Oncogene (2015) 34, 2011–2021 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 www.nature.com/onc

REVIEW and cancer: biological mechanisms and targeted therapies

YAye1,2,MLi3, MJC Long4 and RS Weiss3

Accurate DNA replication and repair is essential for proper development, growth and tumor-free survival in all multicellular organisms. A key requirement for the maintenance of genomic integrity is the availability of adequate and balanced pools of deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA. Notably, dNTP pool alterations lead to genomic instability and have been linked to multiple human diseases, including mitochondrial disorders, susceptibility to viral infection and cancer. In this review, we discuss how a key regulator of dNTP biosynthesis in mammals, the ribonucleotide reductase (RNR), impacts cancer susceptibility and serves as a target for anti-cancer therapies. Because RNR-regulated dNTP production can influence DNA replication fidelity while also supporting genome-protecting DNA repair, RNR has complex and stage-specific roles in carcinogenesis. Nevertheless, cancer cells are dependent on RNR for de novo dNTP biosynthesis. Therefore, elevated RNR expression is a characteristic of many cancers, and an array of mechanistically distinct RNR inhibitors serve as effective agents for cancer treatment. The dNTP metabolism machinery, including RNR, has been exploited for therapeutic benefit for decades and remains an important target for cancer drug development.

Oncogene (2015) 34, 2011–2021; doi:10.1038/onc.2014.155; published online 9 June 2014

DEOXYRIBONUCLEOSIDE TRIPHOSPHATE (dNTP) POOLS AND RNR AND dNTP BIOSYNTHESIS GENOMIC INTEGRITY dNTPs can be generated through de novo and salvage pathways. In The importance of ribonucleotide reductase (RNR) for genome mammals, RNR catalyzes the rate-limiting step of the de novo maintenance relates to its central role in regulating dNTP levels. In pathway, reducing the 2′ carbon of a ribonucleoside diphosphate mammalian cells, total dNTP pool sizes peak during S-phase to (NDP) to produce the corresponding deoxy (d)NDP. Subsequently, support nuclear DNA (nDNA) replication and are roughly 10-fold dNDPs are phosphorylated by nucleoside diphosphate kinase 16 lower in G0/G1, when dNTPs are needed for DNA repair and (NDPK) yielding dNTPs. Importantly, the de novo biosynthesis of mitochondrial DNA (mtDNA) synthesis.1,2 As DNA polymerase deoxythymidine triphosphate (dTTP) is much more complex, substrates, dNTPs influence several aspects of the replication requiring the conversion of deoxyuridine monophosphate to program, including origin choice, fork speed, inter-origin distance, deoxythymidine monophosphate (dTMP) by thymidylate synthase and dormant origin usage.3–5 Failure of cells to maintain followed by phosphorylation by thymidylate phosphate kinase appropriate dNTP concentrations can be highly detrimental, (TMPK) and then NDPK. Deoxyuridine monophosphate is generated leading to DNA breaks, mutagenesis and cell death.6 During either from deoxyuridine triphosphate (dUTP) or deoxycytidine cancer development, uncoordinated cell proliferation can lead to monophosphate, the biosynthesis of both of which is dependent insufficient dNTPs that cause replication stress and further on RNR-catalyzed reduction of the corresponding nucleoside promote genomic instability.7,8 Conversely, elevated dNTP pools diphosphates.6 RNR are present in all organisms and also contribute to increased mutagenesis.1,9,10 feature-conserved radical-mediated nucleotide reduction.17 Mam- Imbalanced dNTP pools enhance mutagenesis mainly by DNA malian RNRs, the focus of this review, consist of two subunits α and misinsertion and impaired proofreading.6,9 DNA misinsertion can β, that associate to form the holoenzyme (Figure 1). α contains the result from competition between dNTPs for pairing with the catalytic (C) site and two different allosteric sites. β harbors a di-iron template base, and a dNTP present in excess can be readily and tyrosyl radical (Y•) essential for RNR activity. Evidence misincorporated. DNA polymerase proofreading is reduced in suggests that catalytically active RNR is minimally an α2β2 the presence of elevated dNTP concentrations through a complex.18–20 RNR oligomerization is influenced by (d)NTP/ATP phenomenon known as the next-nucleotide effect.11 Under such binding to α allosteric sites. The negative regulator dATP induces conditions, DNA chain extension following a base misinsertion inactive α6 (or α6β2) states, highlighting a role for higher-order proceeds before the mismatched nucleotide can be removed.12–14 oligomeric associations as a regulatory mechanism for RNR.18,21–24 dNTP imbalances can also stimulate frameshift mutations by However, as discussed further below, the precise nature of RNR facilitating correct base-pairing following template slippage or oligomeric states in the presence of allosteric activators remains misalignment.15 unsettled (Supplementary Table S1).

