ribonucleotide reductase and cancer: biological...

REVIEW

Ribonucleotide reductase and cancer: biological mechanismsand targeted therapiesY Aye1,2, M Li3, MJC Long4 and RS Weiss3

Accurate DNA replication and repair is essential for proper development, growth and tumor-free survival in all multicellularorganisms. A key requirement for the maintenance of genomic integrity is the availability of adequate and balanced pools ofdeoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA. Notably, dNTP pool alterations lead to genomic instabilityand 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 enzyme ribonucleotide reductase (RNR),impacts cancer susceptibility and serves as a target for anti-cancer therapies. Because RNR-regulated dNTP production caninfluence DNA replication fidelity while also supporting genome-protecting DNA repair, RNR has complex and stage-specific roles incarcinogenesis. Nevertheless, cancer cells are dependent on RNR for de novo dNTP biosynthesis. Therefore, elevated RNR expressionis a characteristic of many cancers, and an array of mechanistically distinct RNR inhibitors serve as effective agents for cancertreatment. The dNTP metabolism machinery, including RNR, has been exploited for therapeutic benefit for decades and remains animportant 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 ANDGENOMIC INTEGRITYThe importance of ribonucleotide reductase (RNR) for genomemaintenance relates to its central role in regulating dNTP levels. Inmammalian cells, total dNTP pool sizes peak during S-phase tosupport nuclear DNA (nDNA) replication and are roughly 10-foldlower in G0/G1, when dNTPs are needed for DNA repair andmitochondrial DNA (mtDNA) synthesis.1,2 As DNA polymerasesubstrates, dNTPs influence several aspects of the replicationprogram, including origin choice, fork speed, inter-origin distance,and dormant origin usage.3–5 Failure of cells to maintainappropriate dNTP concentrations can be highly detrimental,leading to DNA breaks, mutagenesis and cell death.6 Duringcancer development, uncoordinated cell proliferation can lead toinsufficient dNTPs that cause replication stress and furtherpromote genomic instability.7,8 Conversely, elevated dNTP poolsalso contribute to increased mutagenesis.1,9,10

Imbalanced dNTP pools enhance mutagenesis mainly by DNAmisinsertion and impaired proofreading.6,9 DNA misinsertion canresult from competition between dNTPs for pairing with thetemplate base, and a dNTP present in excess can be readilymisincorporated. DNA polymerase proofreading is reduced inthe presence of elevated dNTP concentrations through aphenomenon known as the next-nucleotide effect.11 Under suchconditions, DNA chain extension following a base misinsertionproceeds before the mismatched nucleotide can be removed.12–14

dNTP imbalances can also stimulate frameshift mutations byfacilitating correct base-pairing following template slippage ormisalignment.15

RNR AND dNTP BIOSYNTHESISdNTPs can be generated through de novo and salvage pathways. Inmammals, RNR catalyzes the rate-limiting step of the de novopathway, reducing the 2′ carbon of a ribonucleoside diphosphate(NDP) to produce the corresponding deoxy (d)NDP. Subsequently,dNDPs are phosphorylated by nucleoside diphosphate kinase(NDPK) yielding dNTPs.16 Importantly, the de novo biosynthesis ofdeoxythymidine triphosphate (dTTP) is much more complex,requiring the conversion of deoxyuridine monophosphate todeoxythymidine monophosphate (dTMP) by thymidylate synthasefollowed by phosphorylation by thymidylate phosphate kinase(TMPK) and then NDPK. Deoxyuridine monophosphate is generatedeither from deoxyuridine triphosphate (dUTP) or deoxycytidinemonophosphate, the biosynthesis of both of which is dependenton RNR-catalyzed reduction of the corresponding nucleosidediphosphates.6 RNR enzymes are present in all organisms andfeature-conserved radical-mediated nucleotide reduction.17 Mam-malian RNRs, the focus of this review, consist of two subunits α andβ, that associate to form the holoenzyme (Figure 1). α contains thecatalytic (C) site and two different allosteric sites. β harbors a di-ironcofactor and tyrosyl radical (Y•) essential for RNR activity. Evidencesuggests that catalytically active RNR is minimally an α2β2complex.18–20 RNR oligomerization is influenced by (d)NTP/ATPbinding to α allosteric sites. The negative regulator dATP inducesinactive α6 (or α6β2) states, highlighting a role for higher-orderoligomeric associations as a regulatory mechanism for RNR.18,21–24

However, as discussed further below, the precise nature of RNRoligomeric states in the presence of allosteric activators remainsunsettled (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] 6 January 2014; revised 25 April 2014; accepted 26 April 2014; published online 9 June 2014

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NDP reduction to dNDP takes place in the α C site and requiresthe unpaired electron initially localized as Y• in β (Figure 1).19

Although the di-iron center is present in each βmonomer, a singleY• is generated per β2.17 Protein radical formation occurs viareaction of the diferrous ions with molecular oxygen in a multistepprocess.17,19 How diferrous ions are loaded into β2, includingpotential involvement of iron chaperones, remains unknown.In vitro, β2 can exist in three distinct states: apo-β2 lacks both thedi-iron center and Y•; met-β2 contains the di-iron center but withY• reduced; and holo-β2 houses a diferric–Y• cofactor (the‘metallocofactor’) and is the only state that, when complexedwith α2, is active (Supplementary Figure S1). The β Y• is transferredthrough a proton-coupled electron transfer chain to generate atransient thiyl radical (S•) within the α C site.19 S• initiatesreduction of the NDP ribose ring, ultimately generating dNDPand resulting in disulfide bond formation on α. Thioredoxin orglutaredoxin re-reduces the oxidized cysteines on α, facilitatingturnover.25

