dna damage helle

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Cancer therapy usually involves exposing the body toagents that kill cancer cells more efficiently than normaltissue cells. Such therapies must therefore exploit specificmolecular and cellular features of the cancer they are aim-ing to eliminate. Most cancer cells proliferate more rapidlythan their normal counterparts so most cancer drugstarget the cell cycle. Cell division can be targeted directlyby inhibitors of the mitotic spindle, thus preventing equaldivision of DNA to the two daughter cells. The growth sig-nals that result in entry into the cell cycle can be targetedby hormonal manipulation, therapeutic antibodies anddrugs that inhibit growth signalling pathways. However,the most common means of targeting the cell cycle is toexploit the effect of DNA-damaging drugs. DNA damagecauses cell-cycle arrest and cell death either directly orfollowing DNA replication during the S phase of the cellcycle. Cellular attempts to replicate damaged DNA cancause increased cell killing, thus making DNA-damagingtreatments more toxic to replicating cells than to non-

replicating cells. However, the toxicity of DNA-damagingdrugs can be reduced by the activities of several DNArepair pathways that remove lesions before they becometoxic. The efficacy of DNA damage-based cancer therapycan thus be modulated by DNA repair pathways. In addi-tion, some of these pathways are inactivated in some cancertypes. These two features make DNA repair mechanisms apromising target for novel cancer treatments.

DNA-damaging agents in cancer treatment

Many cancer drugs employed in the clinic have beenused for several decades and are highly efficient in kill-ing proliferating cells (FIG. 1). High levels of DNA damage

cause cell-cycle arrest and cell death. Furthermore, DNAlesions that persist into the S phase of the cell cycle canobstruct replication fork progression, resulting in theformation of replication-associated DNA double-strandbreaks (DSBs). DSBs are generally considered to be themost toxic of all DNA lesions1,2.

Common types of DNA damage that interfere withreplication fork progression are chemical modifications(adducts) of DNA bases, which are created by reactivedrugs that covalently bind DNA either directly or afterbeing metabolized in the body. These alkylating agents aregrouped in two categories: monofunctional alkylatingagents with one active moiety that modifies single basesand bifunctional alkylating agents that have two reactivesites and crosslink DNA with proteins or, alternatively,crosslink two DNA bases within the same DNA strand(intra-strand crosslinks) or on opposite DNA strands(inter-strand crosslinks). Inter-strand crosslinks pose asevere block to replication forks.

DNA synthesis is sometimes targeted by inhibitorsof DNA replication, such as aphidicolin, which directlyinhibits DNA polymerases3, whereas the radical scav-enger hydroxyureainhibits ribonucleotide reductase,which is required for production of the dNTPs that areused for DNA synthesis4. These two replication inhibi-tors can be regarded as DNA-damaging agents becausethey impair replication fork progression and cause DNAlesions, including DSBs5,6.

Antimetabolites, such as5-fluorouracil(5FU) and thio-purines, resemble nucleotides, nucleotide precursors orcofactors required for nucleotide biosynthesis and actby inhibiting nucleotide metabolism pathways, thus

*Radiation Oncology &

Biology, University of Oxford,Old Road Campus Research

Building, off Roosevelt Drive,

Headington, Oxford,

OX3 7DQ, UK.Department of Genetics

Microbiology and Toxicology,

Stockholm University, Svante

Arrhenius vg 16, S-106 91

Stockholm, Sweden.

Correspondence to T.H.

e-mail: thomas.helleday@rob.

ox.ac.uk

doi:10.1038/nrc2342

Published online

7 February 2008

Alkylating agents

Electrophilic compounds that

are reactive either directly or

following metabolism and bind

covalently to electron-rich

atoms in DNA bases (that is,

oxygen and nitrogen).

Antimetabolites

Compounds with similar

chemical structures to

nucleotide metabolites that

interfere with nucleotide

biosynthesis or are

incorporated into DNA.

DNA repair pathways as targetsfor cancer therapyThomas Helleday*, Eva Petermann*, Cecilia Lundin*, Ben Hodgson*and

Ricky A. Sharma*

Abstract | DNA repair pathways can enable tumour cells to survive DNA damage that is

induced by chemotherapeutic treatments; therefore, inhibitors of specific DNA repair

pathways might prove efficacious when used in combination with DNA-damaging

chemotherapeutic drugs. In addition, alterations in DNA repair pathways that arise duringtumour development can make some cancer cells reliant on a reduced set of DNA repair

pathways for survival. There is evidence that drugs that inhibit one of these pathways in

such tumours could prove useful as single-agent therapies, with the potential advantage

that this approach could be selective for tumour cells and have fewer side effects.

R E V I E W S

NATURE REVIEWS |CANCER VOLUME 8 |MARCH 2008 |193
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Non-homologous end

joining

Connection and resealing of

the two ends of a DNA double-

strand break without the need

for sequence homology

between the ends.

Homologous recombinationA process that can copy a DNA

sequence from an intact DNA

molecule (often the newly

synthesized sister chromatid)

to repair or bypass replication

lesions.

Base-excision repair

A repair pathway that replaces

missing or modified DNA

bases, such as those produced

by alkylating agents or in

spontaneously degraded DNA,

with the correct DNA base.

Nucleotide-excision repairA process that removes large

DNA adducts or base

modifications that distort the

double helix and uses the

opposite strand as template

for repair.

Alkyltransferases

A class of enzymes that

directly reverse DNA base

modifications that are induced

by alkylating agents by

transferring the alkyl group

from the base onto the

protein.

depleting cells of dNTPs. They can also impair replicationfork progression by becoming incorporated into the DNA7.In general, the molecular mechanisms through whichanti-metabolites induce cell death are poorly understood.

Another means of interfering with replication is toexploit DNA strand breaks that arise naturally during theprocess of DNA synthesis. Topoisomerases are a groupof enzymes that resolve torsional strains imposed onthe double helix during DNA replication. They inducetransient DNA breaks to relax supercoiled DNA or allowDNA strands to pass through each other8. Resealing ofthese breaks can be prevented by the use of topoisomer-ase poisons that trap the enzymes in complex with theDNA. The nature of the damage that is caused dependson which type of enzyme is targeted. Topoisomerase IIpoisons cause DSBs, and topoisomerase I poisons causepositive supercoils in advance of replication forks134and replication-associated DSBs1,2. This is a strategycommonly used for cancer treatment.

Ionizing radiation and radiomimetic agents such asbleomycincause replication-independent DSBs that cankill non-replicating cells. In addition, such treatmentscan also rapidly prevent DNA replication by activation of

cell-cycle checkpoints to avoid formation of toxic DNAreplication lesions9.

