Ribonucleotide Reductase Large Subunit (RRM1) As a Novel Therapeutic Target

Author Manuscript Published OnlineFirst on April 25, 2017; DOI: 10.1158/1078-0432.CCR-17-0263 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Clinical Cancer Research Ver3.0 (revised)

2 Research Article: Biology of Human tumors

3 Title: Ribonucleotide reductase large subunit (RRM1) as a novel therapeutic target

4 in multiple myeloma

6 Authors: Morihiko Sagawa1,4, Hiroto Ohguchi1, Takeshi Harada1, Mehmet K. Samur2,

7 Yu-Tzu Tai 1, Nikhil C. Munshi1,3, Masahiro Kizaki4, Teru Hideshima1 and Kenneth C.

8 Anderson1

9 Affiliations: 1Jerome Lipper Multiple Myeloma Center, Department of Medical

10 Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston,

11 Massachusetts. 2Department of Biostatistics and Computational Biology, Dana-Farber

12 Cancer Institute and Harvard School of Public Health, Boston, Massachusetts. 3West

13 Roxbury Division, VA Boston Healthcare System, West Roxbury, Massachusetts.

14 4Department of Hematology, Saitama Medical Center, Saitama Medical University,

15 Kawagoe, Saitama, Japan.

17 Running title: Targeting RRM1 as a novel treatment for multiple myeloma

18 Key words: Multiple Myeloma, RRM1, DNA damage response, p53, Clofarabine

20 Financial Support: NIH grants; SPORE P50-100707 (KCA), R01-CA050947 (KCA),

21 and R01-CA178264 (TH and KCA).

Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 25, 2017; DOI: 10.1158/1078-0432.CCR-17-0263 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Corresponding Author: Kenneth C. Anderson, M.D., Jerome Lipper Multiple

2 Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute,

3 Harvard Medical School, M557, 450 Brookline Avenue, Boston, Massachusetts 02215.

4 Telephone; 617-632-2144, Fax; 617-632-2140, E-mail:

5 [email protected],

6 Conflict of Interest: The authors declare no potential conflicts of interest.

8 Word count: 3875 (excluding references), Abstract word count: 250

9 Number of figures: 6, Supplementary figures: 4, Supplementary tables: 1

10 Number of reference: 41

12 Translational Relevance

13 Ribonucleotide reductase, an enzyme required for DNA synthesis and repair, is

14 overexpressed in many cancers and associated with poor prognosis. Here we

15 investigated the biologic significance of ribonucleotide reductase subunit M1 (RRM1)

16 in multiple myeloma (MM) cells. We demonstrate that RRM1 knockdown and an

17 RRM1 inhibitor clofarabine (CLO), alone and especially when combined with

18 melphalan, triggers significant MM cell growth inhibition both in vitro and in vivo in a

19 mouse human MM xenograft model. Importantly, activation of both DNA damage

20 response and p53 pathways mediate combination treatment-induced anti-MM activity.

21 Our findings provide the rationale for clinical investigation of RRM1 inhibitor in

22 combination with DNA damaging agents as a novel treatment strategy in MM.

1 Abstract

2 Purpose: To investigate the biologic and clinical significance of ribonucleotide

3 reductase (RR) in multiple myeloma (MM).

4 Experimental Design: We assessed the impact of RR expression on patient outcome in

5 MM. We then characterized the effect of genetic and pharmacological inhibition of

6 RRM1 on MM growth and survival using siRNA and clofarabine (CLO), respectively,

7 both in vitro and in vivo mouse xenograft model.

8 Results: Newly diagnosed MM patients with higher RRM1 expression have shortened

9 survival. Knockdown of RRM1 triggered significant growth inhibition and apoptosis in

10 MM cells, even in the context of the bone marrow microenvironment. Gene expression

11 profiling showed upregulation of DNA damage response genes and p53 regulated genes

12 after RRM1 knockdown. Immunoblot and QRT-PCR analysis confirmed that γ-H2A.X,

13 ATM, ATR, Chk1, Chk2, RAD51, 53BP1, BRCA1, and BRCA2 were

14 upregulated/activated. Moreover, immunoblots showed that p53, p21, Noxa, and Puma

15 were activated in p53 wild-type MM cells. Clofarabine (CLO), a purine nucleoside

16 analog that inhibits RRM1, induced growth arrest and apoptosis in p53 wild-type cell

17 lines. Although CLO did not induce cell death in p53 mutant cells, it did trigger

18 synergistic toxicity in combination with DNA damaging agent melphalan. Finally, we

19 demonstrated that tumor growth of RRM1-knockdown MM cells was significantly

20 reduced in a murine human MM cell xenograft model.

21 Conclusions: Our results therefore demonstrate that RRM1 is a novel therapeutic target

22 in MM in preclinical setting, and provide the basis for clinical evaluation of RRM1

1 inhibitor, alone or in combination with DNA damaging agents, to improve patient

2 outcome in MM.

1 Introduction

2 Multiple myeloma (MM) is a plasma cell disorder characterized by excess malignant

3 plasma cells in the bone marrow (BM), increased monoclonal gammaglobulin in blood

4 and/or urine, and end organ damage in kidney and bone [1]. Although proteasome

5 inhibitors (bortezomib, carfilzomib, ixazomib), immunomodulatory drugs (lenalidomide,

6 pomalidomide) and monoclonal antibodies (daratumumab and elotuzumab) [2, 3] have

7 achieved remarkable clinical responses and improved patient outcome, relapse of

8 disease is common, highlighting the need for novel treatment strategies [4, 5].

9 Ribonucleotide reductase (RR) is an enzyme that catalyzes the conversion of

10 ribonucleotide diphosphate to deoxynucleotide diphosphate, which is further

11 phosphorylated into deoxynucleotide triphosphate. Deoxynucleotide triphosphate, is a

12 direct substrate of DNA polymerases, and therefore plays a central role in de novo DNA

13 synthesis during cell replication, DNA repair, and cell growth [6, 7]. The RR enzyme

14 primarily exists as a heterodimeric tetramer of large and catalytic subunit RRM1, with

15 small and regulatory subunit RRM2 [6]. RRM1 expression is ubiquitous, while RRM2

16 expression is cell cycle dependent [6].