1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA; 2Department of Biochemistry, Weill Cornell Medical College, New York, NY, USA; 3Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA and 4Graduate Program in Biochemistry, Brandeis University, Waltham, MA, USA. Correspondence: Assistant Professor Y Aye, 224 Baker Laboratory of Chemistry & Chemical Biology, Ithaca, NY 14853, USA or Associate Professor RS Weiss, Department of Biomedical Sciences, Cornell University, Veterinary Research Tower, Ithaca, NY 14853, USA. E-mail: [email protected] or [email protected] Received 6 January 2014; revised 25 April 2014; accepted 26 April 2014; published online 9 June 2014 Ribonucleotide reductase and cancer Y Aye et al 2012 repair.30–33 dNTPs for mtDNA synthesis can also come from salvage pathways localized within mitochondria,34,35 and RNR activity has been identified within mitochondria as well.36 In humans, p53R2 mutations result in mitochondrial disease, including mtDNA depletion syndrome, a lethal condition in which patients have only 1–4% residual mtDNA in muscle;30,37 mitochondrial neurogastrointestinal encephalopathy, a neuro- degenerative disorder associated with mtDNA depletion;38 and autosomal-dominant progressive external ophthalmoplegia, characterized by accumulation of multiple mtDNA deletions in postmitotic tissues.39 Furthermore, p53R2 knockout mice show mtDNA depletion and die due to renal failure.30,40 Together, these findings indicate that the de novo dNTP biosynthesis mediated by RNR is essential to maintain not only nuclear but also mitochondrial genome integrity.

RNR ACTIVITY REGULATION The critical importance of dNTP levels demands that RNR is tightly regulated through several distinct mechanisms, including allosteric and oligomeric regulation, as well as alterations of the level and localization of RNR subunits. Allosteric modulation involves effector binding to two separate allosteric sites in α,a specificity (S) site that regulates substrate choice and an activity (A) site that regulates enzyme activity (Figure 1).29 Substrate specificity is determined by the binding of ATP, dATP, dTTP or Figure 1. RNR and de novo dNTP biosynthesis. Mammalian RNR dGTP (deoxyguanosine triphosphate) to the S site; the mechanism enzymes function to reduce the 2' carbon of NDPs to generate 29,41 dNDPs that are subsequently phosphorylated by nucleoside dipho- has been extensively reviewed. Overall RNR activity is sphate kinase, yielding dNTPs for nuclear and mitochondrial DNA controlled by (d)ATP binding to the A site, with elevated dATP: replication and repair. RNR consists of an α subunit encoded by Rrm1 ATP ratios associated with inhibition. α hexamerization is coupled and a β subunit encoded by Rrm2 or p53R2. The subunits interact to to dATP-induced inhibition.21,22,24,41 form hetero-oligomers, with the active enzyme believed to adopt A second major mechanism for RNR regulation occurs through α β α 2 2 quaternary state. contains the catalytic site (C), an activity site the cell cycle stage-specific control of RNR protein levels. In (A) that governs overall RNR activity through interactions with dATP 42–44 fi mammals, RNR activity is induced during S phase. Rrm1 (inhibitory) or ATP (stimulatory) and a speci city site (S) that expression is mainly regulated transcriptionally, being negligible determines substrate choice. Each β subunit contains a μ-oxo- 45 bridged di-nuclear iron center (Fe-O-Fe) and protein tyrosyl radical in G0/G1 and peaking in S phase. RRM1 protein, however, (Y-O•) that is transferred to the α C site for catalysis. The simplified remains constant throughout the cell cycle owing to its long half- 42,46,47 schematic depictions of RNR holoenzyme structures shown here life. RRM2 is regulated both transcriptionally and by protein were guided by structural studies.18,21 degradation. Similar to that of Rrm1, Rrm2 transcription is minimal 48 in G0/G1 and peaks in S phase. Unlike RRM1, RRM2 abundance correlates with its mRNA level.42,44 Upon S phase exit, RRM2 NDP reduction to dNDP takes place in the α C site and requires protein is degraded through two ubiquitin , the Skp1/ the unpaired electron initially localized as Y• in β (Figure 1).19 Cullin/F-box (SCF) complex and the anaphase-promoting complex 27,28 Although the di-iron center is present in each β monomer, a single (APC). During G2 phase, RRM2 is recognized by cyclin F, an SCF Y• is generated per β2.17 Protein radical formation occurs via ubiquitin F-box protein, and targeted for degradation.28 reaction of the diferrous ions with molecular oxygen in a multistep During mitosis/G1 phase, RRM2 is degraded by the Cdh1-APC process.17,19 How diferrous ions are loaded into β2, including complex that recognizes a KEN box motif at the RRM2 potential involvement of iron chaperones, remains unknown. N-terminus.27 By contrast, p53R2, which lacks a KEN box, is In vitro, β2 can exist in three distinct states: apo-β2 lacks both the continuously expressed throughout the cell cycle. After DNA di-iron center and Y•; met-β2 contains the di-iron center but with damage, p53R2 is transcriptionally induced in a p53-dependent Y• reduced; and holo-β2 houses a diferric–Y• cofactor (the manner and with RRM1 forms an active holoenzyme, providing ‘metallocofactor’) and is the only state that, when complexed dNTPs for DNA repair.26,29,33 with α2, is active (Supplementary Figure S1). The β Y• is transferred RNR subcellular localization affords an additional layer of through a proton-coupled electron transfer chain to generate a regulatory control, although this aspect of mammalian RNR transient thiyl radical (S•) within the α C site.19 S• initiates biology remains controversial. The bulk of RRM1 and RRM2 reduction of the NDP ribose ring, ultimately generating dNDP constitutively localize to the cytoplasm and produce dNTPs that and resulting in disulfide bond formation on α. Thioredoxin or diffuse into the