Cell-cycle checkpoints are regulated by effector kinases,such as ataxia telangiectasia mutated (ATM) and ATMand Rad3-related (ATR)1012, which regulate the activitiesof downstream checkpoint proteins, such as checkpointkinases 1 (CHK1) and 2 (CHK2). Defects in DNA dam-age checkpoint pathways result in sensitivity to a range ofanticancer treatments, for example, loss of ATM results insensitivity to ionizing radiation13. The triggering of thesecheckpoints and subsequent DNA repair activity largelydetermines the efficacy of anticancer drugs in causingtumour regression.

Efficient repair of chemotherapy lesions

Direct DSBs are mainly repaired by non-homologousend joining14, whereas replication-associated DSBsare repaired by homologous recombination(HR)15andrelated replication repair pathways. DNA adducts, suchas those created by alkylating agents, may be excisedand repaired before they are confronted by the replica-tion machinery. This is achieved by base-excision repair,excising a single damaged DNA base or a short strandcontaining the damaged base16or nucleotide-excisionrepair(NER), which excises a single-stranded DNAmolecule of approximately 2430 base pairs contain-ing the DNA lesion17,18. Damaged DNA can also berepaired without removal of the damaged base, in aprocess that directly reverses the DNA alkylation19. TheO-6-methylguanine-DNA methyltransferase (MGMT)is an alkyltransferase that removes alkylations on the O6position of guanine produced by anticancer drugs suchas temozolomide20, and the DNA dioxygenasesABH2(also known as ALKBH2) and ABH3 (also knownas ALKBH3) revert 1-methyladenine and 3-methyl-

cytosine back to adenine or cytosine respectively21. Therepair of alkylated lesions is thought to be quick, withthe majority of lesions probably being repaired withinone hour. If the lesions are removed before the initia-tion of replication, the efficiency of alkylating agents inkilling the tumour is significantly reduced. Thus, modu-lation of DNA repair clearly influences the efficacy ofalkylating agents, and resistance to alkylating agents isoften explained by increased expression and/or activityof DNA repair proteins.

Whereas most DNA repair pathways mediateresistance to DNA damage, mismatch repairis actuallyrequired for the toxicity of several anticancer drugs(FIG. 1). This has been explained by the futile repaircycle model in which mismatch repair removes thenewly inserted intact base instead of the damaged base,triggering subsequent rounds of futile repair which canbe deleterious to the cell22. It is also possible that mis-match repair might have an important role in triggeringcheckpoint signalling and apoptosis, which might medi-ate increased cytotoxicity23. It has been established thata defect in mismatch repair is associated with resistanceto many, but not all, DNA-damaging anticancer agents,such as monofunctional alkylating agents and cispla-tin, as well as the antimetabolite 6-thioguanine7,22,24. Itshould be noted that mismatch repair acts directly atreplication forks and can therefore not prevent them

from encountering damage.Collapse of replication forks during DNA synthesis

can be avoided by bypassing DNA lesions in a processcalled translesion synthesis25,26. This process is carried outby switching the regular polymerases, and , whichare responsible for leading and lagging strand synthesis,respectively27,28, to polymerases with different substratespecificities, thus enabling them to bypass different typesof damaged bases29.

Once a replication fork stalls or collapses, otherrepair pathways are required to permit resumption ofreplication. Collapsed replication forks are recognizedby the checkpoint machinery, which will in turn trigger

At a glance

Several cancer chemotherapy drugs work by producing excessive DNA damage that

causes cell death directly or following DNA replication. Survival is promoted through

repair of these lesions by a number of DNA repair pathways.

The efficacy of anticancer drugs is highly influenced by cellular DNA repair capacity.

Inhibitors of DNA repair increase the efficacy of DNA-damaging anticancer drugs in

preclinical models. Small-molecule inhibitors of DNA repair have been combined

with conventional chemotherapy drugs in several phase III clinical trials.

Tumour development can be associated with perturbed DNA damage response and

repair pathways. This perturbation results in reduced DNA repair capacity and

increased genetic instability in tumour cells. Defects in one DNA repair pathway can

be compensated for by other pathways. Such compensating pathways can be

identified in synthetic lethality screens and then specifically targeted for treatment

of DNA repair-defective tumours.

Evidence indicates that inhibitors of DNA repair pathways can work as single agents

for the targeted treatment of DNA repair-defective cancers. This hypothesis is

currently being tested in phase II trials in which patients with breast or ovarian

cancers that are defective in homologous recombination are being treated with a

poly(ADP-ribose) polymerase inhibitor.

Tumours often exhibit replication stress as a consequence of oncogene-induced

growth signals or hypoxia-induced replication arrest. We propose that DNA repair

inhibitors could be used to prevent the repair of replication lesions present in tumourcells and convert them into fatal replication lesions that specifically kill cancer cells.

R E V I E W S

194 |MARCH 2008 |VOLUME 8 www.nature.com/reviews/cancer
http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39139http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=472http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=545http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=1111http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=11200http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=4255http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=121642http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=221120http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39515http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39515http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=43669http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=43669http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39515http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39515http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=221120http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=121642http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=4255http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=11200http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=1111http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=545http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=472http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39139


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|

OH

Toxic lesions

Single-strand breaks

Double-strand breaks

Base damage

Includes mismatchrepair-mediatedtoxicity

No

Major repair pathways

HR

HR

HR

HR

HR

YesAlkylsulphonates

Nitrosourea compounds

Temozolomide

b Monofunctional alkylators

c Bifunctional alkylators

Nitrogen mustard

Mitomycin C

CisplatinNER

NER

AT

Yes

?

Cancer treatment

Yes BER

BER

BER

d Antimetabolites

5-Fluorouracil (5FU)

Thiopurines

Folate analogues

NHEJ

NHEJ

NHEJ

SSBR

SSBR

e Topoisomerase inhibitors

Camptothecins

Etoposide (VP16)

f Replication inhibitors

Aphidicolin

Hydroxyurea

a Radiotherapy and radiomimetics

Ionizing radiation

Bleomycin

TLS

TLS

No

No

FA

FA

FA

FA

O2G

RecQ

RecQ

RecQ

RecQ


Replication lesions


Single-strand breaks

Replication lesions


DNA crosslinks

Bulky adducts

Replication lesions

Base damage

Bulky adducts

Replication lesions

ENDO

ENDO

ENDO

ENDO

Replication lesions

Uncharacterized

Base damage

Pt

H3CS

O

OO

H3CS

OCH3

OO

CH3

N

N

NH

N

SH

H2N

H3NPt

NH3

Cl Cl

DNA dioxygenases

A class of enzymes that

directly reverse DNA base

methylations through an

oxidation mechanism. The

human DNA dioxygenase

ABH2 is thought to act at

replication forks.

