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The ATM–Chk2 and ATR–Chk1Pathways in DNA Damage Signaling

and Cancer

s in CANCERt 2010, Elsev

Joanne Smith,* Lye Mun Tho,*,{ Naihan Xu,* andDavid A. Gillespie*,{

RESEARCHier Inc. All rights reserv

*Beatson Institute for Cancer Research, Garscube Estate,

Glasgow, UK
{Faculty of Medicine, University of Glasgow, Glasgow, UK
I.
I ntroduction
II.
A ctivation of the ATM–Chk2 and ATR–Chk1 DNA Pathways
III.
C heckpoint Functions of the ATM–Chk2 and ATR–Chk1 Pathways IV. T he Three Rs of Damage Signaling: Resection, Recombination, and Repair
V.
A TM–Chk2 and ATR–Chk1 Pathway Alterations in Cancer
VI.
E xploiting Homologous Recombinational Repair (HRR) Defects for Cancer Therapy VII. D NA Damage Signaling as a Barrier to Tumorigenesis
VIII.
C heckpoint Suppression as a Therapeutic Principle
IX.
F uture Perspectives
R
eferences
DNA damage is a key factor both in the evolution and treatment of cancer. Genomic

instability is a common feature of cancer cells, fuelling accumulation of oncogenic muta-tions, while radiation and diverse genotoxic agents remain important, if imperfect, thera-

peutic modalities. Cellular responses to DNA damage are coordinated primarily by two

distinct kinase signaling cascades, the ATM–Chk2 and ATR–Chk1 pathways, which areactivated by DNA double-strand breaks (DSBs) and single-stranded DNA respectively.

Historically, these pathways were thought to act in parallel with overlapping functions;

however, more recently it has become apparent that their relationship is more complex. In

response to DSBs, ATM is required both for ATR–Chk1 activation and to initiate DNArepair via homologous recombination (HRR) by promoting formation of single-stranded

DNA at sites of damage through nucleolytic resection. Interestingly, cells and organisms

survive with mutations in ATM or other components required for HRR, such as BRCA1

and BRCA2, but at the cost of genomic instability and cancer predisposition. By contrast,the ATR–Chk1 pathway is the principal direct effector of the DNA damage and replication

checkpoints and, as such, is essential for the survival of many, although not all, cell types.

Remarkably, deficiency for HRR in BRCA1- and BRCA2-deficient tumors confers sensi-tivity to cisplatin and inhibitors of poly(ADP-ribose) polymerase (PARP), an enzyme

required for repair of endogenous DNA damage. In addition, suppressing DNA damage

and replication checkpoint responses by inhibiting Chk1 can enhance tumor cell killing by

diverse genotoxic agents. Here, we review current understanding of the organization andfunctions of the ATM–Chk2 and ATR–Chk1 pathways and the prospects for targeting

DNA damage signaling processes for therapeutic purposes. # 2010 Elsevier Inc.

0065-230X/10 $35.00ed. DOI: 10.1016/S0065-230X(10)08002-4

73

74 Joanne Smith et al.

I. INTRODUCTION

Cells in multicellular organisms are continuously exposed to DNAdamage arising from a variety of endogenous and exogenous sources.These include reactive oxygen species, ultraviolet light, background radia-tion, and environmental mutagens. To protect their genomes from thisassault, cells have evolved complex mechanisms, collectively referred to asDNA damage responses, that act to rectify damage and minimize the proba-bility of lethal or permanent genetic damage. The cellular response to DNAdamage encompasses multiple repair mechanisms and checkpoint responsesthat can delay cell cycle progression or modulate DNA replication.Collectively, these processes are essential to maintain genome stability.DNA damage responses are orchestrated by multiple signal transduction

processes, key among which are the ATM–Chk2 and ATR–Chk1 pathways.Activation of these pathways is crucial for the proper coordination ofcheckpoint and DNA repair processes; however, they can also modulateother biological outcomes such as apoptosis or cell senescence. In recentyears, it has become evident that DNA damage responses are central bothfor the evolution and therapy of cancer. Inherited defects in DNA damageresponses predispose to cancer by enhancing the accumulation of oncogenicmutations, while genome instability is also common in sporadic cancers.More recently, it has become apparent that oncogenic mutations elicitspontaneous DNA damage that can suppress the evolution of incipientcancer cells. Escape from this tumor suppressive barrier may be a majorfactor in selecting for additional genetic changes during tumor progressionsuch as mutation of the p53 tumor suppressor, the most frequent alterationin human cancer.Conversely, radiation and genotoxic chemotherapies remain a mainstay of

conventional cancer treatment and are likely to remain so for the foreseeablefuture. Such therapies are, however, imperfect and can incur severe sideeffects. As a result, much current interest is focused on understanding hownormal and tumor cells respond to DNA damage and determining whetherDNA damage responses could be exploited or manipulated for therapeuticpurposes. Two concepts in particular have attracted attention in recentyears. First, inherent defects in genome stability mechanisms, such as ho-mologous recombination, can confer tumor sensitivity to specific genotoxicagents or inhibition of complementary repair pathways. Second, evidencesuggests that pharmacological suppression of DNA damage or checkpointresponses can enhance the efficacy of conventional genotoxic agents.Although promising, a full understanding of the biology and functions ofthe DNA damage signaling pathways will be crucial for the future success ofsuch approaches.

DNA Damage and Cancer 75

II. ACTIVATION OF THE ATM–CHK2 ANDATR–CHK1 DNA PATHWAYS

DNA damage responses are controlled by biochemical pathways whoseprincipal components and general organization have been conserved fromyeasts to humans (Rhind and Russell, 2000). In vertebrates, the two mainsignaling pathways activated by DNA damage consist of the ATM–Chk2and ATR–Chk1 protein kinases (Sancar et al., 2004). ATM and ATR arelarge kinases with sequence similarity to lipid kinases of the phosphatidyli-nositol-3-kinase (PI3K) family, but which phosphorylate only protein sub-strates (Abraham, 2001). Key among these substrates are the serine–threonine checkpoint effector kinases, Chk1 and Chk2, which are selectivelyphosphorylated and activated by ATR and ATM respectively to trigger awide range of distinct downstream responses (Bartek and Lukas, 2003).The ATM–Chk2 and ATR–Chk1 pathways respond to different aberrant

DNA structures (Fig. 1); ATM is recruited to and activated primarily atDNA double-strand breaks (DSBs) in conjunction with theMRE11:RAD50:NBS1 (MRN) sensor complex (Lee and Paull, 2005; Suzuki et al., 1999),whereas ATR is activated via recruitment to tracts of single-stranded DNA(ssDNA) in association with its partner protein, ATRIP (Dart et al., 2004;Lupardus et al., 2002; Zou and Elledge, 2003).The basic mechanisms involved in ATM–Chk2 and ATR–Chk1 pathway

activation have been elucidated in considerable detail. ATM and Chk2 areactivated potently by radiation and genotoxins that induce DSBs, but onlyweakly, if at all, by agents that block DNA replication without inducingdamage (Matsuoka et al., 2000). In undamaged cells, ATM is thought toexist as inactive homodimers. In response to DSBs, inactive ATM homo-dimers are rapidly induced to autophosphorylate in trans, resulting in disso-ciation to form partially active monomers (Bakkenist and Kastan, 2003).The exact nature of the primary activating signal that triggers ATM auto-phosphorylation remains unknown; however, it does not appear to be limit-ed to the immediate vicinity of the damage and may be linked to long-rangealterations in chromatin structure (Bakkenist and Kastan, 2003). Serine (S)S1981 was the first autophosphorylation site to be identified; however, thisresidue is not essential for ATM function, at least in mice (Pellegrini et al.,2006), although its modification is tightly linked to ATM activation undermost circumstances (Bakkenist and Kastan, 2003). Subsequent studies havedocumented additional ATM autophosphorylation sites at S367 and S1893that may contribute to the activation process, perhaps explaining whyS1981 is individually nonessential, while acetylation mediated by theTIP60 acetyl-transferase may also play a role (Lavin and Kozlov, 2007).

Claspin

TopBP1

RPA RPA RPA RPA

MDC153BP1

Chk2

DNA damage (DSBs)

Rad50NBS1

MRE11

ATM

g H2AX

P

P

P

Replication arrest

RPA RPA RPA RPA

gH2AX

RAD1

HUS1RAD9

P

Chk1

P P

G2, Intra-S checkpointsFork stabilization

Origin suppressionS-M checkpoint

p53

G1checkpoint

ATMP

TIP60 Ac

Ac

PP

P

P

P

ATR

ATRIP

Fig. 1 Activation of the ATM–Chk2 and ATR–Chk1 pathways The ATM–Chk2 and ATR–Chk1 pathways are activated selectively by DSBs and tracts of ssDNA complexed with RPA

respectively. Phosphorylation events are indicated by (P) in red, acetylation by (Ac) in yellow.

DSBs in chromatin stimulate ATM autophosphorylation and acetylation but full activation also

requires recruitment to sites of damage in conjunction with the MRN complex where ATMmodifies multiple substrates including the downstream effector kinase, Chk2, leading to Chk2

activation and downstream signal transduction. ATR is recruited to tracts of ssDNA-RPA

through its interacting partner, ATRIP, where it phosphorylates and activates Chk1 in conjunc-

tion with the TopBP1 and Claspin mediator proteins. Two additional mediator proteins,Timeless and Tipin, also contribute to ATR–Chk1 activation although their functions are less

well-understood and omitted here for clarity. The ultimate result of ATM–Chk2 and ATR–Chk1

signaling is the activation of multiple DNA damage and replication checkpoint responses thatare summarized in Fig. 2. Please refer to the text for further details and explanation.


