Download The ATM-Chk2 and ATR-Chk1 Pathways in DNA Damage Signaling

The ATM–Chk2 and ATR–Chk1
Pathways in DNA Damage Signaling
and Cancer
Joanne Smith,* Lye Mun Tho,*,{ Naihan Xu,* and
David A. Gillespie*,{
{
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
*Beatson Institute for Cancer Research, Garscube Estate,
Glasgow, UK
Faculty of Medicine, University of Glasgow, Glasgow, UK
Introduction
Activation of the ATM–Chk2 and ATR–Chk1 DNA Pathways
Checkpoint Functions of the ATM–Chk2 and ATR–Chk1 Pathways
The Three Rs of Damage Signaling: Resection, Recombination, and Repair
ATM–Chk2 and ATR–Chk1 Pathway Alterations in Cancer
Exploiting Homologous Recombinational Repair (HRR) Defects for Cancer Therapy
DNA Damage Signaling as a Barrier to Tumorigenesis
Checkpoint Suppression as a Therapeutic Principle
Future Perspectives
References
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 mutations, while radiation and diverse genotoxic agents remain important, if imperfect, therapeutic modalities. Cellular responses to DNA damage are coordinated primarily by two
distinct kinase signaling cascades, the ATM–Chk2 and ATR–Chk1 pathways, which are
activated 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 DNA
repair 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 sensitivity 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 and
functions of the ATM–Chk2 and ATR–Chk1 pathways and the prospects for targeting
DNA damage signaling processes for therapeutic purposes. # 2010 Elsevier Inc.
Advances in CANCER RESEARCH
Copyright 2010, Elsevier Inc. All rights reserved.
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0065-230X/10 $35.00
DOI: 10.1016/S0065-230X(10)08002-4
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Joanne Smith et al.
I. INTRODUCTION
Cells in multicellular organisms are continuously exposed to DNA
damage arising from a variety of endogenous and exogenous sources.
These include reactive oxygen species, ultraviolet light, background radiation, and environmental mutagens. To protect their genomes from this
assault, cells have evolved complex mechanisms, collectively referred to as
DNA damage responses, that act to rectify damage and minimize the probability of lethal or permanent genetic damage. The cellular response to DNA
damage encompasses multiple repair mechanisms and checkpoint responses
that 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 of
checkpoint and DNA repair processes; however, they can also modulate
other biological outcomes such as apoptosis or cell senescence. In recent
years, it has become evident that DNA damage responses are central both
for the evolution and therapy of cancer. Inherited defects in DNA damage
responses predispose to cancer by enhancing the accumulation of oncogenic
mutations, while genome instability is also common in sporadic cancers.
More recently, it has become apparent that oncogenic mutations elicit
spontaneous DNA damage that can suppress the evolution of incipient
cancer cells. Escape from this tumor suppressive barrier may be a major
factor in selecting for additional genetic changes during tumor progression
such as mutation of the p53 tumor suppressor, the most frequent alteration
in human cancer.
Conversely, radiation and genotoxic chemotherapies remain a mainstay of
conventional cancer treatment and are likely to remain so for the foreseeable
future. Such therapies are, however, imperfect and can incur severe side
effects. As a result, much current interest is focused on understanding how
normal and tumor cells respond to DNA damage and determining whether
DNA damage responses could be exploited or manipulated for therapeutic
purposes. Two concepts in particular have attracted attention in recent
years. First, inherent defects in genome stability mechanisms, such as homologous recombination, can confer tumor sensitivity to specific genotoxic
agents or inhibition of complementary repair pathways. Second, evidence
suggests that pharmacological suppression of DNA damage or checkpoint
responses can enhance the efficacy of conventional genotoxic agents.
Although promising, a full understanding of the biology and functions of
the DNA damage signaling pathways will be crucial for the future success of
such approaches.
DNA Damage and Cancer
75
II. ACTIVATION OF THE ATM–CHK2 AND
ATR–CHK1 DNA PATHWAYS
DNA damage responses are controlled by biochemical pathways whose
principal components and general organization have been conserved from
yeasts to humans (Rhind and Russell, 2000). In vertebrates, the two main
signaling pathways activated by DNA damage consist of the ATM–Chk2
and ATR–Chk1 protein kinases (Sancar et al., 2004). ATM and ATR are
large kinases with sequence similarity to lipid kinases of the phosphatidylinositol-3-kinase (PI3K) family, but which phosphorylate only protein substrates (Abraham, 2001). Key among these substrates are the serine–
threonine checkpoint effector kinases, Chk1 and Chk2, which are selectively
phosphorylated and activated by ATR and ATM respectively to trigger a
wide 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 at
DNA double-strand breaks (DSBs) in conjunction with the MRE11: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 are
activated potently by radiation and genotoxins that induce DSBs, but only
weakly, if at all, by agents that block DNA replication without inducing
damage (Matsuoka et al., 2000). In undamaged cells, ATM is thought to
exist as inactive homodimers. In response to DSBs, inactive ATM homodimers are rapidly induced to autophosphorylate in trans, resulting in dissociation to form partially active monomers (Bakkenist and Kastan, 2003).