Mismatch repair

A process that acts during

DNA replication to correct

base-pairing errors made by

the DNA polymerases.

Figure 1 |Overview of DNA repair pathways involved in repairing toxic DNA lesions formed by cancer

treatments. The DNA-damaging agents that are used in cancer treatment induce a diverse spectrum of toxic DNA

lesions. These lesions are recognized by a variety of DNA repair pathways which are lesion-specific but are

complementary in some respects. a| Ionizing radiation and radiomimetic drugs induce double-strand breaks

(DSBs) that are predominantly repaired by non-homologous end joining (NHEJ). b, c| Monofunctional alkylators (b)

and bifunctional alkylators (c) induce DNA base modifications, which interfere with DNA synthesis. Lesionsproduced by some alkylators are processed into toxic lesions in a mismatch repair-dependent manner. The base-

excision repair (BER) and nucleotide-excision repair (NER) pathways are, together with alkyltransferases (ATs),

major repair pathways, whereas other repair pathways repair toxic replication lesions, such as those produced by

interstrand crosslinks. d| Antimetabolites interfere with nucleotide metabolism and DNA synthesis, causing

replication lesions which have not yet been characterized. Mismatch repair mediates the toxicity of some

antimetabolites (for example, thiopurines). The repair pathways involved in repair of antimetabolite-induced

lesions are, apart from BER, poorly characterized. e| Topoisomerase poisons trap topoisomerase I or II in transient

cleavage complexes with DNA, thus creating DNA breaks and interfering with replication. f| Replication inhibitors

induce replication fork stalling and collapse, resulting in indirect DSBs. The relative contributions of the major

repair pathways to the respective types of DNA damage outlined are indicated by the sizes of the boxes. This is

based on the extent of sensitivity of repair-deficient cells to anticancer drugs in each category. ENDO, endonuclease-

mediated repair; FA, Fanconi anaemia repair pathway; HR, homologous recombination; O2G, DNA dioxygenases;

RecQ, RecQ-mediated repair; SSBR, DNA single-strand break repair; TLS, translesion synthesis.

R E V I E W S



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Translesion synthesis

A mechanism during DNA

replication in which the

standard DNA polymerase is

temporarily exchanged for a

specialized polymerase that

can synthesize DNA across

base damage on the template

strand.

Fanconi anaemia repair

pathway

Proteins of this pathway,

including BRCA2, are mutated

in the hereditary disorder

Fanconi anaemia (FA), resulting

in hypersensitivity to inter-

strand crosslinks. Evidence

suggests that the FA pathway

promotes the repair of stalled

replication forks, possibly by

activating HR and facilitating

ATR- and ATM-dependent

checkpoint signalling.

Endonuclease-mediated

repair

A repair pathway that

introduces a DNA single-strand break in a DNA

structure to facilitate

continuous repair.

RecQ-mediated repair

A repair pathway that unwinds

complex DNA structure to

facilitate repair.

Therapeutic index

The therapeutic index

describes the ability of a

treatment strategy to kill

cancer cells in preference to

cells in normal tissues.

cell-cycle arrest12, DNA repair30or cell death throughapoptosis or senescence3133. Although we know littleof the nature of replication lesions, there is an increas-ing body of information concerning pathways thatrepair them. HR has a central role in the repair of mostreplication lesions formed by anticancer drugs5,6,15,34.There are several ways by which HR is used to restart

replication. The sequence identity between two newlysynthesised DNA molecules can be used to restart rep-lication behind the replication block. Also, recombina-tion can be used to bypass DNA lesions in a processcalled template switching35. Other repair pathwaysactive at replication forks involve the Fanconi anaemia(FA) repair pathway36, endonuclease-mediated repair, suchas that mediated by the MUS81endonuclease37, andRecQ-mediated repair, which involves DNA helicasessuch as Bloom syndrome (BLM)38, Werner syndrome(WRN)39,40and other members of the RecQ family ofhelicases41. Several of the proteins in these pathwayshave been found to be directly linked with HR42or theresolution of recombination products such as Holliday

junctions38,40,43,44. However, cells that are defective inthese pathways show distinct differences from HR-defective cells, indicating that they represent differentbut overlapping repair pathways45.

Cells defective in a specific DNA repair pathwayexhibit sensitivity to drugs producing DNA lesions thatare normally repaired by that pathway. This sensitivityhas been exploited to isolate hamster cell lines showinghypersensitivity to cancer treatments such as etoposide,mitomycinCand ionizing radiation, and also to allowcloning of genes involved in DNA repair46. The DNArepair pathways involved in the repair of damage causedby various anticancer agents are summarized in FIG. 1.

These DNA repair pathways can have increased activityin tumour cells, resulting in resistance to chemotherapeu-tic drugs47. Importantly, these DNA repair pathways canbe inhibited pharmacologically to potentially increase theefficacy or specificity of anticancer agents.

DNA repair inhibitors in combination therapy

The basic understanding of DNA repair, from theprinciples of the DNA lesions created to the pathwaysthat are capable of repairing these lesions, has increasedconsiderably during recent years. This knowledgepermits rational combination of cytotoxic agents andinhibitors of DNA repair to enhance tumour-cell killing.

Understanding lesions and repair pathways enables theuse of DNA-repair inhibitors to exploit tumour defectsor cancer-specific replication lesions (BOX 1). Severalinhibitors of DNA repair have been developed as clinicalagents and clinical trials are ongoing (TABLE 1).

Sensitizers to alkylating agents. Despite the adverse sideeffects caused by alkylating agents on bone marrow andother normal tissues, drugs such ascyclophosphamide,ifosfamide, chlorambucil, melphalanand dacarbazineremain some of the most commonly prescribedchemotherapies in adults and children with varioussolid and haematological malignancies, particularly incombination with anthracyclines and steroids in multi-agent regimens. More recently, a DNA alkylator andmethylator developed in the 1980s, temozolomide(anoral prodrug that crosses the bloodbrain barrier), haschanged clinical practice in the treatment of high-gradegliomas in adults and children48,49.