ATMmonomers are then recruited to DSBs via interactions with the MRNsensor complex (Lee and Paull, 2007), stimulating full activation andproviding a platform that enables ATM to act locally on multiple substratesat the site of damage. Local substrates include the variant histone, H2AX,forming the DNA damage-associated �-H2AX histone mark (Fernandez-Capetillo et al., 2004), the MRN complex itself (discussed in more detailbelow), the cohesin SMC1 (Kitagawa et al., 2004), and the downstreameffector kinase Chk2 (Lukas et al., 2003). ATM phosphorylates Chk2 on a


specific threonine (T) residue, T68, located within an N-terminal serine/threo-nine-glutamine (SQ/TQ)-rich motif (Ahn et al., 2000). Once phosphorylated,the SQ/TQ motif of one Chk2 molecule is recognized by the phosphopeptide-binding Fork-head associated (FHA) domain of another, leading to transienthomodimerization, intermolecular activation loop autophosphorylation, andfull activation (Ahn et al., 2002; Cai et al., 2009; Oliver et al., 2006).Once activated, Chk2 is thought to dissociate from sites of damage and

disperse as a monomer throughout the nucleus to act on multiple substratesinvolved in cell cycle progression, apoptosis, and gene transcription (Lukaset al., 2003). Known substrates of Chk2 include the p53 tumor suppressorprotein (Chehab et al., 2000; Shieh et al., 2000) and its regulator MDMX(Chen et al., 2005), Cdc25 family phosphatases (Blasina et al., 1999;Chaturvedi et al., 1999;Matsuoka et al., 1998), the BRCA1 tumor suppressor(Lee et al., 2000), and transcription factors such as FOXM1 (Tan et al., 2007)and E2F1 (Stevens et al., 2003).It is important to note that ATM also targets other substrates at sites of

damage in addition to those mentioned above, including NBS1, BRCA1,MDC1, and p53BP1 among others (Lavin, 2008). In addition, ATM acts onother substrates which do not necessarily concentrate at sites of damage. Forexample, ATM plays an important role in activating the p53 response toDNA damage both by phosphorylating p53 itself and its stability regulators,MDM2 and MDMX (Chen et al., 2005; Lavin and Kozlov, 2007); however,this is considered to take place in the nucleoplasm rather than specifically atDSBs (Lavin, 2008). In addition, there is increasing evidence that ATM mayalso have substrates and functions in the cytoplasm (Lavin, 2008).By contrast, ATR–Chk1 signaling is activated most strongly when DNA

replication is impeded, for example as a result of nucleotide depletion orreplication-blocking DNA damage lesions such as those inflicted by ultravi-olet (UV) light (Abraham, 2001). When replication is blocked, DNA poly-merases become uncoupled from the replicative helicase (Byun et al., 2005),generating tracts of ssDNA that rapidly become coated with the trimericssDNA-binding protein complex, Replication Protein A (RPA). ATR isrecruited to and activated at such tracts in association with its partnerprotein, ATRIP, which interacts directly with ssDNA complexed with RPAvia the 70kD RPA1 subunit (Zou and Elledge, 2003).Replication fork stalling generates ssDNA directly; however, this structure

can also arise through the action of nucleotide excision repair (NER) or atdysfunctional telomeres. In addition, it is important to note that the ATR–Chk1 pathway is also activated in response to DSBs when ssDNA is gener-ated as a result of nucleolytic strand resection (discussed in more detailbelow). Conversely, replication of damaged DNA can result in DSBs whenleading-strand DNA polymerases encounter single-strand nicks or abasicsites. As a result, the ATM–Chk2 and ATR–Chk1 pathways are frequently


activated simultaneously in cells exposed to diverse genotoxic stresses,including ionizing radiation and most or all cytotoxic chemotherapy agents.Unlike ATM, there is currently no evidence that ATR activation involves

autophosphorylation or indeed any other posttranslational modification(Abraham, 2001). Instead, efficient ATR activation and downstream phos-phorylation of Chk1 depends on the actions of two mediator proteins,TopBP1 and Claspin. TopBP1, which is recruited to ssDNA-RPA via thePCNA-like RAD9: RAD1: HUS1 checkpoint clamp (Delacroix et al., 2007),contains a domain that stimulates ATR activity, although exactly how thisoccurs is unclear (Kumagai et al., 2006;Mordes et al., 2008). A secondmedia-tor, Claspin,which likely associateswith active replication forks during normalreplication (Lee et al., 2003), is then subject to ATR-dependent phosphoryla-tion within a short, repeated motif which, once modified, binds Chk1 andserves as a platform for ATR-mediated phosphorylation and activation (Guoet al., 2000). Phosphorylationwithin theClaspinChk1-bindingmotifs dependson ATR kinase activity (Kumagai and Dunphy, 2003); however, the modifiedresidues do not occur within consensus SQ/TQ) ATR target sites. So far thekinase directly responsible for this final crucial step in Chk1 activation has notbeen unambiguously identified; proposed candidates include ATR,Chk1 itself,and Cdc7 (Bennett et al., 2008; Chini and Chen, 2006; Kim et al., 2008).Interestingly, recent studies have also revealed a requirement for two addi-

tional mediators, Timeless and Tipin (Timeless-interacting protein), both fornormal replication and for ATR–Chk1 activation in response to replicationstress (Kondratov and Antoch, 2007). Timeless binds to both ATR and Chk1whereas Tipin can interact with Claspin (Kemp et al., 2010). Recent dataindicate that like ATRIP, Tipin binds to a specific subunit of the RPA complex(although RPA2 rather than RPA1) and is required for stable association ofboth Timeless and Claspin with tracts of ssDNA-RPA (Kemp et al., 2010).In addition to checkpoint activation, Timeless and Tipin also seem to berequired for replication fork stabilization and restart (Errico et al., 2007).Interestingly, the Drosophila homologue of Timeless is a circadian rhythmregulator, althoughwhether this function is also conserved inmammals is lessclear (Kondratov and Antoch, 2007).Phosphorylated Claspin then recruits Chk1 (Jeong et al., 2003) to ssDNA-

RPA complexes, bringing it into close proximity with active ATR (Kumagaiand Dunphy, 2003) and enabling ATR to phosphorylate Chk1 directly atmultiple S/T-Q sites within the C-terminal regulatory domain, most notablyat serines (S) S317 and S345, which are widely monitored as surrogatemarkers of activation. Phosphorylation of these sites, and in particularserine (S) S345, is essential for Chk1 biological activity, although exactlyhow these modifications regulate Chk1 catalytic remains poorly understood(Niida et al., 2007; Walker et al., 2009). ATR-mediated phosphorylation isreported to stimulate Chk1 kinase activity by relieving inhibition by the


C-terminal regulatory domain (Oe et al., 2001; Walker et al., 2009); how-ever, it may also promote release of Chk1 from chromatin (Smits et al.,2006). Chk1 also undergoes autophosphorylation during activation(Kumagai et al., 2004); however, this does not occur within the activationloop (Chen et al., 2000), and the exact target sites and functional conse-quences of this modification have not yet been clearly established.Once activated, Chk1 is thought to dissociate from Claspin to act on both

nuclear and cytoplasmic substrates (Lukas et al., 2003). Important Chk1substrates involved in cell cycle control include positive and negative regula-tors of Cdk inhibitory phosphorylation, such as Cdc25A (Falck et al., 2002),Cdc25C (Blasina et al., 1999), and Wee1 (Lee et al., 2001). Chk1-mediatedphosphorylation inhibits the activity of both Cdc25A and Cdc25C underconditions of genotoxic stress, although by different mechanisms; phosphor-ylation of Cdc25A targets the protein for degradation (Falck et al., 2002),while phosphorylated Cdc25C is sequestered in an inactive form throughassociation with 14-3-3 proteins (Peng et al., 1997). Wee1 kinase activity, bycontrast, is stimulated by Chk1-mediated phosphorylation (Lee et al., 2001).Chk1 is also thought to modulate recombination by phosphorylating Rad51(Sorensen et al., 2005) and BRCA2 (Bahassi et al., 2008), and to mediateDNA damage-induced repression of gene transcription through phosphory-lation of histoneH3 (Shimada et al., 2008). Although predominantly nuclear,a proportion of active Chk1 also localizes at the centrosome, where it isthought to control the timing of activation of the mitotic Cdk1/cyclinB complex, and thus the onset of mitosis, both after damage and duringunperturbed cell cycles (Kramer et al., 2004).As with ATM, ATR is also thought to act on many other substrates in

addition to Chk1, including BRCA1, mini-chromosome maintenance(MCM) proteins, and components of the RPA complex (Cimprich andCortez, 2008). In addition, global proteomic analyses suggest that ATMand ATR probably phosphorylate many other substrates; however, in mostcases, the functional significance of these modifications has not yet beenestablished (Matsuoka et al., 2007). In contrast to ATM and Chk2, however,ATR and Chk1 are thought to be active at low levels even during unper-turbed cell cycles, particularly during S-phase (Syljuasen et al., 2005), po-tentially explaining why they are essential in many cell types.

III. CHECKPOINT FUNCTIONS OF THE ATM–CHK2AND ATR–CHK1 PATHWAYS

DNA damage or DNA synthesis inhibition in vertebrate cells evokes theactivation of multiple, mechanistically distinct checkpoint responses thatfacilitate repair and promote cell survival (Kastan and Bartek, 2004).

S MG1 G2

DNA damage checkpoints

Replication checkpoints

Originfiring

Forkstabilization

Intra-Scheckpoint

S-M checkpoint

G1 checkpoint G2 checkpoint

Fig. 2 Multiple DNA damage and replication checkpoints in vertebrate cells DNA damage

and DNA synthesis inhibition evoke multiple, mechanistically distinct checkpoint responses in

vertebrate cells that are controlled by the ATM–Chk2 and ATR–Chk1 pathways. In response toDNA damage these can delay entry to S-phase (G1 checkpoint), slow the replication of damaged

DNA (intra-S checkpoint), or prevent entry to mitosis while damage persists (G2 checkpoint).

When DNA synthesis is inhibited disinct checkpoint responses are triggered that serve tostabilize stalled replication forks (fork stabilization), suppress the firing of latent replication

(origin suppression), and delay the onset of mitosis until DNA replication is complete (S-M

checkpoint). Please refer to the text for further details and explanation.