nucleus for DNA replication.49,50 However, there is re-reduces the oxidized cysteines on α, facilitating an increasing evidence that RRM1 and RRM2 accumulate at DNA turnover.25 damage sites in the nucleus.28,51–53 This accumulation is In mammals, α is encoded by the gene Rrm1, whereas two dependent on interaction between RRM1 and the DNA damage different , Rrm2 and p53R2, encode distinct β isoforms. response protein Tip60.51,52 p53R2 was identified as a p53-inducible, DNA damage-responsive The work forging a link between RNR translocation and local gene.26 Mouse p53R2 and RRM2 share 81% identity and domain dNTP production at DNA damage sites is appealing. However, a conservation except for amino-terminal residues required for cell cautionary note is required, as dNTP synthesis requires multiple cycle stage-specific RRM2 degradation.27–29 RRM1–RRM2 holoen- enzymes. The reports linking DNA damage to translocation of zyme provides dNTPs for S-phase nDNA replication and repair in dNTP production machinery have focused on only one or two key proliferating cells, whereas RRM1–p53R2 contributes dNTPs for players, typically RNR subunits. It is unknown whether the whole nDNA repair in quiescent cells as well as mtDNA replication and dNTP production pathway responds en masse to DNA damage or

Oncogene (2015) 2011 – 2021 © 2015 Macmillan Publishers Limited Ribonucleotide reductase and cancer Y Aye et al 2013 whether increased dNTP synthesis actually occurs at DNA repair although no dNTP pool alterations could be identified.67 Elevated sites. Evidence does exist that the de novo thymidylate synthesis RNR activity has also been identified in hydroxyurea (HU)-resistant, pathway forms a scaffolded multienzyme complex at replication RRM2-overexpressing mouse cell lines. In one study with sites in the nucleus.54 With respect to RNR, it is possible that fibroblasts, a 3–15-fold increase in RNR activity was accompanied translocation of this rate-limiting enzyme to DNA damage sites is by moderate changes in dNTP pools.69 By contrast, the sufficient to affect local dNTP pools provided all other enzymes HU-resistant, RRM2-overexpressing mouse mammary tumor TA3 required for dNTP synthesis are within the nucleus. An alternative cell line shows a 40-fold increase in enzymatically active RRM2, hypothesis related to DNA damage-induced RNR translocation is estimated based on increased Y• content, but no detectable dNTP that RNR has non-nucleotide reduction properties that promote pool changes relative to parental cells.43,44 Therefore, the elevated DNA repair. Parsing out the mechanisms at play during DNA mutagenesis associated with RNR activity alterations has not been damage responses will require careful analysis and ultimately new definitively linked to dNTP pool perturbations. Failure to detect ways to accurately measure local dNTP fluxes in cells. dNTP pool changes in cells with altered RNR activity could be because even relatively small changes in dNTP pools can be highly mutagenic.70 Alternatively, a specific interaction between RNR and RNR AND DNA DAMAGE RESPONSES the DNA replication or repair machinery may be involved as noted RNR genes were among the first DNA damage-inducible genes above, so that biologically important localized pool alterations are to be identified.55 In mammalian cells, genotoxins such as obscured during analysis of total intracellular dNTP levels. It is also chlorambucil and ultraviolet light induce Rrm1 and Rrm2 noteworthy that measurement of holocomplex activity in vivo is expression by approximately 10-fold.56,57 RRM2 protein stability challenging, because cell lysis can disrupt complex equilibria is also DNA damage responsive,44 at least in part, through between subunits and alter cellular (d)NTP allosteric modulator ATR-dependent downregulation of cyclin F-mediated RRM2 concentrations. degradation.28 The discovery of p53R2 further connected DNA damage responses and RNR activity and also resolved the question of how DNA repair in quiescent cells could be supported RNR IN CANCER when RRM2 is undetectable. p53 binds the first p53R2 intron Uncontrolled proliferation is a defining feature of cancers and and stimulates p53R2 expression.26,58 In addition, DNA damage- must be supported by a sufficient dNTP supply. Cancer cells induced phosphorylation by ATM stabilizes p53R2 and confers undergo metabolic reprogramming such that glucose is no longer resistance to DNA damage.59 Inhibition of p53R2 expression in metabolized to maximize ATP production as in normal cells but cells that have an intact p53-dependent DNA damage checkpoint instead is used to drive the production of macromolecules for cell reduces RNR activity, DNA repair and cell survival after genotoxin replication, including dNTPs.71 Studies of rat hepatomas in the exposure.26,60 p53R2-null mouse fibroblasts also show severely 1970s established that RNR activity is highly correlated with cancer attenuated dNTP pools under oxidative stress.40 growth rate; 200-fold differences in enzyme activity were One aspect of the DNA damage response influenced by observed between fast- and slow-growing tumor cells.72 RRM2 RNR activity is repair pathway choice.61,62 RNR-mediated dNTP overexpression has been observed in gastric, ovarian, bladder and production after DNA damage fuels DNA synthesis during colorectal cancers.73–77 RRM2 expression is correlated with tumor homologous recombinational repair, whereas RNR inhibition grade for both breast and epithelial ovarian cancers, suggesting a promotes break repair by non-homologous end joining, role for RNR in supporting rapid cell division of high-grade which does not require extensive DNA synthesis.62 However, tumors.78,79 Similarly, RRM2 levels are low in benign skin lesions RNR-mediated dNTP pool increases are accompanied by higher but are significantly higher in malignant melanoma, with high mutation rates, which may result from reduced fidelity RRM2 expression