A class of agents currently being tested in clinicaltrials in combination with temozolomide therapy con-

sists of the pseudosubstrates for MGMT. The lead com-pounds in this class have been O6-benzylguanine50andlomeguatrib (AstraZeneca, London, UK); the latter isalso known as O6-(4-bromothenyl)guanine or PaTrin-2.Resistance to O6-alkylating agents can be overcome inpreclinical models by depletion of MGMT51and a rela-tionship exists between MGMT activity and resistanceto chloroethylating nitrosoureas and methylating agentsin tumour cells grown in vitro and in xenograft mod-els52. O6-Benzylguanine and lomeguatrib have recentlybeen tested in phase III clinical trials and biologicallyeffective doses have been established for both agents53.In the case of O6-benzylguanine, a phase I clinical trialnot only defined the maximum tolerated dose (MTD)of a single dose of temozolomide when combined withO6-benzylguanine, but it also determined the doseof O6-benzylguanine that was effective in producingcomplete depletion of tumour MGMT activity for48 h54. However, results obtained so far indicate that,when used in combination with cytotoxic chemo-therapy, myelosuppression is significantly enhancedby O6-benzylguanine and lomeguatrib, necessitatingsignificant reductions in the doses of alkylating agentsprescribed from those used in standard chemotherapy55.On account of this lack of selectivity for malignant tis-sue versus normal bone marrow, no improvement in thetherapeutic indexhas so far been demonstrated in clinical

trials of these agents.The combination of temozolomide with inhibitors

of poly(ADP-ribose) polymerase 1 (PARP1) is currentlyunder investigation in several clinical trials (TABLE 1).PARP1 is required for the efficient base-excision repairof apurinic sites, the intermediate DNA lesions inducedby temozolomide, and inhibition of PARP1 retards therepair of these lesions. It was originally shown in 1980by Durkacz and Shall that specific inhibitors of PARPcan prevent the rejoining of DNA strand breaks that arecaused by dimethyl sulphate, resulting in demonstrablecytotoxicity in vitro56. However, in the absence of PARP1inhibition, apurinic sites are not generally regarded

Box 1 | Strategies using DNA repair inhibitors in cancer treatment

DNA repair inhibitors can be used in combination with a DNA-damaging anticancer

agent. This will increase the efficiency of the cancer treatment by inhibiting DNA

repair-mediated removal of toxic DNA lesions.

DNA repair inhibitors can be used as monotherapy to selectively kill cancer cells

with a defect in the DNA damage response or DNA repair. Synthetic lethal

interactions between a tumour defect and DNA repair pathway can be used to

identify novel treatment strategies.

In the future, DNA repair inhibitors could potentially be used to amplify tumour-

specific replication lesions to selectively kill cancer cells. This strategy would take

advantage of cancer-specific replication stress caused by oncogenes or the tumour

microenvironment.

R E V I E W S

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=80198http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=641http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=7486http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39207http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=42674http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=42674http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=42674http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39748http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39748http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=40024http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=43113http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=42973http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39768http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=41671http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=142http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=142http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=41671http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39768http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=42973http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=43113http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=40024http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39748http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=42674http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39207http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=7486http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=641http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=80198


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as major contributors to the cytotoxicity induced bytemozolomide. The success of the treatment rationaleadopted by current clinical trials of GPI-21016 (GuilfordPharmaceuticals, Baltimore, Maryland, USA), INO-1001(Inotek Pharmaceuticals, Beverly, Massachusetts, USA)and AG-014699 (Pfizer GRD, La Jolla, California, USA)depends on the overall biological role of and necessityfor PARP in cancer cells that are trying to repair theDNA damage induced by temozolomide. The role ofPARP in DNA repair has not been fully elucidated andadditional roles for PARP in DNA damage signalling orrepair might explain the increased toxicity of combina-tion treatments57,58.

Platinum chemotherapies.Cisplatin, carboplatinandoxaliplatinhave become three of the most commonly

prescribed chemotherapeutic drugs used to treat solidcancers in patients59. Platinum resistance, either intrin-sic or acquired during cyclical treatment, is a majorclinical problem as additional agents that can be addedto therapy in order to circumvent tumour resistance donot currently exist.

Platinum chemotherapy is now being tested with PARPinhibition in two clinical trials (TABLE 1). The rationale forcombining PARP inhibition with platinum chemotherapyis based on preclinical observations that PARP inhibitorspreferentially kill neoplastic cells andinduce completeor partial regression of a wide variety of human tumourxenografts in nude mice treated with platinum chemo-

therapy6062. For example, ABT-888 (Abbott Laboratories,Chicago, Illinois, USA), a potent inhibitor of PARP1 andPARP2,has been shown to potentiate the regressionofestablished tumours induced by temozolomide, cisplatin,carboplatin or cyclophosphamide therapy in rodent ortho-topic and xenograft models63. However, as stated above,the full function of PARP in DNA repair is not clear57,58,and as a result the biological mechanisms of chemosensiti-zation of cancer cells to platinum chemotherapy by PARPinhibition remain to be resolved. Interestingly, as a mono-therapy in these preclinical models,ABT-888 exhibits nosignificant anticancer activity.

DNA demethylating agents such as 2 -deoxy-5-azacytidine (decitabine; MGI Pharma, Bloomington,Minnesota, USA) have been combined with cisplatinor carboplatin to reverse drug resistance caused by the

silencing of mismatch repair genes by hypermethylation.The toxicity of agents such as cisplatin depends at leastpartly on functional mismatch repair (FIG. 1). Preclinicaldata from xenograft models and translational studiesfrom drug-resistant cells and tissues that are mismatchrepair-deficient owing toMLH1hypermethylation havedemonstrated increased chemotherapeutic efficacywhen a demethylating agent is combined with platinumchemotherapy64,65. Decitabine is currently being testedin combination with carboplatin in a phase II clinicaltrial in patients with ovariancancer(see Decitabineand Carboplatin in Relapsed Ovarian Cancer in Furtherinformation).

Table 1 | Ongoing clinical trials of small-molecule inhibitors of the DNA damage response and related signalling pathways

Agent(company)

Target moleculeor pathway

Monotherapyor combinationtherapy agent(s)

Phase of clinical trialplanned, ongoing orrecently completed

Reference

AZD-2281(Astra Zeneca)

PARP GemcitabineCarboplatinTopotecan

Monotherapy

Phase IPhase IPhase I

Phase II

http://www.astrazenecaclinicaltrials.com/article/525925.aspx

AG014699(Pfizer)

PARP TemozolomideantibodyTemozolomide

Phase I

Phase II

http://www.eddn.org/clinicalTr_caResUK.html

INO-1001 (Inotek) PARP Temozolomide Phase I http://www.inotekcorp.com/content/ino-1001.asp

BSI-201(Bipar Sciences)

PARP MonotherapyGemcitabinecarboplatin

Phase IPhase II

http://www.biparsciences.com/BSI201.html

ABT-888 (AbbottLaboratories)