As shown in Fig. 2, DNA damage induces cell cycle delays at the G1/S andG2/M transitions (the G1 and G2 checkpoints), and a transient decrease inthe rate of DNA synthesis (the intra-S checkpoint). Of these, the G1 check-point is unique in depending primarily on the function of the p53 tumorsuppressor protein and its downstream target, the cyclin-dependent kinaseinhibitor p21CIP1 (Kastan and Bartek, 2004). G2 arrest, by contrast, isimposed by blocking activation of the mitotic Cdk1-cyclinB complex bypreventing removal of the inhibitory threonine 14/tyrosine 15 (T14/ Y15)phosphorylation of Cdk1 (O’Connell et al., 2000). This is achieved, at leastin large part, via inhibition of Cdc25 family phosphatases which play animportant role in reversing this inhibitory phosphorylation to rapidlyactivate the Cdk1-cyclin B complex and trigger the onset of mitosis(Boutros et al., 2007). The molecular mechanism of the intra-S checkpointis less well defined; however, it can involve both active replicationfork slowing and suppression of replication origin firing (Grallert andBoye, 2008; Seiler et al., 2007).When DNA synthesis is blocked, additional replication checkpoint

responses are required to stabilize stalled replication forks and prevent theformation of new forks by suppressing late replication origin firing (Branzei


and Foiani, 2009). Because these functions are essential if cells are to resumereplication and complete S-phase when circumstances permit, they aresometimes collectively termed the “replication recovery checkpoint.” Inaddition, it is essential that cells arrested in S-phase do not attempt mitosisuntil replication is complete. The mitotic delay triggered by DNA synthesisinhibition, which is also likely mediated through maintenance of inhibitoryphosphorylation of Cdk1 (O’Connell et al., 2000), is generally termed theS-M checkpoint to distinguish it from the G2 arrest induced by DNA damage.Checkpoint mechanisms were first dissected in detail in budding and

fission yeast. Each possesses an ortholog of Chk2; Rad53 in budding yeastand Cds1 in fission yeast, while both also express Chk1 homologs(O’Connell et al., 2000). The checkpoint functions of the yeast effectorkinases are, however, remarkably variable; in budding yeast Rad53 is thedominant effector of both DNA damage and replication checkpoints, where-as in fission yeast DNA damage responses are assigned to Chk1 while Cds1regulates replication checkpoint functions (O’Connell et al., 2000). WhenChk1 and Chk2 were identified in vertebrates, the obvious question was“what would be the ‘division of labor’ compared to these modelorganisms?”Initially it was widely considered that the ATM–Chk2 and ATR–Chk1

pathways acted in parallel, with Chk1 and Chk2 playing overlapping orpartially redundant roles in downstream checkpoint responses (as depictedin Fig. 1). Although some early studies suggested that Chk1 and Chk2shared certain common substrates involved in cell cycle arrest, such asCdc25 family phosphatases (Chaturvedi et al., 1999; Matsuoka et al.,1998), subsequent genetic and biochemical data have increasingly empha-sized ATR–Chk1 as the principal, direct effector of the DNA damageand replication checkpoints, with ATM–Chk2 playing an auxiliary rolespecifically in the response to DSBs (Bartek and Lukas, 2003; Kastan andBartek, 2004).Fundamental differences in the normal physiological functions of

the ATM–Chk2 and ATR–Chk1 pathways were initially evident from thephenotypes of mice and cells deficient for each pathway. Thus, germ-lineinactivation of ATR or Chk1 results in early embryonic lethality, whereasATM- and Chk2-knockout mice are viable, and at least in the case of Chk2,remarkably normal (Brown and Baltimore, 2000; Liu et al., 2000; Takaiet al., 2000). Similarly, ATM- and Chk2-deficient somatic cells can prolifer-ate successfully in culture (Jallepalli et al., 2003; Lavin and Shiloh, 1997; Xuand Baltimore, 1996), whereas acute genetic inactivation of ATR or Chk1leads to rapid cell death (Brown and Baltimore, 2003; Liu et al., 2000; Niidaet al., 2007). Interestingly, DT40 lymphoma cells represent an exception tothis general rule, since they survive genetic inactivation of Chk1 function,albeit with impaired cell growth and survival (Zachos et al., 2003).


ATM-deficient human and mouse cells classically exhibit impaired G1,intra-S, and G2 checkpoint proficiency after DNA damage (Lavin, 2008). Asmentioned previously, ATM is an important determinant of p53 stabiliza-tion and activation, and evidence suggests that the weakened G1 arrest inATM mutant cells is attributable to inefficient p53 activation and down-stream p21CIP1 induction (Kastan et al., 1992). The mechanism of theintra-S checkpoint is more complex. ATM is thought to suppress DNAreplication via two distinct mechanisms; firstly, via direct phosphorylationof NBS1, and secondly through Chk2-mediated degradation of Cdc25Aleading to inhibition of Cdk2, which in association with cyclins E or A isrequired for DNA replication origin firing (Falck et al., 2002).Interestingly, G2 checkpoint impairment in ATM mutant cells after expo-

sure to ionizing radiation is markedly cell cycle phase-dependent. Thus,ATM-deficient cells in G2 phase at the time of damage are unable to arrestefficiently, whereas cells damaged in G1 and S-phase experience instead aprolonged arrest on reaching G2 compared to genetically normal counter-parts (Xu et al., 2002). This has been explained by the existence of twomolecularly distinct G2/M checkpoint arrests; “immediate G2 arrest,”which is triggered rapidly in G2 cells, and “G2 accumulation,” which affectscells that reach G2 after traversing S-phase and develops over many hours(Xu et al., 2002). Immediate G2 arrest is ATM-dependent and of relativelyshort duration, whereas G2 accumulation is ATM-independent (but ATR–Chk1-dependent) and much longer-lasting (Xu et al., 2002). Why thisshould be is not fully understood; it may reflect a more stringent requirementfor ATM-dependent DSB processing for rapid and efficient checkpoint acti-vation in G2 than in S-phase (discussed in more detail below), whereas theprolonged G2 accumulation experienced by ATM mutant cells could reflecta defect in DNA repair (Beucher et al., 2009). As for mechanism, Chk2 wasinitially invoked as a downstream effector of ATM-dependent G2 arrest viaphosphorylation and inhibition of the Cdk1-activating Cdc25C phosphatase(Chehab et al., 2000; Matsuoka et al., 1998); however, as discussed below,Chk2-deficient cells do not exhibit consistent defects in G2 checkpointproficiency.As with ATM, Chk2 has been implicated in p53 activation (Chehab et al.,

2000), and consistent with this, Chk2-deficient mice show impaired G1checkpoint arrest and defects in p53-dependent gene transcription afterdamage (Hirao et al., 2002; Takai et al., 2002). They also show a markedreduction in p53-dependent apoptotic responses in adult and developingtissues and increased organismal survival after whole body irradiation(Hirao et al., 2002; Takai et al., 2002). Perplexingly, however, p53 regula-tion is not altered in Chk2-deleted HCT116 cells, a human cancer cell linethat retains wild-type p53 (Jallepalli et al., 2003). Also puzzling is the factthat G2 checkpoint impairment as a result of Chk2 inactivation is observed


in some, but not all, experimental systems. Thus, MEFs and HCT116 cellsdeleted for Chk2 retained normal G2 checkpoint proficiency (Jallepalli et al.,2003; Takai et al., 2002), whereas Chk2 knockout DT40 cells exhibited aweakened and delayed G2 arrest at early times after irradiation (Raineyet al., 2008). Interestingly, this defect was most severe in G2 cells, much asdescribed for ATM (Xu et al., 2002), whereas the slower G2 accumulationresponse was relatively unaffected (Rainey et al., 2008). Variations in theseverity of G2 checkpoint deficiency in cells genetically deficient for Chk2have often been interpreted in terms of compensation by Chk1; however, it isequally possible that the role of Chk2 in this checkpoint simply variesbetween cell types.Phenotypic analysis of checkpoint proficiency in ATR- or Chk1-deficient

mouse embryos and cells is complicated by loss of viability; however, inacti-vation of both genes results in profound G2 and S-M checkpoint defectsafter DNA damage or replication arrest (Brown and Baltimore, 2000;Brown and Baltimore, 2003; Liu et al., 2000; Takai et al., 2000). Consistentwith this, Chk1-deficient DT40 cells (which lack functional p53) showcomplete loss of proficiency for all DNA damage and replication checkpointresponses (Zachos et al., 2003, 2005). Strikingly, these cells show no mea-surable G2 arrest or intra-S checkpoint activation after irradiation at anydose tested (Zachos et al., 2003), in marked contrast to the partial loss of G2checkpoint proficiency that results from deletion of Chk2 (Rainey et al.,2008). Loss of G2 checkpoint proficiency was furthermore associated withfailure to maintain inhibitory T14/Y15 Cdk1 phosphorylation (Zachoset al., 2003), indicating that Chk1 is essential to restrain the mitosis-pro-moting activity of Cdc25 family phosphatases after DNA damage. In addi-tion, when DNA polymerase is inhibited Chk1-deficient cells suffer acombination of progressive replication fork collapse and futile origin firingthat leads ultimately to S-M checkpoint failure and premature entry tomitosis with unreplicated DNA (Zachos et al., 2005).Interestingly, Chk1-deficient DT40 cells also exhibit high levels of sponta-

neous replication fork collapse during unperturbed cell cycles which iscompensated by increased replication origin firing (Maya-Mendoza et al.,2007; Petermann et al., 2006). Although this compensation mechanismevidently allows these cells to replicate successfully and to maintain anS-phase of approximately normal length, replication fork collapse as a resultof Chk1 inhibition is a potential cause of cell death in other cell types(Syljuasen et al., 2005).The effects of inhibiting ATR or Chk1 on DNA damage and replication

checkpoint proficiency have also been widely explored in cells in cultureusing various dominant-negative, siRNA depletion, or chemical inhibitionapproaches. The general consensus that has emerged from suchstudies [reviewed in (Bartek and Lukas, 2003; Kastan and Bartek, 2004;


Stracker et al., 2009)] is broadly consistent with the genetic analysis in miceand DT40 cells summarized above; namely that ATR and Chk1 are crucialboth for the G2 and intra-S checkpoint responses induced by DNA damageand for the S-M and fork stabilization/origin suppression checkpoints trig-gered by replication arrest. Whether the ATR–Chk1 pathway also contri-butes to p53-dependent G1 arrest under any circumstances is less clear.Athough some early biochemical data implicated ATR and Chk1 as poten-tial regulators of p53 (Shieh et al., 2000; Tibbetts et al., 1999), more recentevidence that ATR–Chk1 activation by DNA damage is largely restricted tothe S and G2 phases of the cell cycle makes it seem unlikely to be a majorphysiological determinant of G1 arrest (Jazayeri et al., 2006; Walker et al.,2009).