In mammals, α is encoded by the gene Rrm1, whereas twodifferent genes, Rrm2 and p53R2, encode distinct β isoforms.p53R2 was identified as a p53-inducible, DNA damage-responsivegene.26 Mouse p53R2 and RRM2 share 81% identity and domainconservation except for amino-terminal residues required for cellcycle stage-specific RRM2 degradation.27–29 RRM1–RRM2 holoen-zyme provides dNTPs for S-phase nDNA replication and repair inproliferating cells, whereas RRM1–p53R2 contributes dNTPs fornDNA repair in quiescent cells as well as mtDNA replication and

repair.30–33 dNTPs for mtDNA synthesis can also come fromsalvage pathways localized within mitochondria,34,35 and RNRactivity has been identified within mitochondria as well.36

In humans, p53R2 mutations result in mitochondrial disease,including mtDNA depletion syndrome, a lethal condition inwhich 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 inpostmitotic tissues.39 Furthermore, p53R2 knockout mice showmtDNA depletion and die due to renal failure.30,40 Together, thesefindings indicate that the de novo dNTP biosynthesis mediatedby RNR is essential to maintain not only nuclear but alsomitochondrial genome integrity.

RNR ACTIVITY REGULATIONThe critical importance of dNTP levels demands that RNR istightly regulated through several distinct mechanisms, includingallosteric and oligomeric regulation, as well as alterations of thelevel and localization of RNR subunits. Allosteric modulationinvolves effector binding to two separate allosteric sites in α, aspecificity (S) site that regulates substrate choice and an activity(A) site that regulates enzyme activity (Figure 1).29 Substratespecificity is determined by the binding of ATP, dATP, dTTP ordGTP (deoxyguanosine triphosphate) to the S site; the mechanismhas been extensively reviewed.29,41 Overall RNR activity iscontrolled by (d)ATP binding to the A site, with elevated dATP:ATP ratios associated with inhibition. α hexamerization is coupledto dATP-induced inhibition.21,22,24,41

A second major mechanism for RNR regulation occurs throughthe cell cycle stage-specific control of RNR protein levels. Inmammals, RNR activity is induced during S phase.42–44 Rrm1 geneexpression is mainly regulated transcriptionally, being negligiblein G0/G1 and peaking in S phase.45 RRM1 protein, however,remains constant throughout the cell cycle owing to its long half-life.42,46,47 RRM2 is regulated both transcriptionally and by proteindegradation. Similar to that of Rrm1, Rrm2 transcription is minimalin G0/G1 and peaks in S phase.48 Unlike RRM1, RRM2 abundancecorrelates with its mRNA level.42,44 Upon S phase exit, RRM2protein is degraded through two ubiquitin ligases, the Skp1/Cullin/F-box (SCF) complex and the anaphase-promoting complex(APC).27,28 During G2 phase, RRM2 is recognized by cyclin F, an SCFubiquitin ligase F-box protein, and targeted for degradation.28

During mitosis/G1 phase, RRM2 is degraded by the Cdh1-APCcomplex that recognizes a KEN box motif at the RRM2N-terminus.27 By contrast, p53R2, which lacks a KEN box, iscontinuously expressed throughout the cell cycle. After DNAdamage, p53R2 is transcriptionally induced in a p53-dependentmanner and with RRM1 forms an active holoenzyme, providingdNTPs for DNA repair.26,29,33

RNR subcellular localization affords an additional layer ofregulatory control, although this aspect of mammalian RNRbiology remains controversial. The bulk of RRM1 and RRM2constitutively localize to the cytoplasm and produce dNTPs thatdiffuse into the nucleus for DNA replication.49,50 However, there isan increasing evidence that RRM1 and RRM2 accumulate at DNAdamage sites in the nucleus.28,51–53 This accumulation isdependent on interaction between RRM1 and the DNA damageresponse protein Tip60.51,52

The work forging a link between RNR translocation and localdNTP production at DNA damage sites is appealing. However, acautionary note is required, as dNTP synthesis requires multipleenzymes. The reports linking DNA damage to translocation ofdNTP production machinery have focused on only one or two keyplayers, typically RNR subunits. It is unknown whether the wholedNTP production pathway responds en masse to DNA damage or

Figure 1. RNR and de novo dNTP biosynthesis. Mammalian RNRenzymes function to reduce the 2' carbon of NDPs to generatedNDPs that are subsequently phosphorylated by nucleoside dipho-sphate kinase, yielding dNTPs for nuclear and mitochondrial DNAreplication and repair. RNR consists of an α subunit encoded by Rrm1and a β subunit encoded by Rrm2 or p53R2. The subunits interact toform hetero-oligomers, with the active enzyme believed to adoptα2β2 quaternary state. α contains the catalytic site (C), an activity site(A) that governs overall RNR activity through interactions with dATP(inhibitory) or ATP (stimulatory) and a specificity site (S) thatdetermines substrate choice. Each β subunit contains a μ-oxo-bridged di-nuclear iron center (Fe-O-Fe) and protein tyrosyl radical(Y-O•) that is transferred to the α C site for catalysis. The simplifiedschematic depictions of RNR holoenzyme structures shown herewere guided by structural studies.18,21

Ribonucleotide reductase and cancerY Aye et al

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whether increased dNTP synthesis actually occurs at DNA repairsites. Evidence does exist that the de novo thymidylate synthesispathway forms a scaffolded multienzyme complex at replicationsites in the nucleus.54 With respect to RNR, it is possible thattranslocation of this rate-limiting enzyme to DNA damage sites issufficient to affect local dNTP pools provided all other enzymesrequired for dNTP synthesis are within the nucleus. An alternativehypothesis related to DNA damage-induced RNR translocation isthat RNR has non-nucleotide reduction properties that promoteDNA repair. Parsing out the mechanisms at play during DNAdamage responses will require careful analysis and ultimately newways to accurately measure local dNTP fluxes in cells.