additionally correlating with poor overall of replicative polymerases and/or activation of error-prone survival.80 Increased p53R2 expression has also been reported translesion DNA synthesis at elevated dNTP levels.10,63–65 in cancers, including melanoma, oral carcinoma, esophageal Despite the well-documented increase in RNR subunit levels after squamous cell carcinoma and non-small cell lung cancer DNA damage, there remains uncertainty about the extent to which (NSCLC).81–85 To broadly assess how RNR is affected in a large there is a corresponding change in dNTP pools after genotoxic collection of human cancers, we surveyed RNR gene expression in stress. In non-proliferating cells, a slow, fourfold accumulation of human cancers using the ONCOMINE database (Figure 2). p53R2 after DNA damage results in less than a two-fold increase in Remarkably, RRM2 was among the top 10% most overexpressed dNTP pools, a modest change relative to that which occurs upon genes in 73 out of the 168 cancer analyses. These include sarcoma S-phase entry.47 Moreover, logarithmically growing cells do not and cancers of the bladder, brain and central nervous system, show significant dNTP pool increases upon DNA damage. This may breast, colorectal, liver and lung. Similarly, RRM1 was among the reflect the action of additional mechanisms influencing dNTP levels top 10% most-overexpressed genes in 30 out of the 170 studies, after DNA damage. It also remains possible that dNTP biosynthesis including brain and central nervous system cancer, lung cancer is compartmentalized at damage sites during the DNA damage and sarcoma. By contrast, p53R2 was among the top 10% most response,51 such that measurements of total cellular dNTP levels fail overexpressed genes in only 5 out of the 90 studies. to reveal local changes. Elevated RNR expression in cancers could be secondary to cell cycle alterations, as many neoplasms show an increased S-phase fraction, or the direct result of gene amplification or other genetic RNR DEREGULATION AND GENOMIC INSTABILITY or epigenetic alterations. Therefore, we analyzed RNR gene copy Because proper dNTP levels are essential for genomic integrity, number changes using the TCGA database (Supplementary RNR deregulation is mutagenic.1,10,63 Initial insights into the Figure S2). Among the RNR genes, RRM2B was the most frequently consequences of mammalian RNR deregulation came from affected by copy number changes and typically showed gains, in analysis of mouse T-lymphosarcoma cells selected for deoxygua- accord with a recent report on human breast cancers.86 It should nosine resistance and determined to have a mutation in the A site be noted that the RRM2B locus (8q22.3) is located near the C-MYC (RRM1D57N) that disrupts dATP-mediated enzyme inhibition. oncogene (8q24). Most RRM2B copy number increases in cancers The mutagenized cells from which RRM1D57N was cloned show are accompanied by C-MYC amplification, raising the possibility increased dNTP levels and a 40-fold increase in mutation rate.66–68 that they are simply passenger events. However, RRM2B amplifica- RRM1D57N overexpression in CHO cells was subsequently shown to tion does occur independently of C-MYC amplification at low cause a 15–25-fold increase in spontaneous mutation frequency, frequency in breast, colon, ovarian, prostate and uterine cancers,

© 2015 Macmillan Publishers Limited Oncogene (2015) 2011 – 2021 Ribonucleotide reductase and cancer Y Aye et al 2014 cells leads to reduced transformation and suppression of tumorigenicity and lung metastasis in vivo.87 In addition, RRM1 overexpression in human lung cancer cells induces phosphatase and tensin homolog (PTEN) expression and suppresses migration and invasion, as well as overall tumorigenicity and metastasis following xenotransplantation.88 Rrm1-overexpressing transgenic mice show reduced urethane-induced lung tumorigenesis,89 although in an independent study Rrm1 overexpression was not found to alter the background incidence of spontaneous lung neoplasms in mice.90 In human NSCLC patients who underwent surgical resection but received no other form of treatment, high- level tumor-associated RRM1 expression correlated with longer lifespan and later disease recurrence.91 Co-expression of RRM1 and excision repair protein ERCC1 was significantly associated with disease-free and overall survival, especially in patients who underwent lung cancer surgery at early stages, although aspects of this study were later questioned.92 The published mechanisms behind tumor suppression by α, such as PTEN induction, require further investigation and may indicate that functions other than dNTP production are required for α tumor-suppressor activity. Additional animal studies also are needed to address the distinct outcomes reported concerning how α overexpression impacts lung tumorigenesis. Consistent with a role for RNR in promoting DNA repair, high level RRM1 expression also correlates with poor responses to platinum drugs, which induce DNA damage against which high Figure 2. RNR expression in human cancers. The bar graph illustrates RRM1 levels afford protection, and gemcitabine, which directly altered RNR expression in human cancers. Data were retrieved from 93–102 the ONCOMINE cancer gene expression database (version 4.4.4.4, targets RRM1. Although not all studies have found RRM1 search done on 27 November 2013). The y axis represents the levels to affect patient survival after gemcitabine treatment, there number of analyses with differences in gene expression for the gene is general consensus that low RRM1 levels improve responsiveness of interest. Dark red, red and pink: number of analyses with the gene to gemcitabine therapy. Additionally, RRM1 overexpression of interest among the top 1, 5 and 10% most overexpressed genes, has been reported in gemcitabine-resistant cancer cells.103–106 respectively, in a given study. Dark green, green and light green: Consequently, RRM1 expression has been proposed as a number of analyses with the gene of interest among the top biomarker in patients with advanced NSCLC to individualize 1, 5 and 10% most underexpressed genes, respectively, in a given chemotherapy.107 study. RRM2 is among the top 10% of the most overexpressed genes in 73 out of the 168 analyses, RRM1 is among the top 10% in 30 out of the 170 studies and RRM2B (encoding p53R2) is among the top TUMOR PROMOTION BY RRM2 AND P53R2 10% in 5 out of the 96 cases. In addition, RRM2 is among the top 10% of the most underexpressed genes in 7 out of the 168 studies, In contrast to the tumor-suppressing roles of RRM1, RRM2 and RRM1 is among the top 10% in 6 out of the 170 studies. The has oncogenic activity. For instance, RRM2 cooperates with expression level of the c-MYC proto-oncogene is shown for oncoproteins to increase focus formation and anchorage- comparison. c-MYC is among the top 10% of the most overexpressed independent growth in mouse cells.108,109 Elevated RRM2 expres- genes in 40 out of the 174 analyses and among the top 10% of the sion in human carcinoma cells correlates with higher invasive most underexpressed genes in 17 out of the 174 studies. potential in vitro 110 as well as decreased thrombspondin-1 and increased VEGF production, suggesting that RRM2 can promote as well as glioblastoma. This correlates well with increased p53R2 tumor angiogenesis.111 expression in breast and prostate cancers (Figure 2) and supports The role of p53R2 in mutagenesis and tumorigenesis is less a potential role for p53R2 as a tumor promoter. RRM2 is amplified clear-cut. Based on the p53-inducible nature of p53R2 expression at low frequency in breast, ovarian, prostate and uterine cancers, and its role in DNA repair, it was originally suggested that p53R2 malignancies in which RRM2 gene expression is also increased. would have tumor-suppressor activity.26 Under genotoxic stress, RRM1 also underwent rare copy number changes in cancers, with p53R2 promotes p21 accumulation and G1 arrest, which may the direction of change depending on tumor type. This may facilitate repair and prevent mutation accumulation.112 p53R2 reflect the complex and stage-specific roles RRM1 can have in expression is negatively correlated with colon adenocarcinoma tumorigenesis, as discussed below. metastasis.113 Similarly, elevated p53R2 expression suppresses cancer cell invasiveness and correlates with markedly better survival in colorectal cancer patients.114 Nevertheless p53R2 is RRM1: A TUMOR SUPPRESSOR THAT CAN CONFER highly expressed in some human cancers as noted above, and CHEMORESISTANCE experimental suppression of p53R2 expression impairs cancer cell RNR-mediated dNTP biosynthesis can have varied and potentially proliferation in vitro.81 opposing effects on tumorigenesis. Altered dNTP pools can impair Widespread overexpression of either Rrm2 or p53R2, but not DNA replication fidelity, leading to tumor-promoting mutations. Rrm1, in transgenic mice induces NSCLC but not other tumors.90 On the other hand, RNR-supported dNTP production can protect RNR-induced lung neoplasms arise with relatively long latency, against mutations by facilitating DNA repair. Following malignant histopathologically resemble human papillary adenomas and transformation, the same repair mechanisms can protect cancer adenocarcinomas and are associated with K-ras proto-oncogene cells against potentially lethal stresses, such as those caused by mutations. Rrm2 or p53R2 overexpression causes an elevated genotoxic chemotherapies. These complexities are reflected in the mutation frequency in cultured cells, suggesting that RNR-induced data concerning RRM1 in cancer. Several studies point to RRM1 as neoplasms could arise through a mutagenic mechanism. a suppressor of tumor initiation. RRM1 overexpression in cultured Consistent with this possibility, combining RNR deregulation

Oncogene (2015) 2011 – 2021 © 2015 Macmillan Publishers Limited Ribonucleotide reductase and cancer Y Aye et al 2015 with DNA mismatch repair defects synergistically increases cancers with high-level RRM2 expression. Nevertheless, because mutagenesis and tumorigenesis.90 dNTP biosynthesis is a complex process requiring multiple The available data raise fundamental questions about the enzymes, further work is necessary to fortify the link between transforming activity associated with the RNR-β subunit. Given TMPK inhibition and uracil misincorporation. For instance, the that increased RNR activity can lead to altered dNTP pools, one presence of another enzyme necessary for dNTP production, possibility is that RNR-β overexpression promotes error-prone NDPK, has not been established in the same complexes. Whether DNA synthesis, including increased frequency of base mis- enzymes other than the rate-limiting factor RNR must be present insertions, insertion–deletion events and uracil misincorporation, directly at damage sites to allow local dUTP accumulation remains as RNR also reduces UDP to dUDP. Recent evidence indicates unknown, but one possibility is that without NDPK recruitment the that RNR-generated dNTPs are also necessary for neoplastic cells dNTP precursors dTDP and dUDP would instead accumulate. to avoid oncogene-induced senescence.80,115,116 Senescence induced by activated Ras expression or Myc depletion was found to be associated with repression of Rrm2 expression and could be RNR AND CANCER THERAPY bypassed by treatment with exogenous nucleosides or ectopic Because RNR is the gatekeeper of dNTP homeostasis,29,133 the Rrm2 expression. Thus aberrant RNR expression could enable cells enzyme is long recognized as a cancer therapeutic target. to overcome this important barrier to transformation. Apoptosis Although small-molecule inhibitors continue to represent the is another cancer-related pathway that could be impacted by primary strategy for RNR inhibition since the last comprehensive