IV. THE THREE RS OF DAMAGE SIGNALING:RESECTION, RECOMBINATION, AND REPAIR

In eukaryotes DNA DSBs are repaired via two main mechanisms; nonho-mologous end-joining (NHEJ) and homologous recombination repair(HRR). NHEJ occurs throughout the cell cycle; however, because HRRrequires a sister chromatid to serve as a template, this mechanism is restrict-ed to the S and G2 phases. Unlike NHEJ, HRR requires extensive DNAdamage processing to generate tracts of ssDNA that, once coated withRad51 recombinase, invade the homologous DNA duplex to initiate repair.Such single-stranded tracts are generated by resection of DSBs in a 30–50direction, a reaction initiated by the endonuclease activity of the MRNcomplex (Mimitou and Symington, 2009).Because of its central role in initiating HRR, DNA strand resection at

DSBs is regulated during the cell cycle and this regulation is thought to play amajor role in restricting HRR to S and G2. In yeasts, Cdk activity controlsDNA strand resection via phosphorylation of Sae2 (Wohlbold and Fisher,2009), a putative nuclease that promotes resection in collaboration with theyeast counterpart of MRN (Huertas et al., 2008). Vertebrate cells express anortholog of Sae2 in the form of CtIP, which is also required for DSB resection(Sartori et al., 2007). Resection is also cell cycle-regulated in vertebrate cells,and evidence suggests that Cdks regulate this process at least in part viadirect phosphorylation of CtIP in a manner analogous to yeast (Huertas andJackson, 2009; Yun and Hiom, 2009). Thus, because Cdk-mediated phos-phorylation of CtIP is required for strand resection, increased Cdk activity inS and G2 ensures maximum resection activity in these phases of the cellcycle.


Two other important components required for HRR are the BRCA1 andBRCA2 tumor suppressors. BRCA1 is a multifunctional protein that playsmultiple roles in checkpoint activation, DNA repair, and gene transcriptionin response to DNA damage (Huen et al., 2010). BRCA1 is recruited to sitesof ionizing radiation-induced DNA damage and is required for efficientgeneration of ssDNA at early times after irradiation (Schlegel et al., 2006).Although the mechanism is not fully understood, BRCA1 interacts withMRN and CtIP, suggesting that it too may play a role in strand resection(Chen et al., 2008). BRCA2 by contrast is required for homologous recom-bination downstream of strand resection through its well-established role inloading the Rad51 recombinase protein (Thorslund and West, 2007).As shown in Fig. 3, the recent discovery that ATM is required for strand

resection and downstream activation of ATR–Chk1 in response to DSBsprovides a new framework for understanding the organization and integra-tion of checkpoint and repair pathways (Adams et al., 2006; Cuadrado et al.,2006; Jazayeri et al., 2006;Myers and Cortez, 2006). Although not classicallyconsidered a core homologous recombination (HR) factor, ATM is requiredfor efficient HRR of a subset of DSBs specifically in G2 phase (Beucher et al.,2009). Molecular details are still emerging; however, current thinking is thatinitial recognition of DSBs by theMRN complex leads to recruitment and fullactivation of ATM. Active ATM then promotes the recruitment of CtIP tosites of damage where it interacts with and stimulates the nuclease activity ofMRE11 to initiate strand resection and generate short tracts of ssDNA (Youet al., 2009). These may be extended through the actions of other nucleasesand helicases, such as Exo1 and BLM, to generate more extensive regions ofssDNA that recruit RPA and form both the initiating substrate for HRR andactivating platform for ATR–Chk1 activation (Mimitou and Symington,2009). Exactly how ATM stimulates resection is not yet known; however,NBS1 and CtIP are both subject to ATM-dependent phosphorylation afterdamage, and at least in the case of CTIP, this modification appears to berequired both for recruitment to DSBs and resection (You et al., 2009).The ramifications of this model are extensive. Firstly, it explains why ATM

is required for rapid activation of ATR–Chk1 in response to DSBs but notDNA polymerase inhibition, since the latter generates extensive tracts ofssDNA directly without the need for DNA damage processing (Byun et al.,2005; Myers and Cortez, 2006). Secondly, it accounts for why ATR–Chk1activation in response to irradiation-induced DSBs is largely confined to Sand G2 phase (Jazayeri et al., 2006; Walker et al., 2009), since this is whenlevels of Cdk activity become permissive for efficient strand resection(Cerqueira et al., 2009). Thirdly, it explains why other proteins requiredfor ssDNA generation, such as MRE11, NBS1, and BRCA1, are also re-quired both for rapid ATR–Chk1 activation and G2 checkpoint proficiencyin response to DSBs (Myers and Cortez, 2006; Yarden et al., 2002).

Claspin

Rad51

BRCA1

CtIP

RPA RPA RPA RPA

Chk2

DNA damage (DSBs)

Rad50NBS1

MRE11

ATM

g H2AX

ATR

ATRIP

Chk1

DNA standresection

Replicationarrest

Homologousrecombination

BRCA2

p53

TopBP1

RAD1HUS1

RAD9

G2, Intra-S checkpointsFork stabilization

Origin suppressionS-M checkpoint

G1checkpoint

Fig. 3 ATM is required for DNA strand resection and ATR–Chk1 activation in response to

DSBs In response to DSBs ATM, in conjunction with the MRN complex, CtIP and BRCA1, isrequired for nucleolytic strand resection to generate tracts of ssDNA. Evidence suggests that

ATM stimulates the nuclease activity of MRE11 to initiate resection but that this process may

then be extended through the actions of other nucleases and helicases such as Exo1 and BLM.Once complexed with RPA, such tracts form the platform both for ATR-ATRIP recruitment

leading to Chk1 activation and also the initiating structure for HRR. BRCA2 promotes loading

of Rad51 which displaces RPA leading to strand invasion and subsequent recombination. In

contrast, inhibition of DNA synthesis generates tracts of ssDNA-RPA directly without the needfor stand resection by stalling replication forks and uncoupling the replicative polymerase and

helicase. The various posttranslational modifications involved in ATM–Chk2 and ATR–Chk1

activation shown in Fig. 1 are omitted here for clarity. In contrast to the more conventional

scheme depicted in Fig. 1, in this model ATR and Chk1 are the direct effectors of multiple DNAdamage and replication checkpoints with ATM acting upstream specifically in the context of

DSBs. ATM and Chk2, however, continue to signal DNA damage independently to p53 in a

parallel pathway. Please refer to the text for further details and explanation.



Placing ATM upstream of ATR–Chk1 activation in response to DSBssuggests a new interpretation of their overlapping, yet distinct, functions incheckpoint signaling (as depicted in Fig. 3). In this model the ATR–Chk1pathway is the principal direct effector of the damage and replicationcheckpoints (apart from p53-dependent G1 arrest), with ATM modulatingthe response specifically to DSBs indirectly via its role in strand resection.Thus, in ATMmutant cells, rapid activation of ATR and Chk1 in response toDSBs will be impaired as a consequence of inefficient strand resection. Thisdefect is likely to be particularly significant in G2 phase, when resection isthe principal mechanism of ssDNA generation. This model predicts there-fore that the impact of ATM deficiency on ATR–Chk1 activation andthus G2 checkpoint proficiency in response to DSBs will be greatest in G2phase, consistent with the immediate G2 arrest defect described in ATMmutant cells (Xu et al., 2002). This model also explains why Chk1-deficientDT40 cells lack any detectable G2 checkpoint after irradiation, despite thecontinued presence of functional ATM and Chk2 (Zachos et al., 2003), sincein this scheme Chk1 is the sole direct downstream effector of G2 arrest.Conversely, because DNA polymerase inhibition generates ssDNA directlywithout the need for resection, ATR and Chk1 are essential for replicationcheckpoint responses whereas ATM and Chk2 are not.Where to place Chk2 in this scheme becomes an interesting question.

ATM and Chk2 are activated in response to DSBs at all stages of the cellcycle, including G1, consistent with their established roles in activating p53and triggering G1 arrest (Lavin and Kozlov, 2007; Takai et al., 2002). Chk2is, however, variably required for G2 checkpoint proficiency in different celltypes, and whether it is a direct effector of this checkpoint has been ques-tioned (Antoni et al., 2007). One intriguing possibility, suggested by theapparent epistatic relationship between Chk1 and Chk2 in DT40 cells (i.e.,where G2 checkpoint proficiency is completely abolished in the absence ofChk1 but only impaired in the absence of Chk2), is that Chk2 might alsoparticipate in the DNA strand resection process and thus in regulating theefficiency and cell cycle phase-specificity of ATR–Chk1 activation indirectly.Although there is currently no direct evidence for such a role, furtherinvestigation seems warranted.

V. ATM–CHK2 AND ATR–CHK1 PATHWAYALTERATIONS IN CANCER

The importance of genome stability for preventing carcinogenesis is evi-dent both from human cancer predisposition syndromes that result frominherited loss-of-function mutations in DNA damage response genes and


from the occurrence of sporadic mutations affecting such genes in cancers inotherwise genetically normal individuals (Kastan and Bartek, 2004). Exam-ples of both have been found to affect ATM and Chk2 in human cancer,whereas ATR and Chk1 appear to be mutated only rarely. Important insightsinto the roles of these pathways in tumor formation have also been obtainedfrom experiments using genetically modified mice. In discussing these issues,we also refer in passing to other gene functions, such as the MRN complexand BRCA1/BRCA2, where these are closely linked either to the regulationor downstream functions of the ATM–Chk2 and ATR–Chk1 pathways.Homozygous germ-line loss-of-function mutations affecting ATM cause

the pleiotropic human disease syndrome Ataxia telangiectasia (AT), char-acterized by immunodeficiency, neurodegeneration, radiation hypersensitiv-ity, and spontaneous predisposition to lymphoma (Shiloh and Kastan,2001). Similarly, hypomorphic mutations affecting the genes encoding thefunctionally related MRE11 and NBS1 proteins give rise to the humanconditions Ataxia-like disorder (ATLD), and Nijmegen breakage syndrome(NBS), each of which shares some clinical similarities with AT, although onlyNBS is clearly associated with cancer predisposition (Stewart et al., 1999;Varon et al., 1998). As with AT humans, ATM knockout mice are predis-posed to lymphoma and radiosensitive (Xu et al., 1996), while mice withengineered hypomorphic mutations of NBS1 or RAD50 are also cancerprone (Bender et al., 2002; Kang et al., 2002; Williams et al., 2002). Theprecise cause of cancer predisposition in humans and mice with inheriteddefects in ATM or MRN components is not yet known; however, genomicinstability and an increased mutation rate resulting from repair and check-point defects is presumably an important factor.Interestingly, although AT is considered to be an autosomal recessive