RNR AND DNA DAMAGE RESPONSESRNR genes were among the first DNA damage-inducible genesto be identified.55 In mammalian cells, genotoxins such aschlorambucil and ultraviolet light induce Rrm1 and Rrm2expression by approximately 10-fold.56,57 RRM2 protein stabilityis also DNA damage responsive,44 at least in part, throughATR-dependent downregulation of cyclin F-mediated RRM2degradation.28 The discovery of p53R2 further connected DNAdamage responses and RNR activity and also resolved thequestion of how DNA repair in quiescent cells could be supportedwhen RRM2 is undetectable. p53 binds the first p53R2 intronand stimulates p53R2 expression.26,58 In addition, DNA damage-induced phosphorylation by ATM stabilizes p53R2 and confersresistance to DNA damage.59 Inhibition of p53R2 expression incells that have an intact p53-dependent DNA damage checkpointreduces RNR activity, DNA repair and cell survival after genotoxinexposure.26,60 p53R2-null mouse fibroblasts also show severelyattenuated dNTP pools under oxidative stress.40

One aspect of the DNA damage response influenced byRNR activity is repair pathway choice.61,62 RNR-mediated dNTPproduction after DNA damage fuels DNA synthesis duringhomologous recombinational repair, whereas RNR inhibitionpromotes break repair by non-homologous end joining,which does not require extensive DNA synthesis.62 However,RNR-mediated dNTP pool increases are accompanied by highermutation rates, which may result from reduced fidelityof replicative polymerases and/or activation of error-pronetranslesion DNA synthesis at elevated dNTP levels.10,63–65

Despite the well-documented increase in RNR subunit levels afterDNA damage, there remains uncertainty about the extent to whichthere is a corresponding change in dNTP pools after genotoxicstress. In non-proliferating cells, a slow, fourfold accumulation ofp53R2 after DNA damage results in less than a two-fold increase indNTP pools, a modest change relative to that which occurs uponS-phase entry.47 Moreover, logarithmically growing cells do notshow significant dNTP pool increases upon DNA damage. This mayreflect the action of additional mechanisms influencing dNTP levelsafter DNA damage. It also remains possible that dNTP biosynthesisis compartmentalized at damage sites during the DNA damageresponse,51 such that measurements of total cellular dNTP levels failto reveal local changes.

RNR DEREGULATION AND GENOMIC INSTABILITYBecause proper dNTP levels are essential for genomic integrity,RNR deregulation is mutagenic.1,10,63 Initial insights into theconsequences of mammalian RNR deregulation came fromanalysis of mouse T-lymphosarcoma cells selected for deoxygua-nosine resistance and determined to have a mutation in the A site(RRM1D57N) that disrupts dATP-mediated enzyme inhibition.The mutagenized cells from which RRM1D57N was cloned showincreased dNTP levels and a 40-fold increase in mutation rate.66–68

RRM1D57N overexpression in CHO cells was subsequently shown tocause a 15–25-fold increase in spontaneous mutation frequency,

although no dNTP pool alterations could be identified.67 ElevatedRNR activity has also been identified in hydroxyurea (HU)-resistant,RRM2-overexpressing mouse cell lines. In one study withfibroblasts, a 3–15-fold increase in RNR activity was accompaniedby moderate changes in dNTP pools.69 By contrast, theHU-resistant, RRM2-overexpressing mouse mammary tumor TA3cell line shows a 40-fold increase in enzymatically active RRM2,estimated based on increased Y• content, but no detectable dNTPpool changes relative to parental cells.43,44 Therefore, the elevatedmutagenesis associated with RNR activity alterations has not beendefinitively linked to dNTP pool perturbations. Failure to detectdNTP pool changes in cells with altered RNR activity could bebecause even relatively small changes in dNTP pools can be highlymutagenic.70 Alternatively, a specific interaction between RNR andthe DNA replication or repair machinery may be involved as notedabove, so that biologically important localized pool alterations areobscured during analysis of total intracellular dNTP levels. It is alsonoteworthy that measurement of holocomplex activity in vivo ischallenging, because cell lysis can disrupt complex equilibriabetween subunits and alter cellular (d)NTP allosteric modulatorconcentrations.