17 RR is expressed in different types of cancers, and has been associated with drug

18 resistance, cancer cell growth and metastasis [8]. However, other reports show that

19 RRM1 suppresses metastasis through induction of PTEN, that RRM1 expression

20 correlates with ERCC1, and that higher RRM1 expression in non-small cell lung

21 carcinoma is associated with better disease-free and overall survival [9, 10]. In

22 pancreatic cancer, there was no benefit of gemcitabine therapy after surgery in tumors

1 highly expressing RRM1 group, and higher RRM1 expression was associated with

2 shorter survival [11]. In MM, a genome-scale siRNAs lethality study in MM identified

3 RRM1 [12]; however, the biologic role of RR in MM pathogenesis hasn't yet been

4 further elucidated.

5 In this study, we characterized the biological significance of RR in MM

6 pathogenesis. We show that knockdown of RR, especially RRM1, leads to apoptotic cell

7 death in MM both in vitro and in vivo, even in the presence of BM microenvironment,

8 associated with upregulation of DNA damage response and p53 pathway. Non-specific

9 RRM1 inhibitor clofarabine (CLO) also triggers apoptotic MM cell death, upregulates

10 DNA damage response and p53 pathway, and triggers synergistic MM cytotoxicity

11 when combined with melphalan (MEL). Our data therefore provide the rationale for a

12 novel treatment strategy inhibiting RRM1 to improve patient outcome in MM.

15 Methods

16 Cell culture

17 Human MM cell lines NCI-H929, MM.1S, RPMI8226, and U266 were purchased from

18 American Type Culture Collection (ATCC, Manassas, VA, USA). KMS-11 cells were

19 obtained from Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan).

20 Cell lines have been tested and authenticated by STR DNA fingerprinting analysis

21 (Molecular Diagnostic Laboratory, Dana-Farber Cancer Institute), and used within 3

22 months after thawing. MOLP-8 cells were recently obtained from Deutsche Sammlung

1 von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms

2 and Cell Cultures). OPM2 was provided from Dr. Edward Thompson (University of

3 Texas Medical Branch, Galveston, USA). All MM cell lines were cultured in RPMI1640

4 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 U/mL

5 penicillin, 100 μg/mL streptomycin, and 2 μM L-glutamine (Life Technologies, Grand

6 Island, NY, USA). 293T cell lines was obtained from ATCC and maintained in

7 Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) fetal

8 bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Bone marrow

9 samples were obtained from MM patients after informed consent and approval by the

10 Institutional Review Board of the Dana-Farber Cancer Institute. Mononuclear cells were

11 separated by Ficoll-Paque PLUS (GE Healthcare Life Sciences, Pittsburgh, PA, USA),

12 and MM cells were purified by CD138-positive selection with anti-CD138 magnetic

13 activated cell separation microbeads (Miltenyi Biotec, San Diego, CA, USA).

14 Long-term bone marrow stromal cells (BMSC) were established by culturing

15 CD138-negative bone marrow mononuclear cells for 4-6 weeks in DMEM containing

16 15% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cell

17 lines were tested to rule out mycoplasma contamination using the MycoAlert

18 Mycoplasma Detection Kit (Lonza, Basel, Switzerland).

20 Reagents

21 Clofarabine (CLO) was purchased from Selleck Chemicals (Houston, TX, USA).

22 Melphalan (MEL) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary

1 antibodies for the immunoblot were as follows: anti-RRM1, -RRM2 (Abcam,

2 Cambridge, MA, USA); anti-GAPDH, -caspase-8, -caspase-9, -caspase-3,

3 -phosphorylated (p)-p53, -p21, -PUMA, -γ-H2A.X, -p-ATM, -ATM, -p-ATR, -ATR,

4 -p-Chk1, -Chk1, -p-Chk2, -Chk2, -RAD51, -53BP1, -BRCA1 (Cell Signaling

5 Technology, Danvers, MA, USA); anti-p53 (DO-1) (Santa Cruz Biotechnology, Dallas,

6 TX, USA); anti-Noxa (Millipore/Merck, Darmstadt, Germany); and anti-BRCA2

7 (Bethyl Laboratories, Montgomery, TX, USA).

9 Gene expression analysis using publicly available data sets

10 Gene Expression Omnibus data sets (GSE6477, GSE5900, GSE13591, GSE 39754,

11 GSE2658, and GSE36133) were used for gene expression analyses [13-18]. Both

12 201476_s_at and 201477_s_at are the probes for RRM1, and 201890_at is the probe for

13 RRM2 transcript on Affymetrix Human Genome U133A Array or Human Genome

14 U133 Plus 2.0 Array.

16 siRNA transfection

17 NCI-H929, MM.1S, RPMI8226, and KMS-11 cells were transiently transfected with

18 scramble or targeted siRNA (GE Healthcare Dharmacon, Lafayette, CO, USA) against

19 RRM1, RRM2, and p53. siRNA transfection was performed by electroporation using

20 Nucleofector Kit V (Lonza), according to manufacturer’s instructions.

22 Expression plasmid

1 The human RRM2 cDNA was amplified using PCR and ligated into the HpaI and XhoI

2 sites of pMSCV retroviral expression vector (Clontech, Mountain View, CA, USA).

4 Viral production and infection

5 On day 0, 293T packaging cells were plated at a density of 6 x 105 cells per six-well

6 plates. On day 1, cells were transfected with 500 ng of pMSCVpuro plasmid, 500 ng of

7 pMD-MLV and 100 ng of VSV-G, using TransIT-LT1 Transfection Reagent (Mirus Bio,

8 Madison, WI), according to the manufacturer’s instructions. On day 2, media were

9 replaced and cells were cultured for an additional 24 h to obtain viral supernatants. On

10 day 3, media containing virus were harvested, passed through 0.45-mm cellulose acetate

11 membrane filters and used fresh for infection. Overall, 2 x 106 cells per 1 ml of crude

12 viral supernatants in the presence of 8 μg/ml polybrene (Sigma-Aldrich) were

13 spinoculated at 800 x g for 30 min at room temperature, and then incubated in 5% CO2

14 at 37 °C for 5 h. Media were then replaced. After 24 h of viral infection, cells

15 expressing cDNA were selected with puromycin dihydrochloride (Sigma-Aldrich) at 1

16 μg/ml for 2 days, and clones expressing cDNAs were subjected to rescue experiments.

17 Puromycin concentrations were titrated to identify the minimum concentration of each

18 drug that caused complete cell death of uninfected cells after 2 days.