genetic disorder, individuals heterozygous for ATM mutations show anincreased incidence of cancer possibly related to medical or occupationalradiation exposure (Briani et al., 2006; Swift et al., 1991). In addition, cellsfrom heterozygote individuals show sensitivity to radiation in vitro that isintermediate between those from AT patients and normal individuals (Swiftet al., 1991). Taken together, these findings indicate that ATM is a partiallypenetrant cancer susceptibility gene that might interact with certain envi-ronmental predisposing factors. Somatic mutations affecting ATM have alsobeen documented in sporadic lymphoid malignancies and lung adenocarci-nomas, although at relatively low incidence (Ding et al., 2008; Gumy-Pauseet al., 2004).In contrast to AT, ATLD, and NBS, human individuals heterozygous for

loss-of-function alleles of the BRCA1 and BRCA2 tumor suppressor genesare developmentally normal but suffer from a greatly increased incidence ofbreast and ovarian cancer (O’Donovan and Livingston, 2010). As men-tioned previously, BRCA1 is required for ATR–Chk1 activation, G2 arrest,


and other complex responses to DNA damage (Huen et al., 2010). Ingeneral, however, the tumor suppressive functions of BRCA1 and BRCA2are attributed to their distinct but essential roles in HR-mediated DNArepair (O’Donovan and Livingston, 2010). Because tumorigenesis involvesfunctional inactivation of the remaining functional BRCA1 or BRCA2 allelethrough loss of heterozygosity or other means (Collins et al., 1995;Neuhausen and Marshall, 1994), the tumors that arise in susceptible indi-viduals consist of cells that are deficient for HRR whereas those in normaltissues remain proficient (Turner et al., 2005). Genomic instability as a resultof HRR deficiency is thought to play a key role in the development of suchtumors, presumably by accelerating the accumulation of oncogenicmutations (Tutt et al., 2002); however, as discussed below, the presence orabsence of DNA repair proficiency in normal and tumor tissue respectivelyin such individuals also provides an exploitable therapeutic index.Li Fraumeni syndrome is a multiorgan cancer predisposition condition

that is generally, but not exclusively, due to inherited mutations in p53(Birch, 1994). Heterozygous germ-line mutations in Chk2 were initiallyreported in a subset of Li-Fraumeni kindreds lacking p53 mutations, consis-tent with a functional link between Chk2 and p53 (Bell et al., 1999).However, because these mutant alleles were subsequently also found innormal individuals in the general population they are now consideredunlikely to be the genetic cause of Li Fraumeni syndrome (Antoni et al.,2007). Nevertheless, studies have established that individuals bearing cer-tain mutant Chk2 alleles do suffer from a statistically significant increase inthe incidence of breast, prostate, and other cancers, suggesting that Chk2 isindeed a moderate or low penetrance cancer susceptibility gene in humans(Antoni et al., 2007). Despite this, the tumors that arise in individualsheterozygous for such mutations do not consistently lose the remainingnormal Chk2 allele, indicating that Chk2 does not conform to the conven-tional definition of a tumor suppressor (Antoni et al., 2007). One possibilityis that mutant Chk2 proteins exert a dominant-negative effect by inhibitingthe endogenous, normal Chk2, to phenocopy loss of function in the hetero-zygous state. This would be consistent with the occurrence of occasionalsporadic Chk2 mutations and rare instances of reduced or absent expressionin a variety of different tumor types (Antoni et al., 2007). Alternatively, it isconceivable that Chk2 haploinsufficiency per se synergises with other, as yetunknown, oncogenic events or environmental factors to promote malignantprogression.The impact of Chk2 deficiency on tumorigenesis in mice has also been

examined. Although Chk2 knockout mice are developmentally normal andnot spontaneously cancer prone (Takai et al., 2002), they are more sensitiveto chemical skin carcinogenesis, showing an increase both in overall tumorburden and in the rate at which benign tumors formed after exposure to


chemical carcinogens (Hirao et al., 2002). Whether this increase in tumorsensitivity is attributable to increased genomic instability or perhaps to therelative resistance to stress-induced apoptosis that has been observed inChk2 knockout mice however remains unclear.In humans, homozygous hypomorphic mutations affecting ATR give rise

to Seckel syndrome. Seckel syndrome is associated with a wide range ofdeleterious symptoms, including growth retardation and microcephaly;however, such individuals do not suffer from an increased incidence ofcancer (Kerzendorfer and O’Driscoll, 2009). Seckel syndrome has alsobeen modeled in mice (Murga et al., 2009), and the consequences of acuteconditional genetic inactivation of ATR postdevelopment in adult mice havebeen investigated (Ruzankina et al., 2007). Degenerative and prematureaging-like phenotypes were observed in each case, underscoring the crucialimportance of ATR for normal development, stem cell survival, and tissuehomeostasis; however, neither showed evidence of cancer predisposition(Murga et al., 2009; Ruzankina et al., 2007). In general therefore it appearsthat partial or complete inactivation of ATR function, although clearlydeleterious in at least some cell types in vivo, does not perturb genomestability in such a way as to promote carcinogenesis. Consistent with this,somatic mutations affecting ATR have not been widely found in cancers(Heikkinen et al., 2005), with the exception of rare sporadic stomach andendometrial tumors with microsatellite instability (MSI) (Menoyo et al.,2001; Vassileva et al., 2002; Zighelboim et al., 2009).Germ-line mutations in Chk1 have thus far not been implicated in any

human disease and, as with ATR, somatic mutations affecting Chk1 appearto be rare in human cancers, although some exceptions have been reportedin tumors with MSI (Bertoni et al., 1999; Menoyo et al., 2001). Embryoniclethality in knockout mice precludes direct assessment of the effect ofconstitutive Chk1 inactivation on spontaneous or induced carcinogenesis;however, some evidence that partial loss of function can promote tumorformation has been reported. Thus, mammary tumors induced by an onco-genic WNT transgene developed more rapidly in Chk1 hemizygous micethan wild-type (Liu et al., 2000). Importantly, however, loss of the remainingfunctional Chk1 allele was not observed, suggesting that Chk1 continued tobe important for the proliferation or survival of the tumor cells (Liu et al.,2000).More recently, we have examined the effect of experimentally induced

hemi- or homozygous conditional deletion of Chk1 in mouse skin on theformation of tumors induced by the chemical carcinogens, DMBA and TPA,using a conditional allele of Chk1 combined with a Keratin14-CreERrecombinase transgene (Indra et al., 2000; Lam et al., 2004). This combina-tion allows efficient Chk1 deletion throughout the epidermis in response tosystemic treatment with the synthetic estrogen, tamoxifen (LM Tho and DA


Gillespie, unpublished results). We find that homozygous deletion of Chk1throughout the epidermis is tolerated without acute pathology, presumablybecause Chk1 is not essential in the postmitotic, terminally differentiatedcells that comprise the bulk of the tissue. However, when recombination isinduced immediately prior to carcinogen exposure both the number and sizeof benign papillomas obtained is strongly suppressed (Table 1). Further-more, the small lesions that form in ablated skin always derive from cellsthat escape recombination (LM Tho and DA Gillespie, unpublished results).Although the basis of this tumor suppressive effect is not yet fully under-stood, we hypothesize that Chk1 is probably essential for the proliferationor survival of the epidermal stem cells that are thought to give rise tochemical carcinogen-induced tumors in the skin (Morris, 2004). Consistentwith this, developmental or conditional deletion of Chk1 has been shown tolead to rapid cell death in several other tissues including mammary gland,intestine, and lymphocytes (Greenow et al., 2009; Lam et al., 2004; Zaugget al., 2007).In marked contrast, Chk1 hemizygous skin supports normal papilloma

formation but such hemizygous lesions show an increased probability ofprogressing to malignant carcinoma (Table 1). We deduce from these experi-ments that whereas complete loss of Chk1 function is incompatible with skintumor formation, partial loss of function fosters benign-malignant tumorprogression. This conclusion is consistent with the previously describedacceleration of WNT-induced tumorigenesis and also evidence that Chk1

Table 1 Conditional Deletion of Chk1 in Mouse Epidermis Suppresses ChemicalCarcinogen-Induced Skin Tumorigenesis

Cohort Genotype Treatment Mean no. papillomas

%Conversion

to carcinoma

1 Chk1þ/þ K14-CreER Tamoxifen 17.8 (N ¼ 20) 5.92 Chk1Fl/Fl K14-CreER Vehicle 15.4 (N ¼ 19) 5.1

3 Chk1Fl/Fl K14-CreER Tamoxifen 5.5 (N ¼ 18) 2

4 Chk1Fl/þ K14-CreER Tamoxifen 14.6 (N ¼ 19) 9.7

Cohorts of FVB strain mice were bred to express an epithelial-specific K14-CreER transgene (Indra et al.,

2000) in combination with either wild-type Chk1 (Chk1þ) or a conditional, lox P-modified allele of Chk1

(Chk1Fl) (Lam et al., 2004). At 6 weeks of age mice were treated systemically with Tamoxifen, resulting in

efficient Chk1 deletion throughout the epidermis (L.M. Tho and D.A. Gillespie, unpublished results), or

vehicle control. Mice were then immediately treated with a single dose of the carcinogen, DMBA, followed by

twice weekly applications of the tumor promoter TPA for up to 30 weeks (Abel et al., 2009). Total plateau

papilloma burden was quantified at that time, or at time of sacrifice if earlier, together with the proportion of

papillomas that converted to carcinoma. The reduction in papilloma yield in cohort 3 was highly statistically

significant compared to controls (cohorts 1 and 2; Mann Whitney test p < 0.001). Similarly, the rate of

conversion to carcinoma was significantly elevated in cohort 4 compared to controls (cohorts 1 and 2;

chi-squared test p < 0.025). A more detailed description of these data will be presented elsewhere.


haploinsufficiency can lead to aberrant cell cycle regulation and genomicinstability in vivo (Lam et al., 2004; Liu et al., 2000). Conversely, it has beenreported that Chk1 hemizygous mice suffer from an increased incidence ofanemia associated with increased levels of DNA damage in erythroid pro-genitors, suggesting that Chk1 haploinsufficiency can also result in degener-ative effects in certain cell lineages, perhaps also owing to stem cell death(Boles et al., 2010).The ability therefore of cells and organisms to tolerate partial or complete

loss of function within the ATM–Chk2 and ATR–Chk1 pathways is verydifferent. Impairment or even complete loss of ATM–Chk2 signaling iscompatible with cell and organism survival, although frequently at the costof cancer predisposition, presumably at least in part as a result of genomicinstability and more rapid accumulation of oncogenic mutations. In con-trast, ATR and Chk1 seem to be essential for the proliferation and survivalof many, although not all, cell types, both in vitro and in the developingembryo and adult organism, presumably because they control aspects ofDNA metabolism that when dysfunctional lead to cell death rather thansurvival with mutation. In this scheme Chk1 (and arguably ATR) becomes alogical target for therapeutic strategies based on pharmacological check-point suppression (discussed below), although evidence from mouse modelsthat partial loss of Chk1 function (i.e., haploinsufficiency) may promotetumorigenesis, or other undesirable pathologies (Boles et al., 2010), clearlymerits careful consideration.