PARP Temozolomide Phase I NCT00526617

TRC-102(Tracon Pharma)

BER TemozolomidePemetrexed

Phase IPhase I planned

http://www.traconpharma.com/content/pipeline_overview.html

Lomeguatrib(Astra Zeneca)

MGMT IrinotecanTemozolomide

Phase IPhase II

http://www.astrazenecaclinicaltrials.com/article/525925.aspx

O6-Benzylguanine MGMT Temozolomide Phase II http://clinicalstudies.info.nih.gov/cgi/detail.cgi?A_2006-C-0089.html

Decitabine(MGI Pharma121)

Hypermethylationof mismatchrepair genes

Epirubicin,cisplatin,5-fluorouracilCarboplatin

Phase I

Phase II

http://pfsearch.ukcrn.org.uk/StudyDetail.aspx?TopicID=&StudyID=2192

XL844 (Exilixis122) CHK1, CHK2 Gemcitabine Phase I planned http://www.exelixis.com/pipeline_xl844.shtml

The recent or current stage of development of clinical trials is indicated for individual compounds, which are grouped by molecular target. BER, base excisionrepair; CHK, checkpoint kinase; MGMT, O-6-methylguanine methyltransferase; PARP, poly(ADP-ribose) polymerase.

R E V I E W S

http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39176http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=42374http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=10038http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39299http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=4292http://www.cancer.gov/cancertopics/types/ovarianhttp://www.astrazenecaclinicaltrials.com/article/525925.aspxhttp://www.astrazenecaclinicaltrials.com/article/525925.aspxhttp://www.eddn.org/clinicalTr_caResUK.htmlhttp://www.inotekcorp.com/content/ino-1001.asphttp://www.biparsciences.com/BSI201.htmlhttp://clinicaltrials.gov/ct/show/NCT00526617http://www.traconpharma.com/content/pipeline_overview.htmlhttp://www.traconpharma.com/content/pipeline_overview.htmlhttp://www.astrazenecaclinicaltrials.com/article/525925.aspxhttp://www.astrazenecaclinicaltrials.com/article/525925.aspxhttp://clinicalstudies.info.nih.gov/cgi/detail.cgi?A_2006-C-0089.htmlhttp://clinicalstudies.info.nih.gov/cgi/detail.cgi?A_2006-C-0089.htmlhttp://pfsearch.ukcrn.org.uk/StudyDetail.aspx?TopicID=&StudyID=2192http://pfsearch.ukcrn.org.uk/StudyDetail.aspx?TopicID=&StudyID=2192http://www.exelixis.com/pipeline_xl844.shtmlhttp://www.exelixis.com/pipeline_xl844.shtmlhttp://pfsearch.ukcrn.org.uk/StudyDetail.aspx?TopicID=&StudyID=2192http://pfsearch.ukcrn.org.uk/StudyDetail.aspx?TopicID=&StudyID=2192http://clinicalstudies.info.nih.gov/cgi/detail.cgi?A_2006-C-0089.htmlhttp://clinicalstudies.info.nih.gov/cgi/detail.cgi?A_2006-C-0089.htmlhttp://www.astrazenecaclinicaltrials.com/article/525925.aspxhttp://www.astrazenecaclinicaltrials.com/article/525925.aspxhttp://www.traconpharma.com/content/pipeline_overview.htmlhttp://www.traconpharma.com/content/pipeline_overview.htmlhttp://clinicaltrials.gov/ct/show/NCT00526617http://www.biparsciences.com/BSI201.htmlhttp://www.inotekcorp.com/content/ino-1001.asphttp://www.eddn.org/clinicalTr_caResUK.htmlhttp://www.astrazenecaclinicaltrials.com/article/525925.aspxhttp://www.astrazenecaclinicaltrials.com/article/525925.aspxhttp://www.cancer.gov/cancertopics/types/ovarianhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=4292http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39299http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=10038http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=42374http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=39176


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Synthetic lethality

A genetic phenomenon in

which the combination of two

otherwise non-lethal

mutations results in an

inviable cell. Synthetic lethal

phenotypes are indicative of

an interaction between the

products of the two mutant

genes within the cell.

It has been shown in preclinical models that the majorcisplatin intra-strand crosslinks formed in DNA are rec-ognized and repaired by the mammalian NER pathway66.One biological predictor of clinical outcome for patientswith completely resected non-small-cell lung cancerreceiving adjuvant cisplatin-based chemotherapy isexpression of the excision repair cross-complementationgroup 1 (ERCC1) protein67. An immunohistochemicalstudy of 761 operative specimens of non-small-cell lungcancer tissue taken before a proportion of the patients inthe study received adjuvant chemotherapy showed that asignificant benefit from cisplatin-based adjuvant chemo-therapy was associated with the absence of ERCC1 in56% of the samples studied. The results suggested thatpatients with completely resected non-small-cell lungcancer and ERCC1-negative tumours appeared tobenefit from adjuvant cisplatin-based chemotherapy,whereas patients with ERCC1-positive tumours did not.However, it should be noted that, among the patientswho did not receive adjuvant chemotherapy, those withERCC1-positive tumours survived significantly longer

than those with ERCC1-negative tumours. It is thereforeconceivable from the results of this study that ERCC1might not represent a biomarker specific to cisplatin-based chemotherapy, but it might represent a marker ofprognosis and response to combination chemotherapy(not necessarily specific to cisplatin) in a poor-prognosissubset of patients, as has recently been demonstrated forp53overexpression in a similar patient subgroup68.

Although it has been suggested that ERCC1 is apotential anticancer drug target, the protein has noknown enzymatic activity, making the means of regu-lating its activity harder to decipher. Pharmacologicalmodulation of ERCC1 might therefore be less desirablethan a greater understanding of the clinical relevance ofproteinprotein interactions within the NER machin-ery or between ERCC1 and other repair pathways, aspotential targets for improving the efficacy of platinumchemotherapies. For example, UCN-01(7-hydroxy-staurosporine) is an anticancer agent that potentiatescisplatin and carboplatin toxicity (shown in preclinicalmodels and a phase I clinical trial, respectively), whichhas been shown to interfere with the interaction ofERCC1 and another component of the NER pathway,xeroderma pigmentosum A (XPA), in vitro69.