RNR IN CANCERUncontrolled proliferation is a defining feature of cancers andmust be supported by a sufficient dNTP supply. Cancer cellsundergo metabolic reprogramming such that glucose is no longermetabolized to maximize ATP production as in normal cells butinstead is used to drive the production of macromolecules for cellreplication, including dNTPs.71 Studies of rat hepatomas in the1970s established that RNR activity is highly correlated with cancergrowth rate; 200-fold differences in enzyme activity wereobserved between fast- and slow-growing tumor cells.72 RRM2overexpression has been observed in gastric, ovarian, bladder andcolorectal cancers.73–77 RRM2 expression is correlated with tumorgrade for both breast and epithelial ovarian cancers, suggesting arole for RNR in supporting rapid cell division of high-gradetumors.78,79 Similarly, RRM2 levels are low in benign skin lesionsbut are significantly higher in malignant melanoma, with highRRM2 expression additionally correlating with poor overallsurvival.80 Increased p53R2 expression has also been reportedin cancers, including melanoma, oral carcinoma, esophagealsquamous cell carcinoma and non-small cell lung cancer(NSCLC).81–85 To broadly assess how RNR is affected in a largecollection of human cancers, we surveyed RNR gene expression inhuman cancers using the ONCOMINE database (Figure 2).Remarkably, RRM2 was among the top 10% most overexpressedgenes in 73 out of the 168 cancer analyses. These include sarcomaand cancers of the bladder, brain and central nervous system,breast, colorectal, liver and lung. Similarly, RRM1 was among thetop 10% most-overexpressed genes in 30 out of the 170 studies,including brain and central nervous system cancer, lung cancerand sarcoma. By contrast, p53R2 was among the top 10% mostoverexpressed genes in only 5 out of the 90 studies.Elevated RNR expression in cancers could be secondary to cell

cycle alterations, as many neoplasms show an increased S-phasefraction, or the direct result of gene amplification or other geneticor epigenetic alterations. Therefore, we analyzed RNR gene copynumber changes using the TCGA database (SupplementaryFigure S2). Among the RNR genes, RRM2B was the most frequentlyaffected by copy number changes and typically showed gains, inaccord with a recent report on human breast cancers.86 It shouldbe noted that the RRM2B locus (8q22.3) is located near the C-MYConcogene (8q24). Most RRM2B copy number increases in cancersare accompanied by C-MYC amplification, raising the possibilitythat they are simply passenger events. However, RRM2B amplifica-tion does occur independently of C-MYC amplification at lowfrequency in breast, colon, ovarian, prostate and uterine cancers,


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as well as glioblastoma. This correlates well with increased p53R2expression in breast and prostate cancers (Figure 2) and supportsa potential role for p53R2 as a tumor promoter. RRM2 is amplifiedat low frequency in breast, ovarian, prostate and uterine cancers,malignancies in which RRM2 gene expression is also increased.RRM1 also underwent rare copy number changes in cancers, withthe direction of change depending on tumor type. This mayreflect the complex and stage-specific roles RRM1 can have intumorigenesis, as discussed below.

RRM1: A TUMOR SUPPRESSOR THAT CAN CONFERCHEMORESISTANCERNR-mediated dNTP biosynthesis can have varied and potentiallyopposing effects on tumorigenesis. Altered dNTP pools can impairDNA replication fidelity, leading to tumor-promoting mutations.On the other hand, RNR-supported dNTP production can protectagainst mutations by facilitating DNA repair. Following malignanttransformation, the same repair mechanisms can protect cancercells against potentially lethal stresses, such as those caused bygenotoxic chemotherapies. These complexities are reflected in thedata concerning RRM1 in cancer. Several studies point to RRM1 asa suppressor of tumor initiation. RRM1 overexpression in cultured

cells leads to reduced transformation and suppression oftumorigenicity and lung metastasis in vivo.87 In addition, RRM1overexpression in human lung cancer cells induces phosphataseand tensin homolog (PTEN) expression and suppresses migrationand invasion, as well as overall tumorigenicity and metastasisfollowing xenotransplantation.88 Rrm1-overexpressing transgenicmice show reduced urethane-induced lung tumorigenesis,89

although in an independent study Rrm1 overexpression was notfound to alter the background incidence of spontaneous lungneoplasms in mice.90 In human NSCLC patients who underwentsurgical resection but received no other form of treatment, high-level tumor-associated RRM1 expression correlated with longerlifespan and later disease recurrence.91 Co-expression of RRM1and excision repair protein ERCC1 was significantly associatedwith disease-free and overall survival, especially in patients whounderwent lung cancer surgery at early stages, although aspectsof this study were later questioned.92 The published mechanismsbehind tumor suppression by α, such as PTEN induction, requirefurther investigation and may indicate that functions other thandNTP production are required for α tumor-suppressor activity.Additional animal studies also are needed to address the distinctoutcomes reported concerning how α overexpression impactslung tumorigenesis.Consistent with a role for RNR in promoting DNA repair, high

level RRM1 expression also correlates with poor responses toplatinum drugs, which induce DNA damage against which highRRM1 levels afford protection, and gemcitabine, which directlytargets RRM1.93–102 Although not all studies have found RRM1levels to affect patient survival after gemcitabine treatment, thereis general consensus that low RRM1 levels improve responsivenessto gemcitabine therapy. Additionally, RRM1 overexpressionhas been reported in gemcitabine-resistant cancer cells.103–106

Consequently, RRM1 expression has been proposed as abiomarker in patients with advanced NSCLC to individualizechemotherapy.107

TUMOR PROMOTION BY RRM2 AND P53R2In contrast to the tumor-suppressing roles of RRM1, RRM2has oncogenic activity. For instance, RRM2 cooperates withoncoproteins to increase focus formation and anchorage-independent growth in mouse cells.108,109 Elevated RRM2 expres-sion in human carcinoma cells correlates with higher invasivepotential in vitro 110 as well as decreased thrombspondin-1 andincreased VEGF production, suggesting that RRM2 can promotetumor angiogenesis.111