The exact nature of the primary activating signal that triggers ATM autophosphorylation remains unknown; however, it does not appear to be limited to the immediate vicinity of the damage and may be linked to long-range
alterations in chromatin structure (Bakkenist and Kastan, 2003). Serine (S)
S1981 was the first autophosphorylation site to be identified; however, this
residue is not essential for ATM function, at least in mice (Pellegrini et al.,
2006), although its modification is tightly linked to ATM activation under
most circumstances (Bakkenist and Kastan, 2003). Subsequent studies have
documented additional ATM autophosphorylation sites at S367 and S1893
that may contribute to the activation process, perhaps explaining why
S1981 is individually nonessential, while acetylation mediated by the
TIP60 acetyl-transferase may also play a role (Lavin and Kozlov, 2007).
Joanne Smith et al.
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DNA damage (DSBs)
ATM
P
RPA
RPA
RPA
RPA
RPA
RPA
RPA
P
P
53BP1
g H2AX
RPA
Ac
TIP60
MDC1
Replication arrest
RAD1
NBS1
Rad50
MRE11
HUS1
RAD9
ATRIP
TopBP1
Ac
ATM
g H2AX
P
ATR
P
P
P
Claspin
p53
P P
P
Chk2
P
G1
checkpoint
P
Chk1
G2, Intra-S checkpoints
Fork stabilization
Origin suppression
S-M checkpoint
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 ATM
modifies 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 conjunction 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 that
are summarized in Fig. 2. Please refer to the text for further details and explanation.
ATM monomers are then recruited to DSBs via interactions with the MRN
sensor complex (Lee and Paull, 2007), stimulating full activation and
providing a platform that enables ATM to act locally on multiple substrates
at the site of damage. Local substrates include the variant histone, H2AX,
forming the DNA damage-associated -H2AX histone mark (FernandezCapetillo et al., 2004), the MRN complex itself (discussed in more detail
below), the cohesin SMC1 (Kitagawa et al., 2004), and the downstream
effector kinase Chk2 (Lukas et al., 2003). ATM phosphorylates Chk2 on a
DNA Damage and Cancer
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specific threonine (T) residue, T68, located within an N-terminal serine/threonine-glutamine (SQ/TQ)-rich motif (Ahn et al., 2000). Once phosphorylated,
the SQ/TQ motif of one Chk2 molecule is recognized by the phosphopeptidebinding Fork-head associated (FHA) domain of another, leading to transient
homodimerization, intermolecular activation loop autophosphorylation, and
full 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 substrates
involved in cell cycle progression, apoptosis, and gene transcription (Lukas
et al., 2003). Known substrates of Chk2 include the p53 tumor suppressor
protein (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 on
other substrates which do not necessarily concentrate at sites of damage. For
example, ATM plays an important role in activating the p53 response to
DNA 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 at
DSBs (Lavin, 2008). In addition, there is increasing evidence that ATM may
also 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 or
replication-blocking DNA damage lesions such as those inflicted by ultraviolet (UV) light (Abraham, 2001). When replication is blocked, DNA polymerases become uncoupled from the replicative helicase (Byun et al., 2005),
generating tracts of ssDNA that rapidly become coated with the trimeric
ssDNA-binding protein complex, Replication Protein A (RPA). ATR is
recruited to and activated at such tracts in association with its partner
protein, ATRIP, which interacts directly with ssDNA complexed with RPA
via 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 at
dysfunctional telomeres. In addition, it is important to note that the ATR–
Chk1 pathway is also activated in response to DSBs when ssDNA is generated as a result of nucleolytic strand resection (discussed in more detail
below). Conversely, replication of damaged DNA can result in DSBs when
leading-strand DNA polymerases encounter single-strand nicks or abasic
sites. As a result, the ATM–Chk2 and ATR–Chk1 pathways are frequently
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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 phosphorylation of Chk1 depends on the actions of two mediator proteins,
TopBP1 and Claspin. TopBP1, which is recruited to ssDNA-RPA via the
PCNA-like RAD9: RAD1: HUS1 checkpoint clamp (Delacroix et al., 2007),
contains a domain that stimulates ATR activity, although exactly how this
occurs is unclear (Kumagai et al., 2006; Mordes et al., 2008). A second mediator, Claspin, which likely associates with active replication forks during normal
replication (Lee et al., 2003), is then subject to ATR-dependent phosphorylation within a short, repeated motif which, once modified, binds Chk1 and
serves as a platform for ATR-mediated phosphorylation and activation (Guo
et al., 2000). Phosphorylation within the Claspin Chk1-binding motifs depends
on ATR kinase activity (Kumagai and Dunphy, 2003); however, the modified
residues do not occur within consensus SQ/TQ) ATR target sites. So far the
kinase directly responsible for this final crucial step in Chk1 activation has not
been 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 additional mediators, Timeless and Tipin (Timeless-interacting protein), both for
normal replication and for ATR–Chk1 activation in response to replication
stress (Kondratov and Antoch, 2007). Timeless binds to both ATR and Chk1
whereas Tipin can interact with Claspin (Kemp et al., 2010). Recent data
indicate that like ATRIP, Tipin binds to a specific subunit of the RPA complex
(although RPA2 rather than RPA1) and is required for stable association of
both Timeless and Claspin with tracts of ssDNA-RPA (Kemp et al., 2010).
In addition to checkpoint activation, Timeless and Tipin also seem to be
required for replication fork stabilization and restart (Errico et al., 2007).
Interestingly, the Drosophila homologue of Timeless is a circadian rhythm
regulator, although whether this function is also conserved in mammals is less
clear (Kondratov and Antoch, 2007).