RNR-mediated dNTP biosynthesis. In particular, (d)ATP binding to review, studies have uncovered new approaches to small- Apaf-1 and cytochrome c regulates the formation of the molecule-based subunit-specific activity modulation and highlight apoptosome, which is crucial for caspase activation and down- 134,135 117,118 the potential of gene therapy. Small-molecule RNR inhibitors stream apoptotic events. in active clinical use fall into two classes: nucleoside analogs and Reactive oxygen species (ROS) also may contribute to RNR- redox active metal chelators (Figure 3). The former class targets induced mutagenesis and transformation. Because Y• within RRM2 α α 119 RRM1 ( ), in line with the ability of to bind nucleotides, whereas is short-lived, its lability and reactivity could lead to secondary β 120,121 the latter group targets RRM2 ( ), consistent with the dependence reactive species when RRM2 is overproduced. It remains β 19 119 of on a redox active metallocofactor. The existence of these unclear whether RRM2 can indeed propagate ROS. Involvement fl fi two distinct drug classes re ects the inherent biochemical of ROS would be compatible with the lung speci city of diversities of the two subunits, both of which are required for NDP tumorigenesis in RRM2-overexpressing mice as well as the reduction.29,136 Consequently, most drugs targeting α show little observed synergy between RRM2 deregulation and the mismatch cross reactivity with β and vice versa. repair system, which responds to both base mismatches and oxidative DNA damage.90 Interestingly, the between Rrm2 and activated oncogenes in inducing transformation is RNR-α INHIBITORS 108 independent of ribonucleotide reduction. Moreover, RRM2 and fi α → One of the rst nucleoside analogs targeting to be clinically p53R2-overexpressing cells show an increased frequency of G T approved was gemcitabine (Gemzar, F C; Figure 3a), which transversions, a signature of oxidative DNA damage.90 2 continues to be a frontline therapy against pancreatic, bladder and As noted for RRM1, there also is evidence that RRM2 levels in lung cancers.137 RNR inactivation by gemcitabine has been cancer cells can influence therapeutic responses. Suppression of extensively reviewed.138 The active form, the substrate analog RRM2 expression sensitizes cancer cells to both RNR inhibitors and diphosphate (F CDP), is an irreversible, suicide inactivator of α, cisplatin.110,122 Elevated p53R2 expression correlates with resis- 2 which results in a covalent complex between α and the sugar of F tance to genotoxic therapies such as radiation and 5-flurouracil, 2 CDP.139 Despite being stereotyped as an α-targeting drug, F CDP whereas p53R2 knockdown sensitizes cells to DNA-damaging 2 81,123–125 cannot inactivate α without β; holocomplex assembly is obligatory agents. Interestingly, p53R2 levels in cancer cells do not • α correlate with sensitivity to RNR-targeted therapeutics, such as for transient S formation within the substrate-binding C site on triapine.104,122,126 RRM2 is part of a 12 gene set that is predictive of that then reacts with substrate analog F2CDP, affording a reactive fi intermediate capable of irreversibly alkylating α (Figure 3a). bene t from adjuvant chemotherapy in NSCLC and is associated α with poor prognosis.127 RRM2 overexpression is also a marker of The second approved -targeting drug to have its RNR- targeting mechanism elucidated was clofarabine (Clolar, ClF; poor prognosis in ovarian cancer patients receiving gemcitabine 22,23 or other cytotoxic therapies,128 and low RRM2 expression in Figure 3b and Supplementary Figure S3). ClF belongs to a pancreatic cancers is predictive of greater response to clinically successful class of nucleoside prodrugs, including gemcitabine.129,130 Similarly, the absence of p53R2 expression cytarabine (ara-C), nelarabine, azacitidine, decitabine, cladribine fl 140 is associated with responsiveness to chemoradiotherapy for and udarabine, used to treat hematological malignancies. The esophageal squamous cell carcinoma.131 majority of these antimetabolites are thought to target RNR Another intriguing example of therapeutic response modulation as part of their cytotoxic spectrum; however, the molecular by RNR is found in the case of small-molecule inhibitors of TMPK, mechanisms underlying RNR inhibition remain poorly defined which converts dTMP to dTDP in dTTP biosynthesis.52,132 Both RNR except for ClF. As the triphosphate (ClFTP) is the most abundant and TMPK localize to DNA damage in the nucleus, and their ClF metabolite in cells, it was initially postulated that α inhibition activities impact the relative levels of dUTP (produced via RNR) occurred through α A site binding by ClFTP, similarly to the 140 and dTTP (produced via TMPK) at repair sites. When the dUTP: feedback inhibitor dATP. However, both the diphosphate and dTTP ratio is low, there is minimal uracil misincorporation during triphosphate (ClFD(T)P) emerged to be α inhibitors in vitro. ClFTP is DNA repair. However, if the dUTP:dTTP ratio is elevated, high an A-site-binding reversible inhibitor. RNR-α can be completely levels of uracil misincorporation can lead to futile repair cycles, inhibited by brief exposure to saturating amounts of ClFTP, but DNA breakage and cell death.52 These observations are the basis activity is regained upon prolonged drug exposure, resulting in a for a therapeutic strategy that combines TMPK inhibition with low- steady-state level of 50% activity. Whether this restoration of dose chemotherapy. It follows that tumors with elevated RNR activity is important for ClF’s drug efficacy remains unaddressed. activity, dictated in many cases by RRM2 expression, would be Unexpectedly, the substrate analog diphosphate, ClFDP, is a especially susceptible to such a strategy. This prediction holds true reversible α inhibitor that binds the C site with slow release in initial cell culture analyses and offers hope for a targeted properties. Most strikingly, although ClFDP occupies the same site approach that could be effective against the large fraction of as F2CDP, α does not chemically engage with ClFDP, and the drug

