell death in osteosarcoma and breast cancer cells (35). Conversely, Rao and colleagues reported that selective reduction of BRCA1 mRNA levels using antisense RNA induces more rapid cell growth, decreased susceptibility to apoptosis, and cell transformation in 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.

To assess clinical relevance of RRM1 inhibition in multiple myeloma, we showed that the purine analog clofarabine, known to inhibit RRM1 (26, 27), also induces multiple myeloma cell growth inhibition. Similar to RRM1 knockdown, clofarabine treatment also induced DNA damage response proteins, γ-H2A.X, phosphorylated (p)-ATM, and p-ATR, followed by its downstream effectors, p-Chk1 and p-Chk2. Interestingly, clofarabine-induced apoptosis is more potent in multiple myeloma cells with wild-type TP53 compared with cells with mutant-p53 or null-p53. We also showed that RRM1-induced apoptotic multiple myeloma cell death was more evident in p53 wild-type cells than p53-mutant cells. Similar results were reported by Valdez and colleagues (37). Upon DNA damage, p53 is stabilized, upregulated, and phosphorylated at Ser15, cell-cycle arrest, leading to its antiproliferative activity, and apoptosis (38). Our results further demonstrated that both RRM1 knockdown and clofarabine treatment in NCI-H929 cells with p53 wild-type upregulate/activate p53 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 mediating RRM1-induced multiple myeloma cell death. The prevalence of p53 mutation in newly diagnosed multiple myeloma is quite low (ranging from 0%–20%) and is an independent poor prognostic factor (39), whereas higher percentage of patients with p53 abnormalities (p53 mutation and p53 deletion) are noted in more advanced disease including relapsed refractory multiple myeloma (RRMM) and plasma cell leukemia (40). Therefore, RRM1 knockdown/clofarabine treatment, as a single therapeutic strategy, might be difficult to utilize in RRMM patients, and combination treatment strategy is warranted.

Finally, MEL is a member of the nitrogen mustard class of chemotherapeutic agents which alkylates DNA. It triggers formation of DNA adducts and forms crosslinks. The formation of crosslinks between the two strands of DNA, interstrand crosslinking, is a critical event that correlates with in vitro cytotoxicity (41). A previous in vitro report has combined clofarabine with MEL and described synergistic effects (37), without elucidating its mechanism. Importantly, we here found that the synergistic effects triggered by combining clofarabine with MEL are evident not only in wild-type p53 cells, but also in mutant p53 cells, and, importantly, are associated with induction of γ-H2A.X. Furthermore, we found that BRCA1 and BRCA2 were upregulated upon RRM1 knockdown in p53 wild-type cells as well as p53-mutant (and null) cells. These results suggest that MEL can enhance anti–multiple myeloma activity of RRM1 inhibition–induced multiple myeloma cytotoxicity regardless of p53 status, and BRCA1/2 pathway could be the possible alternative pathway for the enhancement of this combination treatment. Because clofarabine is being used as a tool compound in preclinical setting because of its unfavorable toxicities, combination treatment of clofarabine with MEL may not be suitable for clinical settings. Therefore, development of novel RRM1 inhibitor with less myelotoxicity is needed.

In conclusion, we have here elucidated a novel role of RRM1 in multiple myeloma regulating DNA damage response and p53 pathway. Our studies provide the preclinical rationale for targeting RRM1 to enhance sensitivity of tumor cells to MEL and thereby improve patient outcome in multiple myeloma.

No potential conflicts of interest were disclosed.

Conception and design: M. Sagawa, T. Hideshima, K.C. Anderson

Development of methodology: M. Sagawa, H. Ohguchi, K.C. Anderson

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Harada, Y.-T. Tai

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, 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, N.C. Munshi, M. Kizaki, T. Hideshima, K.C. Anderson

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H. Ohguchi, T. Harada, Y.-T. Tai, K.C. Anderson

Study supervision: M. Kizaki, T. Hideshima, K.C. Anderson

This 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 Cancer Society Clinical Research Professor.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.