Phosphorylated Claspin then recruits Chk1 (Jeong et al., 2003) to ssDNARPA complexes, bringing it into close proximity with active ATR (Kumagai
and Dunphy, 2003) and enabling ATR to phosphorylate Chk1 directly at
multiple S/T-Q sites within the C-terminal regulatory domain, most notably
at serines (S) S317 and S345, which are widely monitored as surrogate
markers of activation. Phosphorylation of these sites, and in particular
serine (S) S345, is essential for Chk1 biological activity, although exactly
how these modifications regulate Chk1 catalytic remains poorly understood
(Niida et al., 2007; Walker et al., 2009). ATR-mediated phosphorylation is
reported to stimulate Chk1 kinase activity by relieving inhibition by the
DNA Damage and Cancer
79
C-terminal regulatory domain (Oe et al., 2001; Walker et al., 2009); however, 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 activation
loop (Chen et al., 2000), and the exact target sites and functional consequences 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 Chk1
substrates involved in cell cycle control include positive and negative regulators of Cdk inhibitory phosphorylation, such as Cdc25A (Falck et al., 2002),
Cdc25C (Blasina et al., 1999), and Wee1 (Lee et al., 2001). Chk1-mediated
phosphorylation inhibits the activity of both Cdc25A and Cdc25C under
conditions of genotoxic stress, although by different mechanisms; phosphorylation of Cdc25A targets the protein for degradation (Falck et al., 2002),
while phosphorylated Cdc25C is sequestered in an inactive form through
association with 14-3-3 proteins (Peng et al., 1997). Wee1 kinase activity, by
contrast, 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 mediate
DNA damage-induced repression of gene transcription through phosphorylation of histone H3 (Shimada et al., 2008). Although predominantly nuclear,
a proportion of active Chk1 also localizes at the centrosome, where it is
thought to control the timing of activation of the mitotic Cdk1/cyclin
B complex, and thus the onset of mitosis, both after damage and during
unperturbed 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 and
Cortez, 2008). In addition, global proteomic analyses suggest that ATM
and ATR probably phosphorylate many other substrates; however, in most
cases, the functional significance of these modifications has not yet been
established (Matsuoka et al., 2007). In contrast to ATM and Chk2, however,
ATR and Chk1 are thought to be active at low levels even during unperturbed cell cycles, particularly during S-phase (Syljuasen et al., 2005), potentially explaining why they are essential in many cell types.
III. CHECKPOINT FUNCTIONS OF THE ATM–CHK2
AND ATR–CHK1 PATHWAYS
DNA damage or DNA synthesis inhibition in vertebrate cells evokes the
activation of multiple, mechanistically distinct checkpoint responses that
facilitate repair and promote cell survival (Kastan and Bartek, 2004).
Joanne Smith et al.
80
DNA damage checkpoints
G1 checkpoint
Intra-S
checkpoint
G1
G2 checkpoint
S
Fork
stabilization
G2
Origin
firing
M
S-M checkpoint
Replication checkpoints
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 to
DNA 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 to
stabilize 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 and
G2/M transitions (the G1 and G2 checkpoints), and a transient decrease in
the rate of DNA synthesis (the intra-S checkpoint). Of these, the G1 checkpoint is unique in depending primarily on the function of the p53 tumor
suppressor protein and its downstream target, the cyclin-dependent kinase
inhibitor p21CIP1 (Kastan and Bartek, 2004). G2 arrest, by contrast, is
imposed by blocking activation of the mitotic Cdk1-cyclinB complex by
preventing removal of the inhibitory threonine 14/tyrosine 15 (T14/ Y15)
phosphorylation of Cdk1 (O’Connell et al., 2000). This is achieved, at least
in large part, via inhibition of Cdc25 family phosphatases which play an
important role in reversing this inhibitory phosphorylation to rapidly
activate the Cdk1-cyclin B complex and trigger the onset of mitosis
(Boutros et al., 2007). The molecular mechanism of the intra-S checkpoint
is less well defined; however, it can involve both active replication
fork slowing and suppression of replication origin firing (Grallert and
Boye, 2008; Seiler et al., 2007).
When DNA synthesis is blocked, additional replication checkpoint
responses are required to stabilize stalled replication forks and prevent the
formation of new forks by suppressing late replication origin firing (Branzei
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81
and Foiani, 2009). Because these functions are essential if cells are to resume
replication and complete S-phase when circumstances permit, they are
sometimes collectively termed the “replication recovery checkpoint.” In
addition, it is essential that cells arrested in S-phase do not attempt mitosis
until replication is complete. The mitotic delay triggered by DNA synthesis
inhibition, which is also likely mediated through maintenance of inhibitory
phosphorylation of Cdk1 (O’Connell et al., 2000), is generally termed the
S-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 yeast
and Cds1 in fission yeast, while both also express Chk1 homologs
(O’Connell et al., 2000). The checkpoint functions of the yeast effector
kinases are, however, remarkably variable; in budding yeast Rad53 is the
dominant effector of both DNA damage and replication checkpoints, whereas in fission yeast DNA damage responses are assigned to Chk1 while Cds1
regulates replication checkpoint functions (O’Connell et al., 2000). When
Chk1 and Chk2 were identified in vertebrates, the obvious question was
“what would be the ‘division of labor’ compared to these model
organisms?”