The role of p53R2 in mutagenesis and tumorigenesis is lessclear-cut. Based on the p53-inducible nature of p53R2 expressionand its role in DNA repair, it was originally suggested that p53R2would have tumor-suppressor activity.26 Under genotoxic stress,p53R2 promotes p21 accumulation and G1 arrest, which mayfacilitate repair and prevent mutation accumulation.112 p53R2expression is negatively correlated with colon adenocarcinomametastasis.113 Similarly, elevated p53R2 expression suppressescancer cell invasiveness and correlates with markedly bettersurvival in colorectal cancer patients.114 Nevertheless p53R2 ishighly expressed in some human cancers as noted above, andexperimental suppression of p53R2 expression impairs cancer cellproliferation in vitro.81

Widespread overexpression of either Rrm2 or p53R2, but notRrm1, in transgenic mice induces NSCLC but not other tumors.90

RNR-induced lung neoplasms arise with relatively long latency,histopathologically resemble human papillary adenomas andadenocarcinomas and are associated with K-ras proto-oncogenemutations. Rrm2 or p53R2 overexpression causes an elevatedmutation frequency in cultured cells, suggesting that RNR-inducedneoplasms could arise through a mutagenic mechanism.Consistent with this possibility, combining RNR deregulation

Figure 2. RNR expression in human cancers. The bar graph illustratesaltered RNR expression in human cancers. Data were retrieved fromthe ONCOMINE cancer gene expression database (version 4.4.4.4,search done on 27 November 2013). The y axis represents thenumber of analyses with differences in gene expression for the geneof interest. Dark red, red and pink: number of analyses with the geneof interest among the top 1, 5 and 10% most overexpressed genes,respectively, in a given study. Dark green, green and light green:number of analyses with the gene of interest among the top1, 5 and 10% most underexpressed genes, respectively, in a givenstudy. RRM2 is among the top 10% of the most overexpressed genesin 73 out of the 168 analyses, RRM1 is among the top 10% in 30 outof the 170 studies and RRM2B (encoding p53R2) is among the top10% in 5 out of the 96 cases. In addition, RRM2 is among the top10% of the most underexpressed genes in 7 out of the 168 studies,and RRM1 is among the top 10% in 6 out of the 170 studies. Theexpression level of the c-MYC proto-oncogene is shown forcomparison. c-MYC is among the top 10% of the most overexpressedgenes in 40 out of the 174 analyses and among the top 10% of themost underexpressed genes in 17 out of the 174 studies.


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with DNA mismatch repair defects synergistically increasesmutagenesis and tumorigenesis.90

The available data raise fundamental questions about thetransforming activity associated with the RNR-β subunit. Giventhat increased RNR activity can lead to altered dNTP pools, onepossibility is that RNR-β overexpression promotes error-proneDNA synthesis, including increased frequency of base mis-insertions, insertion–deletion events and uracil misincorporation,as RNR also reduces UDP to dUDP. Recent evidence indicatesthat RNR-generated dNTPs are also necessary for neoplastic cellsto avoid oncogene-induced senescence.80,115,116 Senescenceinduced by activated Ras expression or Myc depletion was foundto be associated with repression of Rrm2 expression and could bebypassed by treatment with exogenous nucleosides or ectopicRrm2 expression. Thus aberrant RNR expression could enable cellsto overcome this important barrier to transformation. Apoptosisis another cancer-related pathway that could be impacted byRNR-mediated dNTP biosynthesis. In particular, (d)ATP binding toApaf-1 and cytochrome c regulates the formation of theapoptosome, which is crucial for caspase activation and down-stream apoptotic events.117,118

Reactive oxygen species (ROS) also may contribute to RNR-induced mutagenesis and transformation. Because Y• within RRM2is short-lived,119 its lability and reactivity could lead to secondaryreactive species when RRM2 is overproduced.120,121 It remainsunclear whether RRM2 can indeed propagate ROS.119 Involvementof ROS would be compatible with the lung specificity oftumorigenesis in RRM2-overexpressing mice as well as theobserved synergy between RRM2 deregulation and the mismatchrepair system, which responds to both base mismatches andoxidative DNA damage.90 Interestingly, the cooperativity betweenRrm2 and activated oncogenes in inducing transformation isindependent of ribonucleotide reduction.108 Moreover, RRM2 andp53R2-overexpressing cells show an increased frequency of G→ Ttransversions, a signature of oxidative DNA damage.90

As noted for RRM1, there also is evidence that RRM2 levels incancer cells can influence therapeutic responses. Suppression ofRRM2 expression sensitizes cancer cells to both RNR inhibitors andcisplatin.110,122 Elevated p53R2 expression correlates with resis-tance to genotoxic therapies such as radiation and 5-flurouracil,whereas p53R2 knockdown sensitizes cells to DNA-damagingagents.81,123–125 Interestingly, p53R2 levels in cancer cells do notcorrelate with sensitivity to RNR-targeted therapeutics, such astriapine.104,122,126 RRM2 is part of a 12 gene set that is predictive ofbenefit from adjuvant chemotherapy in NSCLC and is associatedwith poor prognosis.127 RRM2 overexpression is also a marker ofpoor prognosis in ovarian cancer patients receiving gemcitabineor other cytotoxic therapies,128 and low RRM2 expression inpancreatic cancers is predictive of greater response togemcitabine.129,130 Similarly, the absence of p53R2 expressionis associated with responsiveness to chemoradiotherapy foresophageal squamous cell carcinoma.131