VI. EXPLOITING HOMOLOGOUS RECOMBINATIONALREPAIR (HRR) DEFECTS FOR CANCER THERAPY

In recent years it has emerged that in addition to predisposing to cancer asa result of increased genomic instability, defective HRR may also rendertumor cells inherently vulnerable to specific conventional anticancer agentsand also to new strategies based on inhibition of complementary repairpathways. Thus, BRCA1- and BRCA2-deficient tumor cells have beenfound to be hypersensitive to cross-linking agents such as cisplatin in vitro(Bhattacharyya et al., 2000; Yuan et al., 1999), most probably because HRis the principal mechanism through which replication forks stalled orcollapsed by such lesions are repaired or restarted. Importantly, evidencesuggests this is also true in vivo and that ovarian cancers arising in BRCA1and BRCA2 mutation carriers may respond better to platinum-based thera-py than similar sporadic tumors (Foulkes, 2006). Remarkably, secondarymutations that restore BRCA1 or BRCA2 function can be a cause both fordrug resistance in ovarian cancer cells in culture and treatment failure in


patients, emphasizing the importance of HRR deficiency in determiningsensitivity to platinum compounds (Sakai et al., 2008,2009; Swisher et al.,2008).Poly (ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that is

activated by DNA single-strand breaks (SSBs) and plays a crucial role inmultiple aspects of the DNA damage response (Rouleau et al., 2010).In response to DNA damage active PARP1 binds to SSBs in DNA andcatalyzes the synthesis of branched, protein-conjugated poly (ADP-ribose)chains. Many of these chains become linked to PARP1 itself as a result ofautomodification, although histones, topoisomerases, and many other pro-teins involved in diverse aspects of DNA metabolism are also substrates forPARP1 (D’Amours et al., 1999). Once formed at sites of damage, such poly(ADP-ribose) chains recruit multiple proteins involved in DNA repair andmodulating chromatin structure (Rouleau et al., 2010). In particular, PARP1both modifies and recruits XRCC1, a key scaffolding factor required forbase excision repair (BER). BER excises damaged or mismatched bases fromDNA and also repairs single-strand gaps, nicks, and abasic sites, lesionswhich arise both spontaneously and as a result of oxidative stress orexposure to alkylating agents.Based on its established role in DNA repair, selective small molecule

inhibitors of PARP1 were initially developed with a view to enhancing thepotency of DNA damaging chemotherapies (Zaremba and Curtin, 2007).Although PARP1 inhibitors are relatively nontoxic to most cancer cellsalone, it was subsequently discovered that tumor cells deficient for BRCA1or BRCA2 were inherently and exquisitely sensitive to such agents even inthe absence of exogenous genotoxic stress (Bryant et al., 2005; Farmer et al.,2005). The basis of this selective sensitization has been explained in terms ofsynthetic lethality, a term applied to genetic functions or pathways whoseindividual loss is tolerated but when combined result in lethality (Kaelin,2005). In this context of course the synthetic lethal interaction occursbetween an inherent genetic deficiency for HRR resulting from BRCA1/BRCA2 mutation and a phenocopy of functional BER deficiency that isimposed through pharmacological inhibition of PARP1.Thus, inhibition of PARP1 leads to accumulation of SSBs and other endog-

enousDNAdamage lesions that would normally be corrected by BER (Jeggo,1998). Such lesions are particularly problematical in S-phase, since whenencountered by replicative polymerases they lead to replication fork collapseand formation of DSBs. HRR is thought to be the main mechanism throughwhich such DSBs can be repaired and replication restarted, enabling HRRproficient cells to tolerate PARP inhibition (Li and Heyer, 2008). However,because BRCA1- and BRCA2-deficient tumor cells are impaired both forHRR and BER under conditions of PARP inhibition, replication-associatedDSBs cannot be repaired and thus escalate to lethal proportions (Bryant et al.,


2005; Farmer et al., 2005). As with resistance to platinum compounds,acquired resistance to PARP inhibition can arise through reversionmutationsthat restore BRCA2 function, at least in cell lines (Edwards et al., 2008).Importantly, this synthetic lethal principle can be extended to therapy;

several recent trials have shown that Olaparib, a potent oral PARP inhibitor,has significant antitumor activity as monotherapy specifically in ovariancancers arising in BRCA1 and BRCA2 mutation carriers, and remarkably,this activity is achieved without the toxicity associated with conventionalgenotoxic chemotherapy (Fong et al., 2009, 2010). In addition, much pre-clinical evidence suggests that PARP inhibition may also synergize withconventional genotoxic chemotherapies, including alkylating agents, plati-num compounds, topoisomerase inhibitors, and radiation, both in BRCA1and BRCA2 mutant and sporadic tumors (Ratnam and Low, 2007). As aresult, Olaparib and many other PARP inhibitors are currently undergoingtrials both as monotherapies and in combination with conventional agents(Rouleau et al., 2010).The evident promise of PARP inhibition as a selective therapy against

tumors with defects in HRR raises the obvious question of how widespreadthis phenotype is in sporadic cancers arising in genetically normal indivi-duals. Cell culture studies show that experimental inhibition of many diversegene functions involved either directly in HRR or in DNA damage signalingmore generally (including Rad51, RPA1, NBS1, ATM, ATR, Chk1, Chk2,and certain Fanconi anemia gene products) all result in sensitization to PARPinhibition (McCabe et al., 2006). In addition, human and mouse cellsgenetically deficient for ATM are also sensitized to PARP inhibition (Loseret al., 2010; Williamson et al., 2010). As already mentioned, inheritedmutations in several of these genes result in cancer predisposition; however,there is little evidence that such genes are frequently mutated in sporadiccancers.The PTEN tumor suppressor, by contrast, is one of the most frequently

mutated or inactivated genes in human cancer and leads to upregulation ofthe PI3-kinase-PKB/ Akt growth and survival signaling pathway (Chalhouband Baker, 2009). Loss of PTEN has been known for some time to compro-mise genome stability (Puc et al., 2005); however, recent data have revealedunexpected connections with HR and DNA repair. Remarkably, humanHCT116 colon carcinoma cells genetically deleted for PTEN are both defi-cient for HR and sensitized to PARP inhibitors (Mendes-Pereira et al., 2009).Similar effects have been documented in PTEN-deficient murine astrocytesand human glioblastoma cell line (McEllin et al., 2010). The mechanismthrough which PTEN affects HR is not yet fully understood; however, tworecent studies have shown that Akt, which is upregulated as a result of PTENloss, can inhibit both strand resection and recruitment of essential HRfactors such as BRCA1 and CtIP at radiation-induced DSBs (Tonic et al.,


2010; Xu et al., 2010). The prospect that frequently activated oncogenicsignaling pathways conventionally linked to cell proliferation and survivalmay interact with DNA damage response and repair processes suggest thatimpaired HRR may be widespread in sporadic cancer and that PARP inhi-bitors could have more generic application than currently thought.

VII. DNA DAMAGE SIGNALING AS A BARRIER TOTUMORIGENESIS

Predisposition syndromes demonstrate that genomic instability can pro-mote cancer; however, in recent years an alternative paradigm has emergedin which DNA damage and the resulting downstream signaling processes actas a tumor suppression mechanism (Halazonetis et al., 2008). This conceptoriginated with the observation that premalignant or early stage lesions inseveral cancer types, including bladder, breast, lung, and colon, frequentlyshowed evidence of DNA damage as judged by the presence of multiplesurrogate or direct markers of damage signaling such as �-H2AX, andphosphorylated, active forms of ATM, Chk2, and p53 (Bartkova et al.,2005). These markers were not present in proliferating normal tissue, andstrikingly, their prevalence diminished again in the more malignant, laterstages of disease (Bartkova et al., 2005). Crucially, the signs of DNA damagein early stage lesions preceded the appearance of overt genomic instability orof p53 mutations, both of which are frequent in fully malignant tumors(Bartkova et al., 2005). Based on these observations it was proposed thatspontaneous DNA damage occurs early in the evolution of cancer and thatthe resulting DNA damage signaling processes direct incipient cancer cells toterminal, nonproductive fates such as senescence or apoptosis. It was fur-thermore postulated that this might provide a selective pressure for specificsecondary genetic alterations, such as p53 mutation, that would allow cellsto escape these fates and survive (Bartkova et al., 2005).Although these initial findings were largely correlative, subsequent studies

have provided substantial support for this scenario (Bartkova et al., 2006;Di Micco et al., 2006). Thus, while activated oncogenes are known to drivethe proliferation of malignant tumor cells, expression of the same oncogenesin naı̈ve, genetically normal cells in culture often results not in cell transfor-mation but instead in growth inhibition through cell senescence or apoptosis(Braig and Schmitt, 2006) . Oncogene-induced senescence (OIS) in culturedcells is associated with physical DNA damage and a robust DNA damageresponse (Di Micco et al., 2006), providing a tractable model to test the roleof downstream signaling processes in imposing this phenotype. Remarkably,depletion or inhibition of several key DNA damage signaling components,