Initially it was widely considered that the ATM–Chk2 and ATR–Chk1
pathways acted in parallel, with Chk1 and Chk2 playing overlapping or
partially redundant roles in downstream checkpoint responses (as depicted
in Fig. 1). Although some early studies suggested that Chk1 and Chk2
shared certain common substrates involved in cell cycle arrest, such as
Cdc25 family phosphatases (Chaturvedi et al., 1999; Matsuoka et al.,
1998), subsequent genetic and biochemical data have increasingly emphasized ATR–Chk1 as the principal, direct effector of the DNA damage
and replication checkpoints, with ATM–Chk2 playing an auxiliary role
specifically in the response to DSBs (Bartek and Lukas, 2003; Kastan and
Bartek, 2004).
Fundamental differences in the normal physiological functions of
the ATM–Chk2 and ATR–Chk1 pathways were initially evident from the
phenotypes of mice and cells deficient for each pathway. Thus, germ-line
inactivation of ATR or Chk1 results in early embryonic lethality, whereas
ATM- and Chk2-knockout mice are viable, and at least in the case of Chk2,
remarkably normal (Brown and Baltimore, 2000; Liu et al., 2000; Takai
et al., 2000). Similarly, ATM- and Chk2-deficient somatic cells can proliferate successfully in culture (Jallepalli et al., 2003; Lavin and Shiloh, 1997; Xu
and Baltimore, 1996), whereas acute genetic inactivation of ATR or Chk1
leads to rapid cell death (Brown and Baltimore, 2003; Liu et al., 2000; Niida
et al., 2007). Interestingly, DT40 lymphoma cells represent an exception to
this general rule, since they survive genetic inactivation of Chk1 function,
albeit with impaired cell growth and survival (Zachos et al., 2003).
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Joanne Smith et al.
ATM-deficient human and mouse cells classically exhibit impaired G1,
intra-S, and G2 checkpoint proficiency after DNA damage (Lavin, 2008). As
mentioned previously, ATM is an important determinant of p53 stabilization and activation, and evidence suggests that the weakened G1 arrest in
ATM mutant cells is attributable to inefficient p53 activation and downstream p21CIP1 induction (Kastan et al., 1992). The mechanism of the
intra-S checkpoint is more complex. ATM is thought to suppress DNA
replication via two distinct mechanisms; firstly, via direct phosphorylation
of NBS1, and secondly through Chk2-mediated degradation of Cdc25A
leading to inhibition of Cdk2, which in association with cyclins E or A is
required for DNA replication origin firing (Falck et al., 2002).
Interestingly, G2 checkpoint impairment in ATM mutant cells after exposure to ionizing radiation is markedly cell cycle phase-dependent. Thus,
ATM-deficient cells in G2 phase at the time of damage are unable to arrest
efficiently, whereas cells damaged in G1 and S-phase experience instead a
prolonged arrest on reaching G2 compared to genetically normal counterparts (Xu et al., 2002). This has been explained by the existence of two
molecularly distinct G2/M checkpoint arrests; “immediate G2 arrest,”
which is triggered rapidly in G2 cells, and “G2 accumulation,” which affects
cells that reach G2 after traversing S-phase and develops over many hours
(Xu et al., 2002). Immediate G2 arrest is ATM-dependent and of relatively
short duration, whereas G2 accumulation is ATM-independent (but ATR–
Chk1-dependent) and much longer-lasting (Xu et al., 2002). Why this
should be is not fully understood; it may reflect a more stringent requirement
for ATM-dependent DSB processing for rapid and efficient checkpoint activation in G2 than in S-phase (discussed in more detail below), whereas the
prolonged G2 accumulation experienced by ATM mutant cells could reflect
a defect in DNA repair (Beucher et al., 2009). As for mechanism, Chk2 was
initially invoked as a downstream effector of ATM-dependent G2 arrest via
phosphorylation 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 checkpoint
proficiency.
As with ATM, Chk2 has been implicated in p53 activation (Chehab et al.,
2000), and consistent with this, Chk2-deficient mice show impaired G1
checkpoint arrest and defects in p53-dependent gene transcription after
damage (Hirao et al., 2002; Takai et al., 2002). They also show a marked
reduction in p53-dependent apoptotic responses in adult and developing
tissues and increased organismal survival after whole body irradiation
(Hirao et al., 2002; Takai et al., 2002). Perplexingly, however, p53 regulation is not altered in Chk2-deleted HCT116 cells, a human cancer cell line
that retains wild-type p53 (Jallepalli et al., 2003). Also puzzling is the fact
that G2 checkpoint impairment as a result of Chk2 inactivation is observed
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83
in some, but not all, experimental systems. Thus, MEFs and HCT116 cells
deleted for Chk2 retained normal G2 checkpoint proficiency (Jallepalli et al.,
2003; Takai et al., 2002), whereas Chk2 knockout DT40 cells exhibited a
weakened and delayed G2 arrest at early times after irradiation (Rainey
et al., 2008). Interestingly, this defect was most severe in G2 cells, much as
described for ATM (Xu et al., 2002), whereas the slower G2 accumulation
response was relatively unaffected (Rainey et al., 2008). Variations in the
severity of G2 checkpoint deficiency in cells genetically deficient for Chk2
have often been interpreted in terms of compensation by Chk1; however, it is
equally possible that the role of Chk2 in this checkpoint simply varies
between cell types.