20 Growth-inhibition assay

21 The growth-inhibitory effect was assessed by measuring 3-(4,5-Dimethyl-2-thiazolyl)

22 -2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma-Aldrich) dye absorbance, as

1 previously described [19]. The synergistic effect was assessed by combination index

2 using the CompuSyn software program (ComboSyn Inc., Paramus, NJ, USA).

4 Immunoblot analysis

5 Cells were treated, harvested, washed with PBS, and lysed in RIPA buffer (Boston

6 BioProducts, Ashland, MA, USA) containing protease inhibitor cocktail (Roche,

7 Indianapolis, IN, USA). Protein concentration was measured with Bio-Rad Protein

8 Assay (Bio-Rad Laboratories, Hercules, CA, USA). Whole-cell lysates were subjected

9 to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred

10 to nitrocellulose membrane (Bio-Rad Laboraories), immunoblotted with antibodies

11 described above, and visualized using ECL Western Blotting Detection Reagents (GE

12 Healthcare Life Sciences), as previously described [20].

14 Annexin V/propidium iodide staining

15 Apoptotic cell death was assessed by FITC Annexin-V Apoptosis Detection Kit (BD

16 Biosciences, San Diego, CA, USA), according to manufacturer's instructions. Cells

17 stained with annexin V and propidium iodide were analyzed with BD FACS Canto II

18 (BD Biosciences) using the FACS DIVA software (BD Biosciences), as previously

19 described [21].

21 Cell cycle analysis

22 Cells were harvested and fixed with 70% ethanol for 20 min on ice. After washing with

1 PBS twice, cells were incubated with 5 μg/ml RNase (Roche) in PBS for 20 min at

2 room temperature, and then resuspended in PBS containing 10 μg/ml propidium iodide

3 (Sigma-Aldrich). The stained cells were analyzed with BD FACS Canto II (BD

4 Biosciences), and the percentage of cells in G1, S and G2/M phases was determined

5 using the ModFit LT software (Verity Software House, Topsham, ME, USA).

7 Enzyme-linked immunosorbent assay (ELISA)

8 To isolate nuclear and cytoplasmic proteins, cells were treated, harvested, washed with

9 PBS, and lysed in Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA), according to

10 manufacturer’s instructions. DNA-binding activity of p53 was quantified by ELISA

11 using Trans-AM p53 Transcription Factor Assay Kit (Active Motif), according to

12 manufacturer’s instructions.

14 RNA extraction and quantitative real-time polymerase chain reaction (QRT-PCR)

15 Total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA

16 was synthesized from 1 μg of total RNA with oligo(dT) primers using the SuperScript

17 III First-Strand Synthesis System (Thermo Fisher Scientific, Waltham, MA, USA).

18 Real-time PCR was performed in 96-well plates using the Applied Biosystems 7300

19 Real-Time PCR System (Thermo Fisher Scientific). The PCR mixture contained 10 ng

20 of reverse-transcribed RNA, 100 nM of forward and reverse primers, and SYBR Green

21 PCR Master Mix (Thermo Fisher Scientific), in a final volume of 20 μL. The conditions

22 were 95 °C for 10 min, followed by 40 cycles of 15 sec at 95 °C and 1 min at 60 °C.

1 The relative amount of each transcript was calculated using the relative standard curve

2 method. GAPDH mRNA was used as the invariant control, and values were normalized

3 by GAPDH expression. Specific primers for each gene transcript are shown in

4 Supplementary Table.

6 Affymetrix gene expression analysis

7 Total RNAs for microarray analysis were extracted from NCI-H929 cells transfected

8 with siRNA targeting RRM1, RRM2, or scramble siRNA in biological duplicate using

9 RNeasy Mini Kit (Qiagen). Total RNA (1 μg) was processed, and labeled cRNA was

10 hybridized to Human Genome U133 plus 2.0 arrays (Affymetrix, Santa Clara, CA,

11 USA) according to the standard Affymetrix protocols, as previously described [22].

12 Expression data can be found at http://www.ncbi.nlm.nih.gov/geo/ under accession

13 number GSE93425.

15 Subcutaneous xenograft model

16 Five-week-old male CB17 severe combined immunodeficient (SCID) mice (Charles

17 River Laboratories, Inc., Wilmington, MA, USA) were used for this study. Three x 106

18 viable MM.1S cells transduced with the corresponding siRNA (siRRM1 or scramble)

19 were suspended in 100 μL of PBS, and then inoculated subcutaneously into the left

20 flank of 200-cGy-irradiated mice. Tumor growth was monitored twice a week using an

21 electronic caliper, and the tumor volume was calculated using the formula: (length x

22 width2) x2-1, where length is greater than width. Animal studies were performed under a

1 protocol approved by the Dana-Farber Institutional Animal Care and Use Committee

2 and followed the ARRIVE guidelines [23].

4 Statistical analysis

5 Student’s t-test or analysis of variance followed by Dunnett’s test was used to compare

6 differences between the treated group and relevant control group. Correlation of RRM1

7 and RRM2 expression with overall survival was measured using the Kaplan-Meier

8 method, with Cox proportional hazard regression analysis for group comparison. A

9 value of p < 0.05 was considered significant.

12 Results

13 RRM1 and RRM2 are highly expressed in MM cells.

14 We first investigated the expression of RRM1 and RRM2 in primary MM cells. Our

15 evaluation of RRM1 and RRM2 messenger RNA (mRNA) expression in 3 independent

16 publicly available data sets [13-15] revealed that RRM1 transcript levels are

17 significantly higher in MM than in healthy donor in all data sets, and in monoclonal

18 gammopathy of undetermined significance (MGUS) in 2 out of 3 data sets (Figure 1A-C,

19 upper panels); and that RRM2 transcript levels are also significantly higher in 2 out of 3

20 data sets (Figure 1A-C, lower panels). These results are consistent with previous studies

21 in other cancers (Supplementary Figure S1). We also evaluated another two publicly

22 available data sets of 170 [16] and 350 [17] newly diagnosed patients and found that

1 patients with higher expressions of RRM1 and RRM2 had significantly shorter overall

2 survival (Figure 1D and Supplementary figure S2). We also examined RRM1 and

3 RRM2 protein expression in MM cells. We found that both RRM1 and RRM2 were

4 detected in six human MM cell lines and three patient MM cells (Figure 1E).