Another intriguing example of therapeutic response modulationby RNR is found in the case of small-molecule inhibitors of TMPK,which converts dTMP to dTDP in dTTP biosynthesis.52,132 Both RNRand TMPK localize to DNA damage in the nucleus, and theiractivities impact the relative levels of dUTP (produced via RNR)and dTTP (produced via TMPK) at repair sites. When the dUTP:dTTP ratio is low, there is minimal uracil misincorporation duringDNA repair. However, if the dUTP:dTTP ratio is elevated, highlevels of uracil misincorporation can lead to futile repair cycles,DNA breakage and cell death.52 These observations are the basisfor a therapeutic strategy that combines TMPK inhibition with low-dose chemotherapy. It follows that tumors with elevated RNRactivity, dictated in many cases by RRM2 expression, would beespecially susceptible to such a strategy. This prediction holds truein initial cell culture analyses and offers hope for a targetedapproach that could be effective against the large fraction of

cancers with high-level RRM2 expression. Nevertheless, becausedNTP biosynthesis is a complex process requiring multipleenzymes, further work is necessary to fortify the link betweenTMPK inhibition and uracil misincorporation. For instance, thepresence of another enzyme necessary for dNTP production,NDPK, has not been established in the same complexes. Whetherenzymes other than the rate-limiting factor RNR must be presentdirectly at damage sites to allow local dUTP accumulation remainsunknown, but one possibility is that without NDPK recruitment thedNTP precursors dTDP and dUDP would instead accumulate.

RNR AND CANCER THERAPYBecause RNR is the gatekeeper of dNTP homeostasis,29,133 theenzyme is long recognized as a cancer therapeutic target.Although small-molecule inhibitors continue to represent theprimary strategy for RNR inhibition since the last comprehensivereview, studies have uncovered new approaches to small-molecule-based subunit-specific activity modulation and highlightthe potential of gene therapy.134,135 Small-molecule RNR inhibitorsin active clinical use fall into two classes: nucleoside analogs andredox active metal chelators (Figure 3). The former class targetsRRM1 (α), in line with the ability of α to bind nucleotides, whereasthe latter group targets RRM2 (β), consistent with the dependenceof β on a redox active metallocofactor.19 The existence of thesetwo distinct drug classes reflects the inherent biochemicaldiversities of the two subunits, both of which are required for NDPreduction.29,136 Consequently, most drugs targeting α show littlecross reactivity with β and vice versa.

RNR-α INHIBITORSOne of the first nucleoside analogs targeting α to be clinicallyapproved was gemcitabine (Gemzar, F2C; Figure 3a), whichcontinues to be a frontline therapy against pancreatic, bladder andlung cancers.137 RNR inactivation by gemcitabine has beenextensively reviewed.138 The active form, the substrate analogdiphosphate (F2CDP), is an irreversible, suicide inactivator of α,which results in a covalent complex between α and the sugar of F2CDP.139 Despite being stereotyped as an α-targeting drug, F2CDPcannot inactivate α without β; holocomplex assembly is obligatoryfor transient S• formation within the substrate-binding C site on αthat then reacts with substrate analog F2CDP, affording a reactiveintermediate capable of irreversibly alkylating α (Figure 3a).The second approved α-targeting drug to have its RNR-

targeting mechanism elucidated was clofarabine (Clolar, ClF;Figure 3b and Supplementary Figure S3).22,23 ClF belongs to aclinically successful class of nucleoside prodrugs, includingcytarabine (ara-C), nelarabine, azacitidine, decitabine, cladribineand fludarabine, used to treat hematological malignancies.140 Themajority of these antimetabolites are thought to target RNRas part of their cytotoxic spectrum; however, the molecularmechanisms underlying RNR inhibition remain poorly definedexcept for ClF. As the triphosphate (ClFTP) is the most abundantClF metabolite in cells, it was initially postulated that α inhibitionoccurred through α A site binding by ClFTP, similarly to thefeedback inhibitor dATP.140 However, both the diphosphate andtriphosphate (ClFD(T)P) emerged to be α inhibitors in vitro. ClFTP isan A-site-binding reversible inhibitor. RNR-α can be completelyinhibited by brief exposure to saturating amounts of ClFTP, butactivity is regained upon prolonged drug exposure, resulting in asteady-state level of 50% activity. Whether this restoration ofactivity is important for ClF’s drug efficacy remains unaddressed.Unexpectedly, the substrate analog diphosphate, ClFDP, is areversible α inhibitor that binds the C site with slow releaseproperties. Most strikingly, although ClFDP occupies the same siteas F2CDP, α does not chemically engage with ClFDP, and the drug


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can be recovered intact from α even following prolongedincubation.22

ClF is a hybrid of its predecessors cladribine and fludarabine,two adenine-containing nucleosides used previously for leukemicreticuloendotheliosis and hairy cell leukemia or leukemia andlymphoma, respectively.140–142 These drugs were developed asanalogs of deoxyadenosine, which selectively kills lymphocytes.143