Phenotypic analysis of checkpoint proficiency in ATR- or Chk1-deficient
mouse embryos and cells is complicated by loss of viability; however, inactivation of both genes results in profound G2 and S-M checkpoint defects
after DNA damage or replication arrest (Brown and Baltimore, 2000;
Brown and Baltimore, 2003; Liu et al., 2000; Takai et al., 2000). Consistent
with this, Chk1-deficient DT40 cells (which lack functional p53) show
complete loss of proficiency for all DNA damage and replication checkpoint
responses (Zachos et al., 2003, 2005). Strikingly, these cells show no measurable G2 arrest or intra-S checkpoint activation after irradiation at any
dose tested (Zachos et al., 2003), in marked contrast to the partial loss of G2
checkpoint proficiency that results from deletion of Chk2 (Rainey et al.,
2008). Loss of G2 checkpoint proficiency was furthermore associated with
failure to maintain inhibitory T14/Y15 Cdk1 phosphorylation (Zachos
et al., 2003), indicating that Chk1 is essential to restrain the mitosis-promoting activity of Cdc25 family phosphatases after DNA damage. In addition, when DNA polymerase is inhibited Chk1-deficient cells suffer a
combination of progressive replication fork collapse and futile origin firing
that leads ultimately to S-M checkpoint failure and premature entry to
mitosis with unreplicated DNA (Zachos et al., 2005).
Interestingly, Chk1-deficient DT40 cells also exhibit high levels of spontaneous replication fork collapse during unperturbed cell cycles which is
compensated by increased replication origin firing (Maya-Mendoza et al.,
2007; Petermann et al., 2006). Although this compensation mechanism
evidently allows these cells to replicate successfully and to maintain an
S-phase of approximately normal length, replication fork collapse as a result
of 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 culture
using various dominant-negative, siRNA depletion, or chemical inhibition
approaches. The general consensus that has emerged from such
studies [reviewed in (Bartek and Lukas, 2003; Kastan and Bartek, 2004;
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Joanne Smith et al.
Stracker et al., 2009)] is broadly consistent with the genetic analysis in mice
and DT40 cells summarized above; namely that ATR and Chk1 are crucial
both for the G2 and intra-S checkpoint responses induced by DNA damage
and for the S-M and fork stabilization/origin suppression checkpoints triggered by replication arrest. Whether the ATR–Chk1 pathway also contributes to p53-dependent G1 arrest under any circumstances is less clear.
Athough some early biochemical data implicated ATR and Chk1 as potential regulators of p53 (Shieh et al., 2000; Tibbetts et al., 1999), more recent
evidence that ATR–Chk1 activation by DNA damage is largely restricted to
the S and G2 phases of the cell cycle makes it seem unlikely to be a major
physiological 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; nonhomologous end-joining (NHEJ) and homologous recombination repair
(HRR). NHEJ occurs throughout the cell cycle; however, because HRR
requires a sister chromatid to serve as a template, this mechanism is restricted to the S and G2 phases. Unlike NHEJ, HRR requires extensive DNA
damage processing to generate tracts of ssDNA that, once coated with
Rad51 recombinase, invade the homologous DNA duplex to initiate repair.
Such single-stranded tracts are generated by resection of DSBs in a 30 –50
direction, a reaction initiated by the endonuclease activity of the MRN
complex (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 a
major role in restricting HRR to S and G2. In yeasts, Cdk activity controls
DNA strand resection via phosphorylation of Sae2 (Wohlbold and Fisher,
2009), a putative nuclease that promotes resection in collaboration with the
yeast counterpart of MRN (Huertas et al., 2008). Vertebrate cells express an
ortholog 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 via
direct phosphorylation of CtIP in a manner analogous to yeast (Huertas and
Jackson, 2009; Yun and Hiom, 2009). Thus, because Cdk-mediated phosphorylation of CtIP is required for strand resection, increased Cdk activity in
S and G2 ensures maximum resection activity in these phases of the cell
cycle.
DNA Damage and Cancer
85
Two other important components required for HRR are the BRCA1 and
BRCA2 tumor suppressors. BRCA1 is a multifunctional protein that plays
multiple roles in checkpoint activation, DNA repair, and gene transcription
in response to DNA damage (Huen et al., 2010). BRCA1 is recruited to sites
of ionizing radiation-induced DNA damage and is required for efficient
generation of ssDNA at early times after irradiation (Schlegel et al., 2006).
Although the mechanism is not fully understood, BRCA1 interacts with
MRN and CtIP, suggesting that it too may play a role in strand resection
(Chen et al., 2008). BRCA2 by contrast is required for homologous recombination downstream of strand resection through its well-established role in
loading 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 DSBs
provides a new framework for understanding the organization and integration of checkpoint and repair pathways (Adams et al., 2006; Cuadrado et al.,
2006; Jazayeri et al., 2006; Myers and Cortez, 2006). Although not classically
considered a core homologous recombination (HR) factor, ATM is required
for efficient HRR of a subset of DSBs specifically in G2 phase (Beucher et al.,
2009). Molecular details are still emerging; however, current thinking is that
initial recognition of DSBs by the MRN complex leads to recruitment and full
activation of ATM. Active ATM then promotes the recruitment of CtIP to
sites of damage where it interacts with and stimulates the nuclease activity of
MRE11 to initiate strand resection and generate short tracts of ssDNA (You
et al., 2009). These may be extended through the actions of other nucleases
and helicases, such as Exo1 and BLM, to generate more extensive regions of
ssDNA that recruit RPA and form both the initiating substrate for HRR and
activating 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 after
damage, and at least in the case of CTIP, this modification appears to be
required 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 not
DNA polymerase inhibition, since the latter generates extensive tracts of
ssDNA directly without the need for DNA damage processing (Byun et al.,
2005; Myers and Cortez, 2006). Secondly, it accounts for why ATR–Chk1
activation in response to irradiation-induced DSBs is largely confined to S
and G2 phase (Jazayeri et al., 2006; Walker et al., 2009), since this is when
levels of Cdk activity become permissive for efficient strand resection
(Cerqueira et al., 2009). Thirdly, it explains why other proteins required
for ssDNA generation, such as MRE11, NBS1, and BRCA1, are also required both for rapid ATR–Chk1 activation and G2 checkpoint proficiency
in response to DSBs (Myers and Cortez, 2006; Yarden et al., 2002).