6 RRM1 are required for MM cell survival

7 To evaluate the biologic function of RRM1 and RRM2, we transduced MM cells with

8 short interfering RNA (siRNA) targeting RRM1, RRM2, or control (scramble) by

9 electroporation. Transduction of RRM1- and RRM2-specific siRNA markedly reduced

10 the respective protein expression in 4 cell lines (p53 wild-type; NCI-H929 and MM.1S,

11 p53 mutant; RPMI8226, p53 null; KMS11) examined (Figure 2A). Importantly,

12 knockdown of RRM1 or RRM2 significantly inhibited MM cell line growth (Figure 2A).

13 Of note, RRM2 knockdown did not enhance cell growth inhibition induced by RRM1

14 knockdown. Along with cell growth inhibition, apoptotic cell death was significantly

15 increased by RRM1 or RRM2 knockdown in NCI-H929 MM cells (Figure 2B).

16 Apoptotic cell death was further confirmed by immunoblots showing cleavages of

17 caspase-3, -8 and -9, and poly (ADP-ribose) polymerase (PARP) in NCI-H929 cells

18 (Figure 2C). Consistent with AnnexinV-PI staining, apoptotic cell death triggered by

19 RRM1 or RRM2 knockdown was modest in RPMI8226 cells (Figure 2C). We also

20 performed cell cycle analysis and found that cells in S-phase were increased when

21 RRM1 and RRM2 were knockdowned. As previously reported [24], this result suggests

22 RRM1-, RRM2-knockdown triggered S-phase arrest (Figure 2D).

1 As seen in Figure 2A, RRM1 knockdown induced upregulation of RRM2, while

2 RRM2 knockdown did not induce upregulation of RRM1. These results suggested that,

3 although precise molecular mechanism has not yet been elucidated, RRM2 could

4 compensate RRM1 knockdown effect, although growth inhibitory assay showed RRM2

5 upregulation could not compensate the RRM1-knockdown effect. Therefore, we further

6 induced RRM2 expression to NCI-H929 and RPMI8226 cells by using retroviral

7 expression vector, and then performed RRM1-knockdown. As shown in Figure 2E and

8 Supplementary Figure S3, RRM2 overexpression could not rescue the growth inhibitory

9 effect of RRM1-knockdown, suggesting that RRM1, but not RRM2, is a survival factor

10 and potential therapeutic target in MM.

11 The bone marrow (BM) microenvironment plays a crucial role in MM

12 pathogenesis by promoting tumor cell proliferation, survival and drug resistance [1]. To

13 examine whether the BM microenvironment protects against the effects of RRM1 or

14 RRM2 knockdown, we next co-cultured siRNA-transfected-NCI-H929 and -RPMI8226

15 cells in the presence or absence of BMSC. We observed that the effects of knockdown

16 of both RRM1 and RRM2 were not attenuated even in the presence of BMSC (Figure

17 2F). These data suggest that the BM microenvironment cannot overcome RRM1 or

18 RRM2 knockdown-mediated MM cell growth inhibition.

19 To demonstrate the in vivo efficacy of RRM1 downregulation, RRM1 knockdown

20 MM.1S cells were implanted in mice. As shown in Figure 2G, cell growth was

21 significantly reduced in RRM1 knockdown cells compared with control cells.

1 DNA damage response and p53 pathways are required for RRM1

2 knockdown-induced MM cell death

3 RR is involved in rate-limiting deoxynucleotide (dNTP) generation and functions to

4 maintain centrosome integrity, as well as provide dNTPs during replication or DNA

5 damage repair [24, 25]. Therefore, RRM1 knockdown may impact DNA damage

6 response and/or repair genes. Indeed, immunoblots showed that RRM1 knockdown

7 triggered DNA damage response in MM cells, including γ-H2A.X, phosphorylated

8 (p)-ATM, and p-ATR, as well as their downstream effectors p-Chk1, and p-Chk2

9 (Figure 3A). We next examined downstream target genes RAD51, 53BP1, BRCA1, and

10 BRCA2. As shown in Figure 3B, quantitative real-time PCR (QRT-PCR) analysis

11 showed that RRM1 knockdown induced these genes in both NCI-H929 and RPMI8226

12 cells. Consistent with QRT-PCR, immunoblots showed that RRM1 knockdown also

13 induced increased RAD51, 53BP1, BRCA1 and BRCA2 protein levels (Figure 3C).

14 To identify novel downstream targets of RRM1 (and RRM2) which mediate MM

15 cell growth, we next performed gene expression profiling before and after RRM1 or

16 RRM2 knockdown in NCI-H929 cells. RRM1-knockdown upregulated 665 genes.

17 Including p53 pathway genes CDKN1A (p21 WAF1), PMAIP1 (Noxa), BBC3 (Puma),

18 SESN1, DDB2, and DRAM1 as long as BRCA1 (Figure 4A and 4B). Of note, MM cells

19 with wild-type p53 showed more significant growth inhibition by RRM1 knockdown

20 than in cells with mutant p53 (Figure 2A).

21 We next used ELISA and immunoblots to examine activation of p53 pathway by

22 RRM1 or RRM2 knockdown in NCI-H929 cells. ELISA showed that p53 was activated

1 by both RRM1 and RRM2 knockdown (Figure 4C). Immunoblot also showed that p53

2 pathway is activated, evidenced by induction of p53 phosphorylation at Ser15, as well

3 as upregulation of p21WAF1, Noxa, and PUMA (Figure 4D). Importantly, p53

4 knockdown partially abrogated the effect of RRM1 knockdown (Figure 4E), further

5 validating p53 as a key molecule in RRM1 knockdown-induced MM cell growth

6 inhibition.

7 Therefore, we speculated that in p53 wild-type cells RRM1 knockdown effect

8 derived upon DNA damage response followed by p53 pathway, while in p53

9 mutant/null cells, alternative pathway, such as BRCA1/2 pathway, might be critical.

11 RRM1 inhibitors trigger growth inhibition in p53 wild-type MM cells

12 To assess the potential clinical relevance of RRM1 inhibition in MM, we next examined

13 the effect of the purine nucleoside antimetabolite CLO, an RRM1 inhibitor that is

14 approved for the treatment of acute lymphocytic leukemia [26-30], on MM cell lines

15 (NCI-H929, MM.1S, MOLP-8, RPMI8226, OPM2, U266, and KMS-11). TP53-wild

16 type cells (NCI-H929, MM.1S, and MOLP-8) were more sensitive to CLO treatment

17 compared to TP53-mutant (RPMI8226, OPM2, and U266) or TP53-null (KMS-11) cells

18 (Figure 5A). To elucidate the molecular mechanism of MM cell death triggered by CLO,

19 we carried out immunoblots and observed time-dependent cleavage of caspase-3, -8 and

20 -9, and PARP (Figure 5B). Similar to RRM1 knockdown, CLO treatment upregulated

21 p53 and its downstream target proteins in NCI-H929 cells, without significant alteration

22 of RRM1 or RRM2 protein expression (Figure 5C). DNA damage response pathway

1 proteins including γ-H2A.X, p-ATM, and effectors p-Chk1, and p-Chk2 were also

2 upregulated by CLO treatment in a time-dependent fashion (Figure 5D).