The triphosphate of cladribine is thought to inhibit RNR, albeitthrough an unknown mechanism.144 Fludarabine primarily inhibitsDNA polymerases, with minimal RNR inhibition. Cladribine andfludarabine feature undesirable chemotypes that make themboth susceptible to glycolytic cleavage, reducing their efficacy.Particularly with fludarabine, hydrolytic and enzymatic cleavageproduces 2-fluoroadenine, which is subsequently converted to thehighly toxic 2-fluoro-adenosine triphosphate.145 ClF represents atriumph of semi-rational design, incorporating the most desirableproperties of cladribine and fludarabine, with minimizedtoxicity.140

Similar to natural ligands, antimetabolites also modulate RNRactivity through changes to α quaternary structure. Although RNRoligomeric regulation remains poorly understood, significantphenotypic differences exist between the natural and non-natural ligand-induced states.41 The α hexameric state inducedby dATP only persists when the A site is saturated with dATP, thusenabling interconversions between the active and inactive statesas a function of cellular dATP concentration. ClFTP also binds theA site and hexamerizes α in vitro.22 However, unlike dATP-inducedα-hexamers, ClFTP-induced α-hexamers persist subsequent toClFTP dissociation.23 Persistent α hexamerization is also initiatedby ClFDP binding to the C site, suggesting that quaternaryregulation is not uniquely associated with α allosteric sites. F2CDP-mediated irreversible RNR inactivation in which the suicidesubstrate F2CDP interacts with the C site is also proposed to leadto α6β6.

139 The kinetic stability of the ClFD(T)P-induced hexamericstates was recently exploited to demonstrate that α exists as adynamic equilibrium of oligomers in cells.23 α from untreated cellsis a mixture of dimer and monomer, whereas ClF-treated cellsyield α that is mainly hexameric.The allosteric activator ATP has also been proposed to affect

α oligomerization independent of β. However, there are differingreports, based on distinct technical approaches, on the functionaloutcome of this process (Supplementary Table S1). Kashlan andCooperman24 described ATP-concentration-dependent formationof αm (m= 2,4,6), whereas Dealwis and colleagues21 showed that3 mM ATP causes α hexamerization. Hofer and colleagues146 alsodetected the presence of α2 and α6 states at 0.1 mM ATP. One wayto reconcile these data is that ATP, in a similar way to dATP,induces weakly associated hexamers that readily collapse tolower-order oligomers at low ATP levels. This model is consistentwith analysis of α in 0.5 mM ATP showing a mixture of lower-order species.22 These observations imply that persistenthexamerization is uniquely induced by antileukemic nucleotidesthat inhibit RNR.Despite their success, nucleoside analogs suffer from

complications.147 Most are administered as inactive prodrugs,necessitating successive phosphorylation by cellular kinases togain activity.140 Depending upon the nucleoside, a differentsteady-state equilibrium between the monophosphate, dipho-sphate and triphosphate is established.148 These variable drugmetabolites can lead to low steady-state concentrations of theactive variant, side effects149 and ultimately toxicity.150 Nucleos(t)ides are also susceptible to numerous catabolic pathways,which can generate dangerous side products.151 In addition,resistance to certain dNTP analogs is linked to differential miRNAexpression.152

Both F2C and ClF are used in combination therapies. Thecombination of F2C and carboplatin is widely used.153 The theorybehind this and related approaches is that functional RNR is

Figure 3. Inhibitors targeting multiple characteristics of RNR.(a, b) Nucleotide analog inhibitors interacting with α. (a) Gemcita-bine (F2C) is approved for the treatment of lung, pancreatic, breastand ovarian cancers. RNR catalytic turnover is triggered by forwardproton-coupled electron transfer, resulting in transient C-S• forma-tion that initiates NDP reduction. The active diphosphate (F2CDP)form, as a substrate analog, can interact with the C-S• to form asubstrate radical that subsequently decomposes, resulting inenzyme inactivation. Inactivation leads to covalent crosslinkingbetween α and the sugar of decomposed F2CDP. Formation ofactivated RNR holocomplex bearing the transient C-S• is aprerequisite for F2CDP inactivation. (b) Clofarabine (ClF) is usedfor the treatment of refractory pediatric leukemias. The activediphosphate and triphosphate [ClFD(T)P] forms are reversibleinhibitors that exclusively target α. Inhibition in both cases iscoupled with the assembly of hexameric, catalytically non-viablequaternary states that are induced by ClFD(T)P either in thepresence or absence of β. See also Supplementary Figure S3.(c, d) Small-molecule inhibitors interacting with β. (c) HU is used inthe treatment of chronic myeloid leukemia, melanoma, head andneck and refractory ovarian cancers. Treatment of β with HU leads tothe formation of catalytically incapable apo-β2 that lacks both Y-O•and the di-iron center. (d) Triapine (3AP) is currently being evaluatedin clinical trials. β− Specific targeting is mediated by the activeform Fe(II)-(3AP) that reduces Y-O•, converting holo-β2 into thecatalytically incapable variant, met-β2 (Y-OH). C-SH, reduced Cys;C-S•, Cys radical; Y-OH, Tyr; Y-O•, Tyr radical.