Joanne Smith et al.
86
DNA damage (DSBs)
CtIP
NBS1
Rad50
MRE11
g H2AX
BRCA1
ATM
DNA stand
resection
Chk2
p53
G1
checkpoint
BRCA2
Rad51
Homologous
recombination
RAD1
RPA
RPA
RPA
RPA HUS1
ATRIP
RAD9
Replication
arrest
TopBP1
ATR
Claspin
Chk1
G2, Intra-S checkpoints
Fork stabilization
Origin suppression
S-M checkpoint
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, is
required 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 need
for 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 DNA
damage 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.
DNA Damage and Cancer
87
Placing ATM upstream of ATR–Chk1 activation in response to DSBs
suggests a new interpretation of their overlapping, yet distinct, functions in
checkpoint signaling (as depicted in Fig. 3). In this model the ATR–Chk1
pathway is the principal direct effector of the damage and replication
checkpoints (apart from p53-dependent G1 arrest), with ATM modulating
the response specifically to DSBs indirectly via its role in strand resection.
Thus, in ATM mutant cells, rapid activation of ATR and Chk1 in response to
DSBs will be impaired as a consequence of inefficient strand resection. This
defect is likely to be particularly significant in G2 phase, when resection is
the principal mechanism of ssDNA generation. This model predicts therefore that the impact of ATM deficiency on ATR–Chk1 activation and
thus G2 checkpoint proficiency in response to DSBs will be greatest in G2
phase, consistent with the immediate G2 arrest defect described in ATM
mutant cells (Xu et al., 2002). This model also explains why Chk1-deficient
DT40 cells lack any detectable G2 checkpoint after irradiation, despite the
continued presence of functional ATM and Chk2 (Zachos et al., 2003), since
in this scheme Chk1 is the sole direct downstream effector of G2 arrest.
Conversely, because DNA polymerase inhibition generates ssDNA directly
without the need for resection, ATR and Chk1 are essential for replication
checkpoint 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 cell
cycle, including G1, consistent with their established roles in activating p53
and triggering G1 arrest (Lavin and Kozlov, 2007; Takai et al., 2002). Chk2
is, however, variably required for G2 checkpoint proficiency in different cell
types, and whether it is a direct effector of this checkpoint has been questioned (Antoni et al., 2007). One intriguing possibility, suggested by the
apparent epistatic relationship between Chk1 and Chk2 in DT40 cells (i.e.,
where G2 checkpoint proficiency is completely abolished in the absence of
Chk1 but only impaired in the absence of Chk2), is that Chk2 might also
participate in the DNA strand resection process and thus in regulating the
efficiency and cell cycle phase-specificity of ATR–Chk1 activation indirectly.
Although there is currently no direct evidence for such a role, further
investigation seems warranted.
V. ATM–CHK2 AND ATR–CHK1 PATHWAY
ALTERATIONS IN CANCER
The importance of genome stability for preventing carcinogenesis is evident both from human cancer predisposition syndromes that result from
inherited loss-of-function mutations in DNA damage response genes and
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Joanne Smith et al.
from the occurrence of sporadic mutations affecting such genes in cancers in
otherwise genetically normal individuals (Kastan and Bartek, 2004). Examples of both have been found to affect ATM and Chk2 in human cancer,
whereas ATR and Chk1 appear to be mutated only rarely. Important insights
into the roles of these pathways in tumor formation have also been obtained
from experiments using genetically modified mice. In discussing these issues,
we also refer in passing to other gene functions, such as the MRN complex
and BRCA1/BRCA2, where these are closely linked either to the regulation
or 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), characterized by immunodeficiency, neurodegeneration, radiation hypersensitivity, and spontaneous predisposition to lymphoma (Shiloh and Kastan,
2001). Similarly, hypomorphic mutations affecting the genes encoding the
functionally related MRE11 and NBS1 proteins give rise to the human
conditions Ataxia-like disorder (ATLD), and Nijmegen breakage syndrome
(NBS), each of which shares some clinical similarities with AT, although only
NBS is clearly associated with cancer predisposition (Stewart et al., 1999;
Varon et al., 1998). As with AT humans, ATM knockout mice are predisposed to lymphoma and radiosensitive (Xu et al., 1996), while mice with
engineered hypomorphic mutations of NBS1 or RAD50 are also cancer
prone (Bender et al., 2002; Kang et al., 2002; Williams et al., 2002). The
precise cause of cancer predisposition in humans and mice with inherited
defects in ATM or MRN components is not yet known; however, genomic
instability and an increased mutation rate resulting from repair and checkpoint defects is presumably an important factor.
Interestingly, although AT is considered to be an autosomal recessive
genetic disorder, individuals heterozygous for ATM mutations show an
increased incidence of cancer possibly related to medical or occupational
radiation exposure (Briani et al., 2006; Swift et al., 1991). In addition, cells
from heterozygote individuals show sensitivity to radiation in vitro that is
intermediate between those from AT patients and normal individuals (Swift
et al., 1991). Taken together, these findings indicate that ATM is a partially
penetrant cancer susceptibility gene that might interact with certain environmental predisposing factors. Somatic mutations affecting ATM have also
been documented in sporadic lymphoid malignancies and lung adenocarcinomas, although at relatively low incidence (Ding et al., 2008; Gumy-Pause
et al., 2004).