4 RRM1 inhibitor with melphalan (MEL) induces synergistic MM cytotoxicity

5 Since CLO enhanced DNA damage response pathway, we next combined CLO with

6 DNA damaging agent MEL to assess for enhanced anti-MM activity. CLO in

7 combination with MEL triggered synergistic cytotoxicity not only in NCI-H929 and but

8 also in RPMI8226 cells (Figure 6A). Consistent with cytotoxicity, CLO with MEL also

9 markedly upregulated AnnexinV-positive cells, and cleavage of caspase-3, -8, -9, and

10 PARP in both cells (Figure 6B-C), suggesting that the enhanced combination

11 treatment-induced cytotoxicity was due to apoptotic cell death. Furthermore, γ-H2A.X,

12 biomarker of DNA double-strand break and DNA damage [31], was activated upon

13 combination treatment (Figure 6D). Since CLO may have off-target effects, we carried

14 out combination treatment of MEL with RRM1 knockdown, and confirmed that MEL

15 enhanced RRM1-knockdown-induced cytotoxicity (Figure 6E), associated with

16 enhanced activation of DNA damage response pathway (Figure 6F). These data indicate

17 that RRM1 inhibition by either knockdown or CLO in combination with MEL triggers

18 synergistic MM cytotoxicity.

21 Discussion

22 As in many other cancers, RR is highly expressed in MM cells. More specifically, we

1 here show that both RRM1 (large subunit) and RRM2 (small subunit) are highly

2 expressed in MM cells, but not in normal cells. Importantly, we demonstrate that RRM1

3 knockdown triggers significant MM cell growth inhibition and apoptosis, while RRM2

4 knockdown shows modest growth inhibitory effects. These data suggest that RRM1, but

5 not RRM2, is a survival factor and potential therapeutic target in MM.

6 Maintenance of genomic stability depends on an appropriate response to

7 DNA damage, and the protein kinases ATM and ATR are the master controllers of such

8 DNA damage pathway responses [32, 33]. We have previously reported that pervasive

9 constitutive and ongoing DNA damage is present in hematological malignancies

10 including MM [34], and others have reported that RRM1 maintains centrosomal

11 integrity during replication stress [24]. Importantly, in this study our gene expression

12 data and QRT-PCR results showed that RRM1-knockdown upregulated DNA damage

13 response genes including RAD51, and 53BP1. Therefore, downregulation of RRM1

14 could inhibit the ability of MM cells to survive in ongoing DNA damage, leading to

15 apoptotic cell death. We have previously reported that YAP1 knockdown can trigger

16 p73-mediated apoptosis in a subset of MM with ongoing DNA damage; however,

17 RRM1-knockdown did not alter YAP1 (Supplementary Figure S4), indicating an

18 alternative mechanism of action triggered by RRM1 inhibition.

19 Interestingly, we showed that BRCA1 and BRCA2 were also upregulated in

20 MM cells by RRM1 knockdown irrespective of p53 status. Harkins et al. reported that

21 inducible expression of BRCA1 leads to apoptotic cell death in osteosarcoma and breast

22 cancer cells [35]. Conversely, Rao et al. reported that selective reduction of BRCA1

1 mRNA levels using antisense RNA induces more rapid cell growth, decreased

2 susceptibility to apoptosis, and cell transformation in NIH3T3 fibroblasts [36]. Taken

3 together, our results suggest that upregulation of BRCA1 mRNA and protein level may

4 account, at least in part, for RRM1 knockdown/inhibition-induced apoptotic MM cell

5 death, putative alternative mechanisms.

6 To assess clinical relevance of RRM1 inhibition in MM, we showed that the

7 purine analog CLO, known to inhibit RRM1 [26, 27], also induces MM cell growth

8 inhibition. Similar to RRM1 knockdown, CLO treatment also induced DNA damage

9 response proteins; γ-H2A.X, phosphorylated (p)-ATM, and p-ATR, followed by its

10 downstream effectors; p-Chk1, and p-Chk2. Interestingly, CLO induced-apoptosis is

11 more potent in MM cells with wild-type TP53 compared to cells with mutant-p53 or

12 null-p53. We also showed that RRM1-induced apoptotic MM cell death was more

13 evident in p53 wild-type cells than p53 mutant cells. Similar results were reported by

14 Valdez et al. [37]. Upon DNA damage, p53 is stabilized, upregulated, and

15 phosphorylated at Ser15, cell cycle arrest, leading to its anti-proliferative activity, and

16 apoptosis [38]. Our results further demonstrated that both RRM1-knockdown and CLO

17 treatment in NCI-H929 cells with p53 wild-type upregulate/activate p53 pathway

18 proteins including activation of p-p53 (Ser 15), stabilization of p53, and upregulation of

19 p21, Noxa, and Puma. These results suggest that p53 pathways play a critical role

20 mediating RRM1-induced MM cell death. The prevalence of p53 mutation in newly

21 diagnosed MM is quite low (ranging from 0-20%), and is an independent poor

22 prognostic factor [39], whereas higher percentage of patients with p53 abnormalities

1 (p53 mutation and p53 deletion) are noted in more advanced disease including relapsed

2 refractory MM (RRMM) and plasma cell leukemia [40]. Therefore,

3 RRM1-knockdown/CLO treatment, as a single therapeutic strategy, might be difficult to

4 utilize in RRMM patients, and combination treatment strategy is warranted.