Attenuators of checkpoint signall ing.An alternativeapproach to modulating DNA repair activity and poten-

tially improving the therapeutic index is to interfere withcell cycle checkpoint signalling. XL844(EXEL-9844) isa small-molecule inhibitor of CHK1 and CHK2. Thisdrug causes inhibition of cell-cycle arrest, progressiveDNA damage, inhibition of DNA repair and, ultimately,tumour cell apoptosis in cancer cells grown in vitro70,although the outcome of inhibiting CHK1 and CHK2in general can vary in a cell type-dependent manner.Although concerns have been raised about the potentialtoxicity to normal cells of an approach which inhibitsboth CHK1 and CHK2 kinases71, preclinical data havesuggested that intermittent dosing with XL844 incombination with the deoxycytidine analoguegemcitabine

is well tolerated by female athymic nude mice used asa xenograft model70. XL844 is currently being testedin a clinical trial (NCT00475917) in combination withgemcitabine, which normally causes cell-cycle arrest andapoptosis by its incorporation into DNA.

Two kinases from the phosphatidylinositol 3-kinase(PI3K)-related protein kinase (PIKKs) family, ATM andATR, are central to cellular responses to DSBs. Whenactivated, ATM and ATR phosphorylate a multitude ofproteins, which initiate a cascade that induces cell-cyclearrest and facilitatesDNA repair. An inhibitor of ATMkinase activity, KU55933 (AstraZeneca), is currentlyin preclinical development. The rationale behind theclinical use of ATM inhibitors depends on the premisethat ATM inhibition should improve the therapeuticindex by hypersensitizing tumour cells to agents thatcause DSBs. Here again, the feasibility of inhibitingATM kinase in patients will depend on the level oftoxicity that such agents cause in normal tissues whenthey are used in combination with ionizing radiation orcytotoxic chemotherapy.

Radiosensitizers. DNA-dependent protein kinase(DNAPK, also known as PRKDC), a member of the PIKKfamily, is important for DSB repair by non-homologousend joining following ionizing radiation72. Cells defectivein DNAPK are highly sensitive to ionizing radiation73indicating that inhibition of DNAPK might sensitizetumours to radiation treatment. Wortmannin, a knowninhibitor of PIKKs at low nanomolar concentrations,has antiproliferative effects and is a radiosensitizer inpreclinical models74, but is unsuitable for clinical appli-cations owing to its inherent toxicity and instability incells75. Other small molecules that reversibly inhibitDNAPK kinase activity at low micromolar concentra-tions have been synthesized. They are currently in tran-sition from late preclinical development to early clinicaltrials. In particular, NU7441 (REF.76)has been shownto sensitize cells to topoisomerase II poisons and canalso function as a radiosensitizer in a manner consistentwith DNAPK inhibition77.

DNA repair inhibitors as monotherapy

As discussed above, most of the current small-moleculeinhibitors of DNA repair have so far been tested in earlyclinical trials as sensitizers of tumour cells to chemo-therapy. However, DNA damage also occurs spontane-ously in cells in the absence of treatments and DNA

repair pathways are therefore essential for the survivalof untreated cells. As several cancers are defective inDNA damage response and repair pathways (TABLE 2),the concept of synthetic lethal interactions can be used toadvocate the use of DNA repair inhibitors as monothera-pies (FIG. 2). DNA repair is an ideal target for inhibition incancer cells as the inhibitors should be exclusively toxicto cancer cells and therefore be associated with minimalside effects for patients (BOX 2).

Indeed, DNA repair inhibitors have been demon-strated to work as single agents in patients with DNArepair-defective tumours. The most notable example sofar is the use of PARP inhibitors to treat patients with

R E V I E W S

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2067http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=7157http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=42305http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=7507http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=469731http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=41213http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=41213http://clinicaltrials.gov/ct/show/NCT00475917jsessionid=94D09AADF5BBF70FC2857F92FBE6C03A?order=12http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5591http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5591http://clinicaltrials.gov/ct/show/NCT00475917jsessionid=94D09AADF5BBF70FC2857F92FBE6C03A?order=12http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=41213http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=469731http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=7507http://www.cancer.gov/Templates/drugdictionary.aspx?CdrID=42305http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=7157http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2067


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Table 2 | Synthetic lethal interactions of DNA repair and cell cycle checkpoint genes implicated in cancer by pathway

Protein Syndrome Primary cancers Biomarker SLIs S. cerevisiaehomologue

SLIs in S.cerevisiae (n)

HR

BRCA1, BRCA2 Breast, ovarian112, 135 PARP1 (REFS 78,79)

RAD54B NHL, colon cancer113 PARP1 (REF. 87) rdh54

RAD51B (RAD51L1) Lipoma, uterineleiomyoma114 PARP1 (REF. 87) rad51 31

CtIP (RBBP8) Colorectal cancer115 sae2 5

NHEJ

MRE11 Ataxiatelangiectasia-like disorder

Colorectal cancer108 IR sensitivity mre11 40

LIG4 LIG4 Leukaemia116 IR sensitivity lig4

Artemis (DCLRE1C) Omenn Lymphoma117 pso2

MMR

MSH2, MLH1, MSH6,PMS1, PMS2,MLH3

Hereditary nonpolyposiscolorectal cancer118120

Microsatelliteinstability

msh2, mlh1,msh6, pms1, mlh3

3

RecQ

BLM Bloom Various121

Increased SCE sgs1 43WRN Werner Various122 Increased

telomeric SCE sgs1 43

RECQL4 RothmundThomson

Skin basal and sqamouscell, osteosarcoma122

sgs1 43

Damage signalling

ATM Ataxia-telangiectasia

Leukaemia123 IR sensitivity PARP1(REFS 124,125),FANC89

tel1 2

NBS1 Nijmegenbreakage

Various126 IR sensitivity xrs2 30

p53 LiFraumeni Various127 -

CHK2 LiFraumeni Various128 dun1/rad53 37

NERXPA, XPC, DDB1,ERCC4,ERCC5, POLH

XP Skin cancers129 UV sensitivity rad14, rad4,rad1, rad2, rad30

11

ERCC2,ERCC3 XP, Cockayne,trichothio-dystrophy

Skin cancers129 rad25, rad3 8

ERCC1 Cerebro-oculo-facio-skeletal

Squamous cell carcinoma,head and neck130

rad10 6

FA

FANCA,FANCC,FANCD2, FANCE,FANCG

Fanconianaemia

Various131 Impaired FANCD2ubiqutination131

ATM89

FANCB, FANCF,FANCL Fanconianaemia


BRIP1 Fanconianaemia

Various131

FANCM Fanconianaemia


mph1 1

BER

POLB Various132 pol4

FEN1 Various133 rad27 104

Synthetic lethalities observed in mammalian cells and the number of synthetic lethal interactions (SLIs) for their Saccharomyces cervisiaehomologues are shown.The genes showing SLIs with DNA repair genes can potentially be used as targets for novel drugs that then may selectively kill cancer cells in monotherapy.A complete list of the SLIs in S. cervisiaecan be found in SupplementaryinformationS1(table). ATM, ataxia telangiectasia mutated; BER, base-excision repair;CHK, checkpoint kinase; ERCC, excision repair cross-complementation; FA, Fanconi anaemia-mediated repair; FANC, Fanconi anemia, complementation group;FEN1, flap structure-specific endonuclease 1; HR, homologous recombination; IR, infrared; MLH, mutL homologue; MMR, mismatch repair; MSH, mutShomologue; NER, nucleotide-excision repair; NHEJ, non-homologous end joining; PARP, poly(ADP-ribose) polymerase; RecQ, RecQ-mediated repair; SCE, sisterchromatid e xchange; UV, ultraviolet; XP, xeroderma pigme ntosum.