In contrast to AT, ATLD, and NBS, human individuals heterozygous for
loss-of-function alleles of the BRCA1 and BRCA2 tumor suppressor genes
are developmentally normal but suffer from a greatly increased incidence of
breast and ovarian cancer (O’Donovan and Livingston, 2010). As mentioned previously, BRCA1 is required for ATR–Chk1 activation, G2 arrest,
DNA Damage and Cancer
89
and other complex responses to DNA damage (Huen et al., 2010). In
general, however, the tumor suppressive functions of BRCA1 and BRCA2
are attributed to their distinct but essential roles in HR-mediated DNA
repair (O’Donovan and Livingston, 2010). Because tumorigenesis involves
functional inactivation of the remaining functional BRCA1 or BRCA2 allele
through loss of heterozygosity or other means (Collins et al., 1995;
Neuhausen and Marshall, 1994), the tumors that arise in susceptible individuals consist of cells that are deficient for HRR whereas those in normal
tissues remain proficient (Turner et al., 2005). Genomic instability as a result
of HRR deficiency is thought to play a key role in the development of such
tumors, presumably by accelerating the accumulation of oncogenic
mutations (Tutt et al., 2002); however, as discussed below, the presence or
absence of DNA repair proficiency in normal and tumor tissue respectively
in 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 initially
reported in a subset of Li-Fraumeni kindreds lacking p53 mutations, consistent with a functional link between Chk2 and p53 (Bell et al., 1999).
However, because these mutant alleles were subsequently also found in
normal individuals in the general population they are now considered
unlikely to be the genetic cause of Li Fraumeni syndrome (Antoni et al.,
2007). Nevertheless, studies have established that individuals bearing certain mutant Chk2 alleles do suffer from a statistically significant increase in
the incidence of breast, prostate, and other cancers, suggesting that Chk2 is
indeed a moderate or low penetrance cancer susceptibility gene in humans
(Antoni et al., 2007). Despite this, the tumors that arise in individuals
heterozygous for such mutations do not consistently lose the remaining
normal Chk2 allele, indicating that Chk2 does not conform to the conventional definition of a tumor suppressor (Antoni et al., 2007). One possibility
is that mutant Chk2 proteins exert a dominant-negative effect by inhibiting
the endogenous, normal Chk2, to phenocopy loss of function in the heterozygous state. This would be consistent with the occurrence of occasional
sporadic Chk2 mutations and rare instances of reduced or absent expression
in a variety of different tumor types (Antoni et al., 2007). Alternatively, it is
conceivable that Chk2 haploinsufficiency per se synergises with other, as yet
unknown, oncogenic events or environmental factors to promote malignant
progression.
The impact of Chk2 deficiency on tumorigenesis in mice has also been
examined. Although Chk2 knockout mice are developmentally normal and
not spontaneously cancer prone (Takai et al., 2002), they are more sensitive
to chemical skin carcinogenesis, showing an increase both in overall tumor
burden and in the rate at which benign tumors formed after exposure to
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Joanne Smith et al.
chemical carcinogens (Hirao et al., 2002). Whether this increase in tumor
sensitivity is attributable to increased genomic instability or perhaps to the
relative resistance to stress-induced apoptosis that has been observed in
Chk2 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 of
deleterious symptoms, including growth retardation and microcephaly;
however, such individuals do not suffer from an increased incidence of
cancer (Kerzendorfer and O’Driscoll, 2009). Seckel syndrome has also
been modeled in mice (Murga et al., 2009), and the consequences of acute
conditional genetic inactivation of ATR postdevelopment in adult mice have
been investigated (Ruzankina et al., 2007). Degenerative and premature
aging-like phenotypes were observed in each case, underscoring the crucial
importance of ATR for normal development, stem cell survival, and tissue
homeostasis; however, neither showed evidence of cancer predisposition
(Murga et al., 2009; Ruzankina et al., 2007). In general therefore it appears
that partial or complete inactivation of ATR function, although clearly
deleterious in at least some cell types in vivo, does not perturb genome
stability 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 and
endometrial 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 appear
to be rare in human cancers, although some exceptions have been reported
in tumors with MSI (Bertoni et al., 1999; Menoyo et al., 2001). Embryonic
lethality in knockout mice precludes direct assessment of the effect of
constitutive Chk1 inactivation on spontaneous or induced carcinogenesis;
however, some evidence that partial loss of function can promote tumor
formation has been reported. Thus, mammary tumors induced by an oncogenic WNT transgene developed more rapidly in Chk1 hemizygous mice
than wild-type (Liu et al., 2000). Importantly, however, loss of the remaining
functional Chk1 allele was not observed, suggesting that Chk1 continued to
be 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 the
formation of tumors induced by the chemical carcinogens, DMBA and TPA,
using a conditional allele of Chk1 combined with a Keratin14-CreER
recombinase transgene (Indra et al., 2000; Lam et al., 2004). This combination allows efficient Chk1 deletion throughout the epidermis in response to
systemic treatment with the synthetic estrogen, tamoxifen (LM Tho and DA
91
DNA Damage and Cancer
Gillespie, unpublished results). We find that homozygous deletion of Chk1
throughout the epidermis is tolerated without acute pathology, presumably
because Chk1 is not essential in the postmitotic, terminally differentiated
cells that comprise the bulk of the tissue. However, when recombination is
induced immediately prior to carcinogen exposure both the number and size
of benign papillomas obtained is strongly suppressed (Table 1). Furthermore, the small lesions that form in ablated skin always derive from cells
that escape recombination (LM Tho and DA Gillespie, unpublished results).