5 Finally, MEL is a member of the nitrogen mustard class of chemotherapeutic

6 agents which alkylates DNA. It triggers formation of DNA adducts, and forms

7 crosslinks. The formation of crosslinks between the two strands of DNA, interstrand

8 crosslinking, is a critical event which correlates with in vitro cytotoxicity [41]. A

9 previous in vitro report has combined CLO with MEL and described synergistic effects

10 [37], without elucidating its mechanism. Importantly, we here found that the synergistic

11 effects triggered by combining CLO with MEL are evident not only in wild-type p53

12 cells, but also in mutant p53 cells; and importantly, are associated with induction of

13 γ-H2A.X. Furthermore, we found that BRCA1 and BRCA2 was upregulated upon

14 RRM1 knockdown in p53 wild-type cells as well as p53 mutant (and null) cells. These

15 results suggest that MEL can enhance anti-MM activity of RRM1 inhibition-induced

16 MM cytotoxicity regardless of p53 status, and BRCA1/2 pathway could be the possible

17 alternative pathway for the enhancement of this combination treatment. Since CLO is

18 being used as a tool compound in preclinical setting because of its unfavorable toxicities,

19 combination treatment of CLO with MEL may not be suitable for clinical settings.

20 Therefore, development of novel RRM1 inhibitor with less myelotoxicity is needed.

21 In conclusion, we have here elucidated a novel role of RRM1 in MM regulating

22 DNA damage response and p53 pathway. Our studies provide the preclinical rationale

1 for targeting RRM1 to enhance sensitivity of tumor cells to MEL and thereby improve

2 patient outcome in MM.

5 Acknowledgments

6 This research was supported by NIH grants SPORE P50-100707 (KCA), R01-CA

7 050947 (KCA), and R01-CA178264 (TH and KCA). K.C.A. is an American Cancer

8 Society Clinical Research Professor.

1 References

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1 Figure Legends

2 Figure 1. RRM1 and RRM2 expression in MM cells.

3 (A-C) RRM1 (upper panels) and RRM2 (lower panels) mRNA expression in MM

4 patient samples. Three independent data sets (A; GSE6477, B; GSE5900, and C;

5 GSE13591) were analyzed for RRM1 and RRM2 expression in normal donors,

6 monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple

7 myeloma (MM), newly diagnosed MM, relapsed MM, and plasma cell leukemia (PCL).

8 *P<0.05, **P<0.01, ***P<0.001. NS, not significant; analysis of variance (ANOVA)

9 followed by Dunnett’s test. (D) Survival analysis in newly diagnosed MM patients

10 related to RRM1 and RRM2 expression (GSE39754). Red line indicates upper 1/3 of

11 each gene expression, while blue line indicates lower 2/3 of each gene expression. (E)

12 Immunoblot analysis of RRM1 and RRM2 in 6 MM cell lines, 3 MM patient samples

13 (CD138 positive cells from bone marrow), and 3 normal donor PBMC samples.

15 Figure 2. In vitro and in vivo effects of RRM1 and RRM2 knockdown in MM cells.

16 (A) RRM1- and RRM2-specific siRNA were used to knockdown respective genes in

17 MM cell lines. Growth inhibition of the cells was measured by MTT assay. The growth

18 of all 4 MM cell lines was significantly reduced at 72 and/or 96 h, especially in siRRM1

19 cells. Blue bar: 48 h, Orange bar: 72 h and gray bar: 96 h. **P<0.01 compared with

20 scramble (control) at the same time period; Student’s t-test. Immunoblots confirmed

21 RRM1 and RRM2 knockdown. Whole-cell lysates were subjected to immunoblot

22 analysis, and GAPDH served as the loading control for each membrane. (B) RRM1 and

1 RRM2 were knockdowned in NCI-H929 and RPMI8226 cells with RRM1- and

2 RRM2-specific siRNA, and the number of apoptotic cells were examined at 72 h. While

3 significant apoptosis was triggered by siRNA knockdown in NCI-H929 cells, whereas

4 only mild apoptosis was observed in RPMI8226 cells **P<0.01 compared with

5 scramble; Student’s t-test. (C) Immunoblot analysis of apoptosis-related proteins in

6 RRM1- and RRM2-knockdown NCI-H929 and RPMI8226 cells. Whole-cell lysates

7 were subjected to immunoblot analysis, and GAPDH served as the loading control for

8 each membrane. (D) RRM1 and RRM2 were knockdowned in NCI-H929 cells with

9 RRM1- and RRM2-specific siRNA, and the cell cycle analysis was performed at 48 h.

10 Increase in the number of cells in S-phase was seen in siRNA knockdown cells. (E)

11 NCI-H929 cells were induced with either control or pMSCV-RRM2 plasmid, and then

12 knockdowned with scramble or RRM1-targeted siRNA. Growth inhibition of the cells

13 was measured by MTT assay. **P<0.01 compared with scramble (control) at the same

14 time period; Student’s t-test. Immunoblots confirmed RRM1 knockdown and RRM2

15 overexpression. GAPDH served as the loading control for each membrane. (F) RRM1

16 and RRM2 were knockdowned in NCI-H929 and RPMI8226 cells with RRM1- and

17 RRM2-specific siRNA, and co-cultured in the presence or absence patients’ bone

18 marrow stroma cell (BMSC) for 72 h. Data indicate that the BM microenvironment

19 could not abrogate the knockdown effect of RRM1 and RRM2. **P<0.01 compared

20 with scramble; Student’s t-test. (G) MM cells transduced with siRRM1 or scramble

21 (3x106 viable cells) were subcutaneously injected into 200 cGy irradiated SCID mice.

22 Data represent mean ±s.e.m. N=5 mice per group. An image of tumors in each group is

1 shown (top panel). *P=0.0159; Student’s t-test. Data are representative of at least two

2 independent experiments except for xenograft experiment.

4 Figure 3. DNA damage response pathway plays essential role in RRM1-knockdown

5 MM cells.

6 (A) Immunoblot analysis of DNA damage response pathway genes in RRM1- and

7 RRM2-knockdown NCI-H929 and RPMI8226 cells. GAPDH served as the loading

8 control for each membrane. (B) Quantitative real-time PCR (QRT-PCR) analysis of

9 RRM1, RRM2, RAD51, 53BP1, BRCA1, and BRCA2 in NCI-H929 and RPMI8226 cells

10 transduced with either siRNA targeting RRM1, RRM2 or scramble (control). Shown are

11 relative signal intensity (scramble=1) normalized by GAPDH. **P<0.01 compared with

12 scramble; Student’s t-test. (C) Immunoblot analysis of RAD51, 53BP1, BRCA1, and

13 BRCA2 in NCI-H929 and RPMI8226 cells transduced with siRNA targeting RRM1,

14 RRM2 or scramble. GAPDH served as the loading control for each membrane, and data

15 are representative of at least two independent experiments.