© 2015 Macmillan Publishers Limited Oncogene (2015) 2011 – 2021 Ribonucleotide reductase and cancer Y Aye et al 2016 can be recovered intact from α even following prolonged incubation.22 ClF is a hybrid of its predecessors cladribine and fludarabine, two adenine-containing nucleosides used previously for leukemic reticuloendotheliosis and hairy cell leukemia or leukemia and lymphoma, respectively.140–142 These drugs were developed as analogs of deoxyadenosine, which selectively kills lymphocytes.143 The triphosphate of cladribine is thought to inhibit RNR, albeit through an unknown mechanism.144 Fludarabine primarily inhibits DNA polymerases, with minimal RNR inhibition. Cladribine and fludarabine feature undesirable chemotypes that make them both susceptible to glycolytic cleavage, reducing their efficacy. Particularly with fludarabine, hydrolytic and enzymatic cleavage produces 2-fluoroadenine, which is subsequently converted to the highly toxic 2-fluoro-adenosine triphosphate.145 ClF represents a triumph of semi-rational design, incorporating the most desirable properties of cladribine and fludarabine, with minimized toxicity.140 Similar to natural ligands, antimetabolites also modulate RNR activity through changes to α quaternary structure. Although RNR oligomeric regulation remains poorly understood, significant phenotypic differences exist between the natural and non- natural ligand-induced states.41 The α hexameric state induced by dATP only persists when the A site is saturated with dATP, thus enabling interconversions between the active and inactive states as a function of cellular dATP concentration. ClFTP also binds the A site and hexamerizes α in vitro.22 However, unlike dATP-induced α-hexamers, ClFTP-induced α-hexamers persist subsequent to ClFTP dissociation.23 Persistent α hexamerization is also initiated by ClFDP binding to the C site, suggesting that quaternary regulation is not uniquely associated with α allosteric sites. F2CDP- mediated irreversible RNR inactivation in which the suicide substrate F2CDP interacts with the C site is also proposed to lead 139 to α6β6. The kinetic stability of the ClFD(T)P-induced hexameric states was recently exploited to demonstrate that α exists as a dynamic equilibrium of oligomers in cells.23 α from untreated cells is a mixture of dimer and monomer, whereas ClF-treated cells yield α that is mainly hexameric. The allosteric activator ATP has also been proposed to affect α oligomerization independent of β. However, there are differing reports, based on distinct technical approaches, on the functional Figure 3. Inhibitors targeting multiple characteristics of RNR. a b α a outcome of this process (Supplementary Table S1). Kashlan and ( , ) Nucleotide analog inhibitors interacting with .( ) Gemcita- 24 bine (F2C) is approved for the treatment of lung, pancreatic, breast Cooperman described ATP-concentration-dependent formation α 21 and ovarian cancers. RNR catalytic turnover is triggered by forward of m (m = 2,4,6), whereas Dealwis and colleagues showed that 146 proton-coupled electron transfer, resulting in transient C-S• forma- 3mM ATP causes α hexamerization. Hofer and colleagues also tion that initiates NDP reduction. The active diphosphate (F2CDP) detected the presence of α2 and α6 states at 0.1 mM ATP. One way form, as a substrate analog, can interact with the C-S• to form a to reconcile these data is that ATP, in a similar way to dATP, substrate radical that subsequently decomposes, resulting in induces weakly associated hexamers that readily collapse to enzyme inactivation. Inactivation leads to covalent crosslinking lower-order oligomers at low ATP levels. This model is consistent between α and the sugar of decomposed F2CDP. Formation of with analysis of α in 0.5 mM ATP showing a mixture of lower- activated RNR holocomplex bearing the transient C-S• is a 22 prerequisite for F2CDP inactivation. (b) Clofarabine (ClF) is used order species. These observations imply that persistent for the treatment of refractory pediatric leukemias. The active hexamerization is uniquely induced by antileukemic nucleotides diphosphate and triphosphate [ClFD(T)P] forms are reversible that inhibit RNR. inhibitors that exclusively target α. Inhibition in both cases is Despite their success, nucleoside analogs suffer from coupled with the assembly of hexameric, catalytically non-viable complications.147 Most are administered as inactive prodrugs, quaternary states that are induced by ClFD(T)P either in the necessitating successive phosphorylation by cellular kinases to presence or absence of β. See also Supplementary Figure S3. gain activity.140 Depending upon the nucleoside, a different c d β c ( , ) Small-molecule inhibitors interacting with .() HU is used in steady-state equilibrium between the monophosphate, dipho- the treatment of chronic myeloid leukemia, melanoma, head and sphate and triphosphate is established.148 These variable drug neck and refractory ovarian cancers. Treatment of β with HU leads to β • metabolites can lead to low steady-state concentrations of the the formation of catalytically incapable apo- 2 that lacks both Y-O 149 150 and the di-iron center. (d) Triapine (3AP) is currently being evaluated active variant, side effects and ultimately toxicity. Nucleos(t) in clinical trials. β − Specific targeting is mediated by the active ides are also susceptible to numerous catabolic pathways, 151 form Fe(II)-(3AP) that reduces Y-O•, converting holo-β2 into the which can generate dangerous side products. In addition, catalytically incapable variant, met-β2 (Y-OH). C-SH, reduced Cys; resistance to certain dNTP analogs is linked to differential miRNA C-S•, Cys radical; Y-OH, Tyr; Y-O•, Tyr radical. expression.152 Both F2C and ClF are used in combination therapies. The 153 combination of F2C and carboplatin is widely used. The theory behind this and related approaches is that functional RNR is