R E V I E W S

http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=25788http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5890http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=3981http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=64421http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2956http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2956http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5378http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5395http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5395http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=27030http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=9401http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=7508http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=1642http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2072http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2073http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2073http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5429http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2068http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2071http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2071http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2175http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2176http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2176http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2177http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2178http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2189http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2187http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2188http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=55120http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=55120http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=83990http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=57697http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5423http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2237http://www.nature.com/nrc/journal/v8/n3/suppinfo/nrc2342.htmlhttp://www.nature.com/nrc/journal/v8/n3/suppinfo/nrc2342.htmlhttp://www.nature.com/nrc/journal/v8/n3/suppinfo/nrc2342.htmlhttp://www.nature.com/nrc/journal/v8/n3/suppinfo/nrc2342.htmlhttp://www.nature.com/nrc/journal/v8/n3/suppinfo/nrc2342.htmlhttp://www.nature.com/nrc/journal/v8/n3/suppinfo/nrc2342.htmlhttp://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2237http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5423http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=57697http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=83990http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=55120http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2188http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2187http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2189http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2178http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2177http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2176http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2175http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2071http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2068http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=5429http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2073http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=2072http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=1642http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=7508http://www.ncbi.nlm.nih.gov/sites/entrez?Db=gene&Cmd=ShowDetailView&TermToSearch=9401http://www.ncbi.nlm.nih.gov/sites/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|

Targeted monotherapyCancer mutation

Replication lesion

Survival

a Initial cancer mutation

Lethal

b Inhibitor of Top3 in cancer cells

Survival

c Drug-resistant mutation d Second therapy in resistant cells

Lethal

SAE2mre11

SGS1

TOP3 mre11

SGS1

TOP3

sgs1

Gene function Drug-resistant mutation

Monotherapy Mutation Monotherapymre11 mre11SAE2

sgs1

inherited breastand ovarian cancers that lack wild-typecopies of the BRCA1or BRCA2genes78,79. BRCA1- andBRCA2-mutated cells are defective in HR repair80,81and show extensive replication-associated lesions82,83.These recombination-defective cells are 1001,000-fold more sensitive to PARP inhibitors than are theheterozygote or the wild-type cell lines, indicatingtheir potential to be exploited as specific treatments ofBRCA1-or BRCA2-defective tumours78,79. One explana-tion for this sensitivity is that PARP inhibitors inducesingle-strand breaks that can result in DSBs as a resultof stalled replication forks. Such lesions would normallybe repaired by HR, but this is prohibited in BRCA1- orBRCA2-deficient cancer cells79,8486. PARP activity is alsorequired for the actions of CHFR(checkpoint proteinwith forkhead-associated and ring finger domains)58andthe reactivation of stalled replication forks57. These func-tions might also explain the hypersensitivity to PARPinhibitors of recombination-defective cells. Translationof these observations has led to phase II clinical trials of

monotherapy using the PARP inhibitor AZD2281(AstraZeneca) currently recruiting patients with breastand ovarian cancer who harbour mutations in BRCA1orBRCA2genes. A separate phase II trial with the PARP1inhibitor AG014699 (Pfizer GRD) is due to open torecruitment of known carriers of BRCA1or BRCA2mutations with locally advanced or metastatic cancers ofthe breast or ovaries. It should be noted however that notall patients with mutations in BRCA1or BRCA2respondto PARP inhibitors as a monotherapy. The reasons forthis are currently under investigation. Cells that aredefective in recombination-related proteins other thanBRCA1 or BRCA2, such as RAD51, RAD54, XRCC2,

XRCC3, DSS1 (also known as SHFM1), replicationprotein A1 (RPA1), ATM, ATR, CHK1, CHK2, NBS1(also known as NBN) and components of the Fanconianaemia repair pathway, also show increased sensitivityto PARP inhibition79,87,88. This suggests that PARP inhibi-tors might also be suitable in treating several types oftumours with defects in HR.

Another synthetic lethal interaction has recentlybeen discovered between the Fanconi anaemia repairpathway and ATM. Two pancreatic tumour lines defec-tive in the Fanconi anaemia pathway were more sensitiveto the ATM inhibitor KU-55933 than isogenic controlcells89. This finding provides a rationale for studyingATM inhibitors in the treatment of Fanconi anaemiarepair-defective pancreatic cancer.

Finding new synthetic lethal DNA repair-based partner-

ships.As mutations in checkpoint and DNA repairpathways are associated with cancer (TABLE 2), it shouldbe straightforward to exploit DNA repair inhibitors

for the treatment of tumours carrying specific defectsin DNA repair or damage signalling (FIG. 2). Genome-wide screens in model organisms, such as the bud-ding yeast Saccharomyces cerevisiae, have helped toidentify protein interaction networks, which serve toelucidate protein functions within highly complex cel-lular processes. Such knowledge can prove extremelyuseful in identifying suitable targets for monotherapy,but perhaps more significantly those that could be usedin combination therapies. We have compiled a list ofreported mutations in DNA damage response genesfound in human tumours and homologous syntheticlethal interactions that have been demonstrated in

Figure 2 |Synthetic lethal interactions to identify molecular targets for inhibitors of DNA repair. A mutation in a

single tumour suppressor gene can result in genetic instability that can accelerate tumour progression90. Here, we use

synthetic lethal interactions in Saccharomyces cerevisiaeto illustrate a hypothetical scenario. a| We start with an

Mre11mutation that is found in mismatch repair-defective tumours108. b| The yeast mre11is synthetic lethal with

topoisomerase III (Top33)109. If the synthetic lethal interaction is conserved from yeast to man, inhibitors of Top3 would,

in theory, kill Mre11-mutated tumour cells. Top3-mutated yeast cells are viable, albeit with genetic instability110,

and thus Top3 inhibitors may specifically kill Mre11-mutated cancer cells while being tolerated in normal cells.

c| Replication repair networks are complex and an additional mutation might override synthetic lethal interactions. In

yeast, an additional mutation in sgs1, upstream of the hypothetically targetedTOP3pathway, reverses the synthetic

lethal phenotype

109

. Thus, in this scenario, an additional tumour mutation in the human homologue of SGS1, BLM, mightresult in resistance to Top3 inhibitors. d| sae2is in turn synthetic lethal with sgs1in yeast111. If this synthetic lethal

interaction is conserved, the human SAE2homologue CtIP (also known as retinoblastoma binding protein 8 (RBBP8))

might, in theory, then be targeted as a second-line therapy in Top3 inhibitor-resistant tumours that gained resistance

owing to a BLM mutation. In summary, understanding of DNA repair networks is important to identify pathways to

target and to circumvent resistance mechanisms.