including ATM, Chk1, Chk2, and p53, relieved OIS to allow more vigorouscell proliferation and efficient transformation of oncogene-expressing cells(Bartkova et al., 2006; Di Micco et al., 2006). Although cell culture experi-ments obviously do not accurately recapitulate all features of tumorigenesisin vivo, it is telling that markers of cell senescence and DNA damagesignaling are both present in early stage or premalignant lesions and bothare lost as tumors progress to more malignant forms (Bartkova et al., 2006).Such studies have stimulated interest in two questions; firstly, how do

oncogenes trigger DNA damage, and secondly, what are the genetic orepigenetic changes which enable tumor cells to survive and proliferate inthe face of such damage? With respect to the first question, telomere erosion,reactive oxygen species, or acquired mutations in genes required for genomestability could all plausibly generate DNA damage during tumorigenesis ortransformation; however, it has been argued that none of these occur suffi-ciently frequently to account for OIS either in vitro or in vivo (Negrini et al.,2010). Instead, current evidence favors the view that dominant, growth-promoting oncogenes, such as Ras or EGFR, which are frequently andrecurrently mutated in sporadic cancers, disturb the DNA replication pro-cess directly in such a way as to generate DNA damage (Halazonetis et al.,2008). Exactly how oncogene-induced replication stress occurs remainsunclear; however, Cdks are potential culprits. Ordered cycles of Cdk activa-tion and deactivation are essential for the proper organization and activa-tion of the replication program through the replication origin licensingsystem (Diffley, 2004). Because Cdk activity is key to uncontrolled prolifer-ation, it is deregulated by oncogene activation and tumor suppressor loss bymany different mechanisms. Amplification of Cdk activity as a result ofoncogenic signaling therefore may erode these ordered cycles, leading toaberrant over- or under-replication and thus to DNA damage (Blow andGillespie, 2008). According to this view, uncontrolled proliferation inevita-bly leads to spontaneous DNA damage and genomic instability (Halazonetiset al., 2008).With respect to mechanisms that may allow escape from oncogene-in-

duced DNA damage, recent high-throughput sequencing studies have gener-ally failed to find evidence for frequent mutations affecting DNA damageresponse genes, such as ATM–Chk2 and ATR–Chk1, in sporadic cancers,although clearly this does not rule out functional inactivation by epigeneticor other means (Negrini et al., 2010). By contrast, missense mutations thatinactivate the p53 tumor suppressor protein are frequent in sporadic cancerand some genetic evidence is consistent with the idea that these could beselected to permit escape from DNA damage-induced OIS or apoptosis(Negrini et al., 2010). One possible explanation for their prevalence is thatmutant p53 proteins can act dominantly, forming complexes with andinhibiting normal p53 or the related p63 and p73 proteins (Brosh and


Rotter, 2009). In contrast, biallelic mutations would presumably be requiredto inactivate the function of most other DNA damage response genes. At allevents, frequent loss of p53 means that many tumor cells lack an effectiveG1 checkpoint. As discussed below, this defect may render such tumorsinherently vulnerable to therapeutic strategies based on checkpointsuppression.Although inactivation of p53 could plausibly mitigate many of the nega-

tive effects of oncogene-induced DNA damage, it does not offer an obviousexplanation for why DNA damage signaling per se should be lost in the laterstages of tumorigenesis (Bartkova et al., 2005). Also, since malignant tumorscommonly exhibit high levels of genomic instability (Negrini et al., 2010), itseems unlikely that the ongoing generation of oncogene-induced DNAdamage ceases as tumors progress, but more likely that its presence is simplyno longer detected. Why should this be? As previously mentioned, severalrecent studies have revealed that upregulation of the PI3K-Akt pathway,which is common in cancer as a result of PTEN inactivation and othermechanisms (Chalhoub and Baker, 2009), inhibits both HRR and check-point activation by suppressing DNA damage processing (Tonic et al., 2010;Xu et al., 2010). One prediction of these findings is that tumor cells withhigh PI3K-Akt pathway activity will show a muted response to endogenousDNA damage, potentially providing another mechanism through whichoncogene-induced DNA damage could be tolerated. However, a secondprediction is that this would inevitably be associated with genomic instabili-ty and an increased mutation rate, since ongoing oncogene-induced DNAdamage would continue undetected but unabated in cells with high PI3K-Akt activity. Finally, it seems possible that tumors which have evolvedthrough such a route might show an altered response to conventionalgenotoxic therapies, since presumably the recognition and signaling oftherapeutic DNA damage would also be suppressed.

VIII. CHECKPOINT SUPPRESSION AS ATHERAPEUTIC PRINCIPLE

Radiation and genotoxic chemotherapies remain the mainstays of cancertreatment. Although new, molecularly targeted, drugs like imatinib haverevolutionized treatment of rare cancers such as chronic myeloid leukemia(Agrawal et al., 2010), there seems little prospect that conventional thera-pies will be replaced in the treatment of other, more common malignanciesin the foreseeable future. Such treatments are, however, of limited efficacyand toxic not only to tumor cells but also to normal tissues, leading to severeside-effects. The realization that radiation and essentially all genotoxic


anticancer agents potently activate the ATM–Chk2 and ATR–Chk1 signal-ing pathways has stimulated much interest in whether these could bemanipulated pharmacologically to enhance the efficacy of conventionaltherapies.Several considerations argue that this may be the case. Firstly, defects in

DNA damage responses, whether inherent or experimentally imposed, canresult in sensitization to genotoxic stress. Examples already mentionedinclude the acute radiosensitivity of ATM-deficient cells, the inherent vul-nerability of BRCA1- and BRCA2-deficient cells to cross-linking agents, andthe synthetic lethality that results when cells with HRR deficiency are subjectto PARP inhibition. Secondly, enhanced DNA damage signaling has beenlinked to radio- and chemo-resistance in leukemia and gliomas (Bao et al.,2006; Nieborowska-Skorska et al., 2006), raising the possibility that thismight be reversed. Finally, it has been argued that the frequent functionalinactivation of p53 in human cancer creates a generic and exploitabledistinction between tumor and normal cells in terms of checkpoint profi-ciency. In essence this hypothesis holds that loss of p53-mediated G1 arrestunder conditions of genotoxic therapy will render tumor cells more depen-dent on checkpoints activated in S and G2 phases than their geneticallynormal counterparts. Obviously this strategy is predicated on theassumption that in tumor cells the primary function of these residualp53-independent checkpoints is protective; that is, that their inhibitionwill either escalate damage, promote the formation of more lethal lesions,or trigger some mechanism of cell death that would not otherwise occur inthe absence of checkpoint suppression.Over the past decade considerable evidence has accumulated to support

this concept. Thus, inhibition of Chk1 using either siRNA depletion or theselective chemical inhibitor, UCN-01, has been shown to potentiate cellkilling by a wide range of genotoxic agents, including ionizing radiation,alkylating agents, nucleoside analogs, cisplatin, and topoisomerase inhibi-tors (Carrassa et al., 2004; Cho et al., 2005; Ganzinelli et al., 2008; Hiroseet al., 2001; Karnitz et al., 2005; Koniaras et al., 2001; Wang et al., 1996;Yu et al., 2002). In many, although not all, of these studies Chk1 inhibitionresulted in a greater degree of sensitization in tumor cells that were deficientfor p53 than in their proficient counterparts, consistent with the idea thatloss of G1 arrest indeed creates a therapeutic index. Sensitization has alsobeen observed as a consequence of inhibiting ATM, ATR, and other down-stream components involved checkpoint regulation such as the Cdk1-reg-ulating kinases, Wee1 andMyt1 (Karnitz et al., 2005; Mukhopadhyay et al.,2005). In comparison, where tested, inhibition of Chk2 has in general beenfound to have little or no effect on cell survival under conditions of damage,emphasizing the fundamental functional distinction between Chk1 andChk2 (Carrassa et al., 2004; Karnitz et al., 2005; Pan et al., 2009).


Although these studies support the general principle that checkpoints areprotective and help tumor cells to survive exposure to genotoxic stress,exactly how checkpoint suppression leads to cell death remains poorlyunderstood. Evidence for amplified levels of damage, mitotic catastrophewith damaged or incompletely replicated DNA, and increased levels ofapoptosis have all been reported (Carrassa et al., 2004; Cho et al., 2005;Ganzinelli et al., 2008; Hirose et al., 2001; Karnitz et al., 2005; Koniaraset al., 2001; Yu et al., 2002). This is likely to be a complex issue; however,since genotoxic agents with distinct mechanisms of action activate differentcombinations of DNA damage and replication checkpoint responses, whilethe effects of some genotoxins may also depend on cell cycle phase and thusvary among individual cells in a population. The cellular consequences ofsuppressing checkpoint responses and the mechanisms of induced cell deaththat contribute to sensitization are therefore likely to be equally variable andcomplex.Some of this complexity is illustrated by studies in Chk1 knockout DT40

lymphoma cells examining the relationship between checkpoint deficiencyand cell survival under different conditions of genotoxic stress. Compared towild-type, Chk1-deficient DT40 cells are markedly sensitive to ionizingradiation, the DNA polymerase inhibitor aphidicolin, and the nucleosideanalog 5-fluorouracil (5-FU); however, the mechanism of cell death arisingfrom checkpoint deficiency is different in each case. Thus, abrogation of theChk1-dependent G2 checkpoint leads to division with lethal damage afterirradiation, presumably because G2 arrest would normally provide a vitalopportunity to repair such damage prior to mitosis (Zachos et al., 2003).By contrast, when DNA polymerase is blocked with aphidicolin, deficientfork stabilization/origin suppression and S-M checkpoints result in massivereplication fork collapse and lethal premature entry to mitosis with unrepli-cated DNA (Zachos et al., 2003, 2005). 5-FU also inhibits DNA replicationthrough its inhibitory action on thymidylate synthase (TS) leading to nucle-otide pool depletion (Longley et al., 2003); however, sensitization in thiscase proved to stem from uncontrolled replication in the presence of drugdue to loss of Chk1-mediated slowing of DNA synthesis, rather than forkcollapse followed by premature entry to mitosis (Robinson et al., 2006).Failure to slow replication resulted in enhanced incorporation of 5-FU intocellular genomic DNA and a large increase in DSBs compared to Chk1proficient cells (Robinson et al., 2006). Thus, the consequences of replica-tion checkpoint suppression vary according to the nature of the genotoxicagent and the mechanism of DNA synthesis inhibition.The many preclinical “proof-of-principle” studies over the past decade or