2016


needed to provide dNTPs to repair DNA damage caused bydrugs such as carboplatin. In line with this hypothesis, theF2C/carboplatin combination is successful in treating carboplatin-resistant tumors.154 ClF is commonly used with ara-C, a dCTPanalog that, following phosphorylation to ara-CTP, can block DNAsynthesis. This combination highlights that nucleoside analogshave complex metabolic pathways: ara-CTP inhibits deoxycytidinekinase (dCK), an enzyme responsible for phosphorylating ara-C toara-CMP.155 Steady-state cellular ara-CTP levels are relatively low,impairing efficacy. RNR inhibition by ClFD(T)P stimulates dCKactivity,156 leading to higher ara-CTP levels.157 A similar effect isobserved in cells treated with F2C and staurosporine, whichincreases dCK activity.158

RNR-β INHIBITORSThe di-iron center and Y• within β are logical targets for anti-cancer drugs. One structurally simple β inhibitor, HU (Figure 3c), isa known metal chelator and radical quencher. It is used in thetreatment of CML,159 AML 160 and glioblastoma.161 Although β is aHU target 162 and β overexpression confers HU resistance,69,163,164

this drug is promiscuous, and other metalloenzymes, such ascarbonic anhydrase and matrix metalloproteinases, are also HUtargets.165 HU attacks both the Y• and di-iron center ofmammalian RNR-β on a similar timescale.166 This contrasts withthe mechanism of HU-induced β-inactivation in bacteria, whichinvolves exclusively Y• quenching, leading to a met enzymestate.167 These data underscore the dual properties of HU as ametal-chelator and single-electron donor.A second β-targeting drug, triapine (3AP; Figure 3d) is currently

in clinical trials for CML and various solid tumors.168 3APrepresents an important case study, because it highlights thecomplexities in deconvoluting inhibition mechanisms. 3AP is themost successful of a group of thiosemicarbazone β inhibitors andis active against HU-resistant tumors.169 Initially, it was assumedthat 3AP inhibited β through iron chelation, either from the βactive site170 or from the labile iron pool.171 However, this theorywas questioned with observations that metal-bound 3AP,particularly Fe(II)-(3AP) complex, under aerobic conditions, iscapable of generating ROS172 that could inhibit β.173 Recentstudies indicate that the Fe(II)-(3AP) complex is the active inhibitorin vitro and can reduce Y• at a rate faster than iron chelation at theβ active site.119 Cultured K562 cells and HU-resistant TA3 cellstreated with 3AP showed no change in iron content within β butunderwent a rapid loss of Y•. No oxidation of β residues oraccumulation 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 toradical-targeting drugs, a finding that opens a range of prospectsfor inhibitor design and optimization.

GENETIC STRATEGIES FOR RNR INHIBITIONAlthough the most effective and widely used approaches for RNRinhibition center on small-molecule inhibitors, targeted knock-down of RNR subunits using small interfering RNA (siRNA) also hasbeen developed. RRM2 knockdown impairs tumor cell prolifera-tion174 and sensitizes cancer cells to DNA-damaging agents aswell as the RNR inhibitors HU and 3AP.122 RRM2 knockdown alsoovercomes cisplatin resistance in cultured cells.54 Similarly, siRNAsdesigned to downregulate RRM1 expression in tumors have beendeveloped to overcome gemcitabine resistance.175 Although theapplication of these siRNA-based strategies in patients iscomplicated by challenges related to delivery176 and stability inplasma,177 encouraging results have been observed. A 20-merphosphorothioate oligodeoxynucleotide, GTI-2040,178 that signifi-cantly reduces RRM2 mRNA levels, is presently in clinical trials forvarious solid tumors.174–176 Delivery of siRNA against RRM2 by

nanoparticles or retroviruses has been shown to suppress cancersof the head, neck and pancreas.179,180

SUMMARY AND FUTURE PERSPECTIVESSince the discovery of RNR activity by Reichard et al.181 in 1961,there have been tremendous advances in understanding thestructure, function and biological significance of this essentialfamily of enzymes.17,19,41 The next stage in understanding RNRfunctions in disease-related contexts likely will require a concertedinterdisciplinary effort that merges biochemical and geneticanalyses and couples in vitro enzymology and structural studieswith relevant cell culture and animal models. Many mechanisticdetails remain to be resolved for mammalian RNR, including thedynamics and functional importance of oligomerization andsubcellular localization, as well as the intricacies of cofactorassembly and metalloenzyme regulation. RNR, encompassingboth conventional reductase and possible moonlighting (non-reductase) functions, clearly has important, subunit-specific rolesin cancer biology, influencing tumor initiation, progression andtherapeutic sensitivity, while also serving as a target for anti-cancer drugs. The extent to which the oncogenic impact of RNRrelates to RNR-mediated mutagenesis, suppression of senescence,ROS production or modulation of apoptosis remains a keyquestion for future studies. Finally, given that RNR is well-established as an effective therapeutic target, cell-based high-throughput phenotypic screening assays represent a promisingavenue for future drug development. Beginning with theidentification of a novel radical-based catalytic mechanism, thestudy of RNR has revealed many intriguing biochemical mechan-isms over the years, and we anticipate that more surprises are instore with the continued analysis of the role of RNR in cancer.

CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGEMENTSWe thank Professors Rick Cerione and Jennifer Surtees for helpful discussion andcomments on the manuscript. YA acknowledges a faculty development grant fromthe ACCEL program supported by NSF (SBE-0547373), an Affinito-Stewart grant fromthe President's Council of Cornell Women and a Milstein sesquicentennial juniorfaculty fellowship. MJCL acknowledges an HHMI international student predoctoralfellowship.

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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)


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ribonucleotide reductase and cancer: biological...

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