R E V I E W S

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Biomarkers

A molecule or substance

whose detection indicates a

particular disease state or

treatment response.

Hypoxia

A subnormal concentration of

oxygen. In cancer tissue,

hypoxia is often the result of

abnormal vasculature.

S. cerevisiae(SupplementaryinformationS1(table)).

This information could be used to identify proteinsencoded by the human homologues of genes showingsynthetic lethal interactions that may represent goodtargets for specific treatment of cancers carrying muta-tions in DNA repair genes. Unfortunately, the poorlydefined biochemical properties of these proteins arestill a significant barrier to the exploitation of theseproteins as potential targets for drug intervention inthe immediate future.

A further limitation to exploiting these pathwaysis the lack of reliable biomarkersto aid the selection ofpatients that might respond to such treatments. This isparticularly important as cancers in patients that have no

DNA repair defect will not respond. The most reliable

biomarkers will probably be those that identify loss ofspecific post-translational modifications present in theDNA damage response and repair pathways, or thosethat indicate increased activity of the targeted pathway.

Future directions

Current chemotherapy regimens demonstrate that pro-duction of excessive replication lesions is a successfulmeans of killing cancer cells. It has been observed thattumour cells themselves exhibit a high level of endogenousreplication lesions that result in genetic instability32,33,90.In theory, DNA repair inhibitors could be used to impairthe repair of replication lesions that are present in tumourcells and convert them into fatal replication lesions thatspecifically kill cancer cells. For example, there is evidencethat the increased expression or activity of oncogenes caninduce replication stress32,33,91,92. In such tumours it mightbe possible to use DNA repair inhibitors to make existingcancer-specific replication lesions more toxic, resultingin fatal replication lesions selectively killing oncogene-expressing cancer cells32,33,91972(BOX 3).

More advanced cancers are exposed to another sourceof replication stress owing to the tumour microenviron-ment. Tumours are often hypoxic, which has been shownto disrupt DNA synthesis98. These conditions cause repli-cation lesions that activate the ATM- and ATR-mediatedcheckpoint response99101. Furthermore, DNA repair is

downregulated in hypoxic cells102, which cumulativelycontributes to the genetic instability observed in thesecells103,104. In hypoxic cancer cells, therefore, inhibitors ofthe checkpoint response could prove to be more efficientthan inhibitors of DNA repair105.

Conclusions

The potential of inhibitors of DNA repair in the futureof cancer therapy is starting to become apparent.Although selective inhibition of DNA repair pathwayscan be used to enhance current chemotherapy, themost attractive use of DNA repair inhibitors might bein using cancer defects for selective cell killing. DNA

Box 2 | Advantages and limitations using DNA repair inhibitors as single agents in treatment of cancers

DNA repair inhibitors can exploit tumour-specific defects in checkpoint signalling and DNA repair to convert

endogenous DNA lesions into fatal replication lesions that selectively kill tumour cells.

A general problem for novel cancer therapies is that they are not sufficiently efficient at killing cancer cells to

replace current cytotoxic therapies. As a result, some enzyme inhibitors (that do not target DNA repair) have failed

in late clinical trial development owing to a general lack of anti-tumour efficacy. Inhibition of DNA repair amplifies

toxic replication-associated DNA lesions that directly result in cell death. DNA repair inhibitors might therefore be

highly efficient at killing tumours.

Cross-talk between DNA repair pathways in normal cells minimizes side effects from the inhibition of a single DNA

repair pathway.

Tumour inactivation of DNA damage signalling and DNA repair are often relatively early events during carcinogenesis,

suggesting that non-toxic DNA repair inhibitors may be considered in the treatment of patients with pre-malignant or

early neoplastic lesions, such as those arising in patients with inherited mutations inBRCA1orBRCA2genes, or

intestinal lesions in patients with defects inMLH1andMSH2genes or their methylation status.

Cross-talk between DNA repair pathways, such as the cross-talk between homologous recombination and non-

homologous end joining in the repair of DNA double-strand breaks or the cross-talk between base-excision repair,

alkyltransferases and DNA dioxygenases in the repair of alkylation damage, is likely to result in acquisition of

resistance mechanisms in tumours, which is a limitation for killing more advanced tumours.

Box 3 | Targetting oncogene-induced replication stress

The transformation of normal cells to a cancerous state is often initiated by the

activation of oncogenes, which provide excessive growth signals106. It was recently

shown that such oncogene activation can induce replication-associated DNA

lesions32,33,91,92, including increased replication origin firing, re-replication and

impaired fork progression32,107. These replication-associated lesions trigger a cell-cycle

checkpoint response that stops the proliferation of pre-cancerous cells early during

neoplastic transformation32,33,9193. This involves checkpoint proteins, such as p53 and

CHK2, that can induce apoptosis or senescence94,95,

Genes encoding proteins in the checkpoint pathways are often mutated during

cancer development96, allowing cells to evade checkpoints and continue to

proliferate. Taken together, these observations suggest that a key feature of cancer

cells that overexpress oncogenes might be higher levels of endogenous replication-associated lesions than those present in normal cells. This in turn would contribute to

genetic instability97that would assist the tumour to induce the genetic changes

required for continuing transformation to malignant phenotypes90. More importantly,

the replication lesions caused by oncogene activation have been found to resemble

those produced by anticancer treatments32, and, like the latter, these lesions would

require repair for the cancer cells to survive. We therefore propose that future DNA

repair inhibitors should be used to make existing cancer-specific replication lesions

more toxic, resulting in fatal replication lesions selectively killing oncogene-

expressing cancer cells. The nature of the replication lesions that are produced

following chemotherapy or oncogene-induced stress are poorly understood.

Although several replication repair pathways have been identified, we currently have

little information regarding their complex interplay. Indeed, more intense basic

research is required in this area to identify novel anticancer targets.

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