so have encouraged efforts within the pharmaceutical industry to developdrugs targeting the ATM–Chk2 and ATR–Chk1 pathways, some of whichhave already entered clinical trials. Thus far these efforts have concentrated


mainly on Chk1, presumably in recognition of its role as the ultimateeffector of both DNA damage and replication checkpoints and the factthat it is thought to be expressed and active in virtually all tumors (Daiand Grant, 2010). Drugs specifically targeting ATM and Chk2 are, however,also under development, although at an earlier stage, and also other check-point-regulating kinases such as Wee1 and Myt1 (Antoni et al., 2007;Ashwell et al., 2008; Dai and Grant, 2010). Thus far no selective inhibitorsof ATR have been reported.UCN-01 was the first Chk1 inhibitor approved for clinical trials; however,

undesirable pharmacological characteristics combined with lack of selectiv-ity limited its utility and led to the development of several new and moreselective agents (Ashwell et al., 2008; Dai and Grant, 2010; Fuse et al.,2005). Several of these have reached clinical trials while others remain underpreclinical development. Current Chk1 inhibitors in Phase I clinical trialsinclude AZD7762 (AstraZeneca), PF-00477736 (Pfizer), XL844 (Exelixis),and SCH 900766 (Schering-Plough). It is important to note that althougheach of these drugs was developed to inhibit Chk1, several also have signifi-cant, and in one case even greater, activity against Chk2. Whether thepotential for dual inhibition of both Chk1 and Chk2 is relevant to thebiological effects of these agents as reviewed below is currently unclear.Each of these agents has shown promising preclinical activities that recapit-

ulatemanyof the effects of experimentalChk1 inhibition onDNAdamage andreplication checkpoint responses described above (Ashwell et al., 2008; Daiand Grant, 2010). In addition to monitoring effects on cell survival, several ofthese studies have sought evidence of drug efficacy in terms of Chk1 inhibitionand to gain insight into potential mechanisms of synergistic cell killing. Bio-chemical readouts of Chk1 activity include turnover of Cdc25A phosphatase,phosphorylation of Cdc25 C on serine 216 (S216), and increased levels ofinhibitory T14/Y15 phosphorylation of Cdk1. To document the biologicalconsequences of Chk1 inhibition, nuclear foci of �-H2AX and Rad51 providea means of quantifying DNA damage and proficiency for HRR respectively,while premature entry tomitosis can be detected by flow cytometry when cellswith incompletely replicatedDNAbecome positive for histoneH3phosphory-lated on serine 10 (pS10) (Zachos et al., 2005).AZD7762 is a potent ATP-competitive inhibitor of Chk1 and Chk2 that is

currently in clinical trials in combination with gemcitabine and irinotecanfor solid malignancies (Morgan et al., 2010; Zabludoff et al., 2008).In preclinical studies AZD7762 has been shown to synergize with ionizingradiation, irinotecan, and gemcitabine in a variety of tumor cell lines andxenografts with evidence of greater potency in p53-deficient cells (Morganet al., 2010; Zabludoff et al., 2008). In each case AZD7762 treatmentresulted in stabilization of Cdc25A, decreased levels of T14/Y15 phosphory-lated Cdk1, and an increase in mitotic entry compared to DNA damaging


agent alone, indicating efficient Chk1 inhibition and checkpoint override(Morgan et al., 2010; Zabludoff et al., 2008). Enhanced cell killing byAZD7762 in the context of ionizing radiation was also associated withincreased and persistent �-H2AX staining and a marked suppression ofRad51 focus formation, indicating that drug treatment resulted in higherlevels of DNA damage and also likely resulted in suppression of HRR(Morgan et al., 2010; Zabludoff et al., 2008).Gemcitabine is an antimetabolite and DNA strand-terminator that inhi-

bits DNA replication via inhibition of both ribonucleotide reductase andDNA polymerase, stalling replication forks and arresting cells in S-phase(Ewald et al., 2008). A more detailed analysis of gemcitabine chemosensiti-zation revealed that AZD7762 treatment caused collapse of stalled replica-tion forks, ectopic replication origin firing, and ultimately premature entryto mitosis with unreplicated DNA and apoptosis (McNeely et al., 2010).Interestingly, siRNA depletion of Cdk1 mitigated cell death under condi-tions of AZD7762 and gemcitabine treatment, suggesting that prematureentry to mitosis as a result of S-M checkpoint failure was a direct cause ofcell death (McNeely et al., 2010). Replication fork collapse also resulted inhigh levels of DSBs and ATM activation, and consistent with this, AZD7762enhanced gemcitabine cytotoxicity particularly effectively in cells with DSBrepair defects (McNeely et al., 2010).PF-00477736 is a potent ATP-competitive inhibitor of Chk1 (Ki 0.49 nM)

with more modest activity towards Chk2 (Ki 47 nM) in vitro. It is incombination trials with gemcitabine for the treatment of advanced solidtumors (Ashwell et al., 2008; Dai and Grant, 2010). In preclinical studiesPF-00477736 has been shown to abrogate both the G2 and intra-S-phasecheckpoints in cells treated with camptothecin and gemcitabine respectively,and to enhance the cytotoxicity of gemcitabine and carboplatin in cell andxenograft assays (Blasina et al., 2008). Unexpectedly, PF-00477736 alsosynergizes strongly with docetaxel in cells and xenografts, releasing cellsfrom mitotic arrest and enhancing apoptosis (Zhang et al., 2009). Themechanism of this synergy is not yet fully understood. High concentrationsof docetaxel can induce DNA damage which could be a factor; however,other findings have shown that Chk1 is required for spindle checkpointfunction and mitotic progression, raising the possibility that Chk1 inhibitorsmight enhance the effects of antimitotic drugs more generally (Carrassaet al., 2009; Peddibhotla et al., 2009; Zachos et al., 2007).XL-844 is a more potent inhibitor of Chk2 (Ki 0.07 nM) than Chk1

(Ki 2.2 nM). XL-844 is in combination trials with gemcitabine for thetreatment of advanced solid tumors and lymphoma (Ashwell et al., 2008;Dai and Grant, 2010). In PANC-1 cells treated with gemcitabine XL-844has been shown to override the S-phase checkpoint leading to increasedlevels of Chk1 phosphorylation (S317) and �-H2AX, indicative of increased


levels of damage, followed by subsequent premature entry into mitosis anddecreased clonogenic survival (Matthews et al., 2007). Similarly, SCH-900766 is a selective Chk1 inhibitor that is in combination trials withgemcitabine for the treatment of solid tumor and lymphoma and cytarabinefor the treatment of acute leukemia. SCH-900766 has been shown to abro-gate both the intra-S and G2 checkpoints resulting in sensitization of tumorcells to IR and alkylating agents, although full details of these effects are notyet in the public domain (Dai and Grant, 2010).Taken together, these studies validate Chk1 as a target whose pharmaco-

logical inhibition can potentiate tumor cell killing by a wide range ofgenotoxic agents in vitro. As depicted in Fig. 4, much remains to be learnedabout the detailed mechanisms involved in chemosensitization, however,Chk1 inhibition can clearly both amplify the extent of damage inflicted bya given agent and promote the formation of more lethal lesions, for exampleby triggering stalled replication fork collapse to form DSBs. In addition,evidence suggests that damage escalation as a result of Chk1 inhibition canenhance tumor cell killing both by conventional routes, for example byincreasing apoptosis, but also by triggering novel mechanisms such as pre-mature entry to mitosis with unreplicated DNA. Clearly, the challenge nowwill be to determine whether these preclinical principles established in thelaboratory using novel Chk1 inhibitor drugs will have utility in the clinic.

IX. FUTURE PERSPECTIVES

Genomic instability has long been recognized as a cardinal feature, andarguably an important cause of, cancer, however, in recent years it has alsoemerged as a potential Achilles heel that offers new therapeutic opportunities.Thus far this prospect has been most evident in cancers that arise in predis-posed individuals, for example in BRCA1 and BRCA2 mutation carriers,where impairment of one particular form of DNA repair specifically intumor cells creates sensitivity to both existing and novel treatments.Although similar loss-of-function mutations do not appear to be common insporadic cancers, genomic instability is, perhaps because DNA damage re-sponse genes are inactivated epigenetically, or the proteins they encode areinhibited by oncogenic signaling processes. This raises the possibility thatindividual sporadic cancers might also be selectively targeted if the functionalbasis and consequences of genomic instability in individual tumors could beunderstood. Alternatively, it may be possible to improve the efficacy, or miti-gate the undesirable side-effects, of existing genotoxic therapies by developingdrugs that inhibit DNA damage responses controlled by the ATM–Chk2 andATR–Chk1 pathways. Evidence suggests that inhibition of the Chk1 protein

IR/topoisomerase inhibitors(DSBs)

Chk1

DNA standresection Gemcitabine, 5-FU,

Platinum compounds(stalled replication forks)

ATR

Damage escalation,Suppression of HRR,Division with damage,

Premature mitosis,Increased apoptosis

Chk1 inhibition

Fork collapseleading to DSBs

ATM

G2 arrest,Intra-S checkpoint,

S-M checkpoint,Fork stabilization,

Origin suppression,

Fig. 4 Chk1 inhibition potentiates cell killing by genotoxic agents by multiple mechansimsActivation of Chk1 in response to DSBs and stalled replication forks in p53-deficient tumor cells

triggers multiple cell cycle checkpoint responses that slow replication, delay the onset of mitosis,

stabilize stalled replication forks, and suppress futile origin firing upon exposure ionizingradiation (IR) or a wide range of genotoxic anticancer agents.. Preclinical studies using

siRNA depletion or a variety of selective inhibitor drugs, some of which are in clinical trials,

has shown that Chk1 inhibition overrides these checkpoint responses leading to damage

escalation and increased tumor cell killing through multiple mechanisms. Please refer to thetext for further details and explanation.


kinase in particularmay have therapeutic potential, particularly in tumors thathave suffered loss of p53 function during their evolution. Such efforts are,however, in their infancy, and as understanding of the biological andmolecularfunctions of these pathways deepens, additional rational therapeutic strategiesbased on genome stability defects will likely emerge.

ACKNOWLEDGMENTS

The authors wish to thank Cancer Research-UK, the Beatson Institute for Cancer Research,

and the Royal College of Radiologists (LMT) for financial support.


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