17 Figure 4. Transcriptional activity of TP53 pathway is crucial in TP53 wild-type

18 MM cells.

19 (A) Scatter plots depicting the relative gene expression in NCI-H929 cells treated with

20 siRRM1, siRRM2, or scramble. Genes related to TP53 and BRCA1 (plotted in red)

21 were eluted together with >1.5 fold change. (B) Heatmap showed induction of

22 TP53-related genes in RRM1- RRM2-knockdown cells compared to scramble. Yellow

1 denoted higher expression, while blue denoted lower expression. (C) Transcription

2 activity levels of TP53 in TP53 wild-type NCI-H929 cells. Fold changes relative to

3 scramble are shown. (D) Immunoblot analysis of TP53 and its related proteins in

4 whole-cell lysates from RRM1- and RRM2-knockdown NCI-H929 cells (E) NCI-H929

5 cells were treated with siRRM1,sip53, or both; left panel shows survival of cells 72 h

6 after knockdown. Right panel shows confirmation of knockdown, and GAPDH served

7 as the loading control for each membrane. Data are representative of at least two

8 independent experiments in Figure 4C-4E. **P<0.01; Student’s t-test.

10 Figure 5. RRM1 inhibitor induces apoptosis in MM cells.

11 (A) Seven MM cell lines (NCI-H929, MM.1S, MOLP8, RPMI8226, OPM2, U266, and

12 KMS-11 cells) were treated with CLO (0-30 μM) for 48 h, and growth was then

13 measured by MTT assay. (B-D) Immunoblot analysis of cell lysates of NCI-H929 cells

14 treated with CLO (5 μM, 3-48 h) GAPDH served as the loading control for each

15 membrane, and data are representative of at least two independent experiments.

17 Figure 6. RRM1 inhibition combined with DNA damaging agent CLO have

18 synergistic effect on MM cells.

19 (A) (left panels) NCI-H929 and RPMI8226 cells were treated with the combination of

20 CLO and MEL for 48 h at the indicated doses, and tumor growth reduction was

21 measured by MTT assay. (right panels) Combination Index (CI) was calculated in each

22 combination therapy. CI under 1 is recognized as synergy. (B) NCI-H929 and

1 RPMI8226 cells were treated with CLO (NCI-H929; 3 μM, RPMI8226; 10 μM) and

2 MEL (20 μM) for 48 h, and the number of apoptotic cells were examined. Combination

3 treatment indicates higher percentage of apoptotic cells. (C,D) Immunoblot analysis of

4 cell lysates after combination treatment with CLO (NCI-H929; 3 μM, RPMI8226; 10

5 μM) and MEL (20 μM) for 48 h. (E) NCI-H929 and RPMI8226 cells were treated with

6 combination of siRNA treatment (siRRM1 or scramble) and MEL for 72 h at the

7 indicated doses, and tumor growth was measured by MTT assay. **P<0.01. NS, not

8 significant; Student’s t-test. (F) Immunoblot analysis of cell lysates after combination

9 siRNA treatment (siRRM1 or scramble) and MEL at the indicated doses and time (same

10 condition as Figure 6E). GAPDH served as the loading control for each membrane, and

11 data are representative of at least two independent experiments.

Figure 1 *** A *** B C ** ** NS *** NS ) *** NS ) * ) *** 2

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

MM PCL MGUS MGUS MGUS New MM ering MM rmal donor Normal donor Relapsed MM Normal dono old No Smoldering MM Sm

*** ** NS NS ** ) NS NS 2 )

) NS *** 14 2 14 2 14 *** NS NS ** ** NS 12 12 12 10 10 10 8 8 8 6 RRM2 expression (Log expression RRM2 RRM2 expression (Log expression RRM2

RRM2 expression (Log expression RRM2 6 4 r US MM PCL MGUS MGUS MG New MM Normal donor Normal donor Relapsed MM Normal dono Smoldering MM Smoldering MM

D RRM1 (OS HR=1.65 cox p value=0.0042) RRM2 (OS HR=1.41 cox p value=0.0015) Survival Survival

High 1/3 High 1/3 Low 2/3 Low 2/3 p-value= 0.039

0.0 0.2 0.4 0.6 0.8 1.0 p-value= 0.021 0.0 0.2 0.4 0.6 0.8 1.0 0 20 40 60 80 0 20 40 60 80 Month Month

#1 H929 .1S MC #3 PMI8226 M.1S NCI- MM.1S R U266 OPM2 KMS-11 Pt #1 Pt #2 Pt #3 MM PBMC PBMC #2PB M RRM1

RRM2

GAPDH

A NCI-H929 MM.1S 120% 120% 100% Author Manuscript Published OnlineFirst100% on April 25, 2017; DOI: 10.1158/1078-0432.CCR-17-0263 80% Author manuscripts have been peer 80%reviewed and** accepted for publication but have not yet been edited. ** **** 60% ** ** scramblesiRRM1 siRRM2 siRRM1+siRRM2 60% scramblesiRRM1 siRRM2 siRRM1+siRRM2 48h ** ** 48h 40% ** ** RRM1 40% RRM1 72h ** ** 72h MTT (% control) (%control) MTT 96h control) (% MTT 20% 96h RRM2 20% RRM2 0% 0%

GAPDH GAPDH

RRM2 siRRM1 siRRM2 scramble siRRM1 siRRM2 scramble

siRRM1+siRRM2 siRRM1+si KMS-11 120% RPMI8226 120% 100% 100% 80% ** 80% ** ** ** ** scramblesiRRM1 siRRM2 siRRM1+siRRM2 ** ** scramblesiRRM1 siRRM2 siRRM1+siRRM2 60% 60% ** ** ** RRM1 48h RRM1 40% ** 40% 72h 48h MTT (% control) (%control) MTT MTT (% control) (%control) MTT 96h RRM2 20% 72h RRM2 20% 96h 0% 0% GAPDH GAPDH

scramble siRRM1 siRRM2 scramble siRRM1 siRRM2

siRRM1+siRRM2 siRRM1+siRRM2 B C

scramble siRRM1 siRRM2 NCI-H929 RPMI8226 scramble siRRM1 siRRM2 scramblesiRRM1 siRRM2 13.4% 38.5% 19.3% 48 72 4872 48 72 48 724872 48 72 (h) NCI-H929 RRM1 9.4% 18.8% 17.3% RRM2 PI 15.9% 16.8% 12.4% caspase-9 RPMI8226

