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

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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 5 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. 16 17 Running title: Targeting RRM1 as a novel treatment for multiple myeloma 18 Key words: Multiple Myeloma, RRM1, DNA damage response, p53, Clofarabine 19 20 Financial Support: NIH grants; SPORE P50-100707 (KCA), R01-CA050947 (KCA), 21 and R01-CA178264 (TH and KCA). 22 1 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. 7 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 11 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. 2 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 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 3 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 inhibitor, alone or in combination with DNA damaging agents, to improve patient 2 outcome in MM. 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 4 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 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 5 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 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. 13 14 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 6 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
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