Oncogene (2015) 2011 – 2021 © 2015 Macmillan Publishers Limited Ribonucleotide reductase and cancer Y Aye et al 2017 needed to provide dNTPs to repair DNA damage caused by nanoparticles or retroviruses has been shown to suppress cancers drugs such as carboplatin. In line with this hypothesis, the of the head, neck and pancreas.179,180 F2C/carboplatin combination is successful in treating carboplatin- resistant tumors.154 ClF is commonly used with ara-C, a dCTP analog that, following phosphorylation to ara-CTP, can block DNA SUMMARY AND FUTURE PERSPECTIVES synthesis. This combination highlights that nucleoside analogs Since the discovery of RNR activity by Reichard et al.181 in 1961, have complex metabolic pathways: ara-CTP inhibits deoxycytidine there have been tremendous advances in understanding the kinase (dCK), an enzyme responsible for phosphorylating ara-C to structure, function and biological significance of this essential ara-CMP.155 Steady-state cellular ara-CTP levels are relatively low, family of enzymes.17,19,41 The next stage in understanding RNR impairing efficacy. RNR inhibition by ClFD(T)P stimulates dCK functions in disease-related contexts likely will require a concerted 156 157 activity, leading to higher ara-CTP levels. A similar effect is interdisciplinary effort that merges biochemical and genetic observed in cells treated with F2C and staurosporine, which analyses and couples in vitro enzymology and structural studies 158 increases dCK activity. with relevant cell culture and animal models. Many mechanistic details remain to be resolved for mammalian RNR, including the RNR-β INHIBITORS dynamics and functional importance of oligomerization and subcellular localization, as well as the intricacies of cofactor • β The di-iron center and Y within are logical targets for anti- assembly and metalloenzyme regulation. RNR, encompassing β cancer drugs. One structurally simple inhibitor, HU (Figure 3c), is both conventional reductase and possible moonlighting (non- a known metal chelator and radical quencher. It is used in the reductase) functions, clearly has important, subunit-specific roles 159 160 161 β treatment of CML, AML and glioblastoma. Although is a in cancer biology, influencing tumor initiation, progression and 162 β 69,163,164 HU target and overexpression confers HU resistance, therapeutic sensitivity, while also serving as a target for anti- this drug is promiscuous, and other metalloenzymes, such as cancer drugs. The extent to which the oncogenic impact of RNR carbonic anhydrase and matrix metalloproteinases, are also HU relates to RNR-mediated mutagenesis, suppression of senescence, targets.165 HU attacks both the Y• and di-iron center of ROS production or modulation of apoptosis remains a key mammalian RNR-β on a similar timescale.166 This contrasts with question for future studies. Finally, given that RNR is well- the mechanism of HU-induced β-inactivation in bacteria, which established as an effective therapeutic target, cell-based high- involves exclusively Y• quenching, leading to a met enzyme 167 throughput phenotypic screening assays represent a promising state. These data underscore the dual properties of HU as a avenue for future drug development. Beginning with the metal-chelator and single-electron donor. fi A second β-targeting drug, triapine (3AP; Figure 3d) is currently identi cation of a novel radical-based catalytic mechanism, the in clinical trials for CML and various solid tumors.168 3AP study of RNR has revealed many intriguing biochemical mechan- represents an important case study, because it highlights the isms over the years, and we anticipate that more surprises are in complexities in deconvoluting inhibition mechanisms. 3AP is the store with the continued analysis of the role of RNR in cancer. most successful of a group of thiosemicarbazone β inhibitors and 169 is active against HU-resistant tumors. Initially, it was assumed CONFLICT OF INTEREST that 3AP inhibited β through iron chelation, either from the β fl active site170 or from the labile iron pool.171 However, this theory The authors declare no con ict of interest. was questioned with observations that metal-bound 3AP, particularly Fe(II)-(3AP) complex, under aerobic conditions, is ACKNOWLEDGEMENTS capable of generating ROS172 that could inhibit β.173 Recent We thank Professors Rick Cerione and Jennifer Surtees for helpful discussion and studies indicate that the Fe(II)-(3AP) complex is the active inhibitor • comments on the manuscript. YA acknowledges a faculty development grant from in vitro and can reduce Y at a rate faster than iron chelation at the the ACCEL program supported by NSF (SBE-0547373), an Affinito-Stewart grant from β 119 . Cultured K562 cells and HU-resistant TA3 cells the President's Council of Cornell Women and a Milstein sesquicentennial junior treated with 3AP showed no change in iron content within β but faculty fellowship. MJCL acknowledges an HHMI international student predoctoral underwent a rapid loss of Y•. No oxidation of β residues or fellowship. accumulation of oxidized cellular proteins could be detected, suggesting that ROS is not important for β inhibition by 3AP. These data collectively imply that human β is highly susceptible to REFERENCES radical-targeting drugs, a finding that opens a range of prospects 1 Mathews CK. DNA precursor metabolism and genomic stability. FASEB J 2006; 20: for inhibitor design and optimization. 1300–1314. 2 Rampazzo C, Miazzi C, Franzolin E, Pontarin G, Ferraro P, Frangini M et al. Regulation by degradation, a cellular defense against deoxyribonucleotide pool GENETIC STRATEGIES FOR RNR INHIBITION imbalances. Mutat Res 2010; 703:2–10. 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