4.4% 5.8% 5.9% caspase-8

Annexin V caspase-3

100% ** PARP 80% ** GAPDH 60%

40% early apoptosis 20%

Apoptosis (%) Apoptosis late apoptosis 0% siRRM1 siRRM2 siRRM1 siRRM2 Scramble Scramble

NCI-H929 RPMI8226

D scramble siRRM1 siRRM2 G1 S G2-M 120%

100%

80%

60%

% of cells of % 40%

20%

siRRM1 siRRM2 Scramble

Figure 2 (continued)

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

80% RRM1 60% RRM2 40% MTT (% control) (%control) MTT 20% GAPDH

0% - - ++ RRM2 cDNA + - -+ scramble - - ++ RRM2 cDNA siRRM1 + - -+ scramble - + - + - + - + siRRM1

F NCI-H929 RPMI8226

200% ** ** 200% ** ** ** ** ** ** 150% 150%

100% 100%

MTT (% control) (%control) MTT 50% control) (% MTT 50%

0% 0% siRRM1 - + - - + - siRRM1 - + - - + - siRRM2 - - + - - + siRRM2 - - + - - + BMSC BMSC

(7 weeks)

2000 Scramble siRRM1

1500 Scramble siRRM1 1000

500

tumor volume (mm3) volume tumor *

0 1 2 3 4 5 6 7 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2017 American Association for Cancer weeks Research. Author Manuscript Published OnlineFirst on April 25, 2017; DOI: 10.1158/1078-0432.CCR-17-0263 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 3

A B scramble siRRM1 siRRM2 NCI-H929 RPMI8226 RRM1 RRM2 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 relativemRNA relative mRNA level relativemRNA ** ** ** ** 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 relative mRNA level relativemRNA Chk1 level relativemRNA 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 level relativemRNA relative mRNA level relativemRNA

0 0 C H929 RPMI8226 H929 RPMI8226

NCI-H929 RPMI8226

1 1 M M2 R scramble siRR siR scramblesiRRM siRRM2

RAD51

53BP1

BRCA1

BRCA2

Downloaded fromGAPDH clincancerres.aacrjournals.org on October 2, 2021. © 2017 American Association for Cancer Research. Figure 4 Author Manuscript Published OnlineFirst on April 25, 2017; DOI: 10.1158/1078-0432.CCR-17-0263 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 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 6 LRMP NCI-H929_siRRM2 PMAIP1

2 2 6 10 14 -2 0 2 NCI-H929_Scramble

C D

NCI-H929 ** 2 **

scramble siRRM1 siRRM2 1.5 RRM1

1 RRM2

0.5 phospho-p53 (Ser15) expression (fold change) change) expression(fold 0 Scramble siRRM1 siRRM2 p53 NCI-H929 p21

E Noxa ** 120% PUMA

100% GAPDH 80%

60%

40% MTT assayMTT (%control)

20%

0% - + - + siRRM1 - - + + sip53 NCI-H929

RRM1

p53

GAPDH

- + - + siRRM1 - - +Downloaded+ sip53 from clincancerres.aacrjournals.org on October 2, 2021. © 2017 American Association for Cancer NCI-H929 Research. Author Manuscript Published OnlineFirst on April 25, 2017; DOI: 10.1158/1078-0432.CCR-17-0263 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 5 A 120%

100% H929 80% MM1S MOLP8 60% RPMI8226

40% KMS11

MTT (% of control) control) of (% MTT OPM2 20% U266

0% 0 0.3 1 3 10 30 (μM)

B C NCI-H929 NCI-H929 control CLO 5 μM control CLO 5 μM 3 8 24 48 3 8 24 48 (h) 3 8 24 48 3 8 24 48 (h)

caspase-9 RRM1

RRM2 caspase-8 phospho-p53 (ser15)

caspase-3 p53

p21 PARP

Noxa GAPDH

Puma

GAPDH

D NCI-H929 control CLO 5 μM 3 8 24 48 3 8 24 48 (h) γ-H2A.X

phospho-ATM (Ser1981)

ATM

phospho-ATR (Ser428)

ATR

phospho-Chk1 (Ser317)

Chk1

phospho-Chk2 (Thr68)

Chk2

Downloaded from clincancerres.aacrjournals.orgGAPDH on October 2, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 25, 2017; DOI: 10.1158/1078-0432.CCR-17-0263 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 6

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

0% 0 0uM 5uM 10uM 20uM CLO 0 0.2 0.4 0.6 0.8 1 Fa 120% RPMI8226

100%

80% 2

60% 1.5 1 40% CI

MTT (% control) control) (% MTT 0.5 20% 0 0 0.2 0.4 0.6 0.8 1 0% Fa 0uM 5uM 10uM 20uM CLO

control CLO MEL CLO+MEL

0.7% 1.5% 1.1% 14.4%

NCI-H929

1.1% 1.0% 0.8% 3.9%

1.5% 1.3% 1.0% 6.0%

RPMI8226

0.9% 1.5% 0.9% 4.5%

Annexin V

C D

NCI-H929 RPMI8226 NCI-H929 RPMI8226 CLO - + - + - + - + CLO - + - + - + - + MEL - - + + - - + + MEL - - + + - - + + γ-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 Downloaded fromG clincancerres.aacrjournals.orgAPDH on October 2, 2021. © 2017 American Association for Cancer Research. GAPDH Author Manuscript Published OnlineFirst on April 25, 2017; DOI: 10.1158/1078-0432.CCR-17-0263 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 6 (continued)

E F NCI-H929 NCI-H929 RPMI8226 ** **** scramble siRRM1 scramble siRRM1 120% N**S ** MEL 0 10 20 0 10 20 0 10 20 0 10 20 (uM) ** MEL 100% RRM1 0uM 80% 10uM γ-H2A.X 60% 20uM

40% phospho-ATM (Ser1981)

MTT (% control) control) (% MTT 20% ATM

0% phospho-ATR (Ser428) Scramble siRRM1

ATR

RPMI8226 phospho-Chk1 (Ser317) **** Chk1 120% ** ** ** NS ** phospho-Chk2 (Thr68) 100% 80% Chk2 60% GAPDH 40%

MTT (% control) control) (% MTT 20% 0% Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2017 American Association for Cancer scramble siRRM1 Research. Author Manuscript Published OnlineFirst on April 25, 2017; DOI: 10.1158/1078-0432.CCR-17-0263 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Ribonucleotide reductase large subunit (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

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2017/04/25/1078-0432.CCR-17-0263.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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