Although the basis of this tumor suppressive effect is not yet fully understood, we hypothesize that Chk1 is probably essential for the proliferation
or survival of the epidermal stem cells that are thought to give rise to
chemical carcinogen-induced tumors in the skin (Morris, 2004). Consistent
with this, developmental or conditional deletion of Chk1 has been shown to
lead to rapid cell death in several other tissues including mammary gland,
intestine, and lymphocytes (Greenow et al., 2009; Lam et al., 2004; Zaugg
et al., 2007).
In marked contrast, Chk1 hemizygous skin supports normal papilloma
formation but such hemizygous lesions show an increased probability of
progressing to malignant carcinoma (Table 1). We deduce from these experiments that whereas complete loss of Chk1 function is incompatible with skin
tumor formation, partial loss of function fosters benign-malignant tumor
progression. This conclusion is consistent with the previously described
acceleration of WNT-induced tumorigenesis and also evidence that Chk1
Table 1 Conditional Deletion of Chk1 in Mouse Epidermis Suppresses Chemical
Carcinogen-Induced Skin Tumorigenesis
Cohort
1
2
3
4
Genotype
Treatment
Mean no. papillomas
% Conversion
to carcinoma
Chk1þ/þ K14-CreER
Chk1Fl/Fl K14-CreER
Chk1Fl/Fl K14-CreER
Chk1Fl/þ K14-CreER
Tamoxifen
Vehicle
Tamoxifen
Tamoxifen
17.8 (N ¼ 20)
15.4 (N ¼ 19)
5.5 (N ¼ 18)
14.6 (N ¼ 19)
5.9
5.1
2
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.
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Joanne Smith et al.
haploinsufficiency can lead to aberrant cell cycle regulation and genomic
instability in vivo (Lam et al., 2004; Liu et al., 2000). Conversely, it has been
reported that Chk1 hemizygous mice suffer from an increased incidence of
anemia associated with increased levels of DNA damage in erythroid progenitors, suggesting that Chk1 haploinsufficiency can also result in degenerative 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 very
different. Impairment or even complete loss of ATM–Chk2 signaling is
compatible with cell and organism survival, although frequently at the cost
of cancer predisposition, presumably at least in part as a result of genomic
instability and more rapid accumulation of oncogenic mutations. In contrast, ATR and Chk1 seem to be essential for the proliferation and survival
of many, although not all, cell types, both in vitro and in the developing
embryo and adult organism, presumably because they control aspects of
DNA metabolism that when dysfunctional lead to cell death rather than
survival with mutation. In this scheme Chk1 (and arguably ATR) becomes a
logical target for therapeutic strategies based on pharmacological checkpoint suppression (discussed below), although evidence from mouse models
that partial loss of Chk1 function (i.e., haploinsufficiency) may promote
tumorigenesis, or other undesirable pathologies (Boles et al., 2010), clearly
merits careful consideration.
VI. EXPLOITING HOMOLOGOUS RECOMBINATIONAL
REPAIR (HRR) DEFECTS FOR CANCER THERAPY
In recent years it has emerged that in addition to predisposing to cancer as
a result of increased genomic instability, defective HRR may also render
tumor cells inherently vulnerable to specific conventional anticancer agents
and also to new strategies based on inhibition of complementary repair
pathways. Thus, BRCA1- and BRCA2-deficient tumor cells have been
found to be hypersensitive to cross-linking agents such as cisplatin in vitro
(Bhattacharyya et al., 2000; Yuan et al., 1999), most probably because HR
is the principal mechanism through which replication forks stalled or
collapsed by such lesions are repaired or restarted. Importantly, evidence
suggests this is also true in vivo and that ovarian cancers arising in BRCA1
and BRCA2 mutation carriers may respond better to platinum-based therapy than similar sporadic tumors (Foulkes, 2006). Remarkably, secondary
mutations that restore BRCA1 or BRCA2 function can be a cause both for
drug resistance in ovarian cancer cells in culture and treatment failure in
DNA Damage and Cancer
93
patients, emphasizing the importance of HRR deficiency in determining
sensitivity 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 in
multiple aspects of the DNA damage response (Rouleau et al., 2010).
In response to DNA damage active PARP1 binds to SSBs in DNA and
catalyzes the synthesis of branched, protein-conjugated poly (ADP-ribose)
chains. Many of these chains become linked to PARP1 itself as a result of
automodification, although histones, topoisomerases, and many other proteins involved in diverse aspects of DNA metabolism are also substrates for
PARP1 (D’Amours et al., 1999). Once formed at sites of damage, such poly
(ADP-ribose) chains recruit multiple proteins involved in DNA repair and
modulating chromatin structure (Rouleau et al., 2010). In particular, PARP1
both modifies and recruits XRCC1, a key scaffolding factor required for
base excision repair (BER). BER excises damaged or mismatched bases from
DNA and also repairs single-strand gaps, nicks, and abasic sites, lesions
which arise both spontaneously and as a result of oxidative stress or
exposure 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 the
potency of DNA damaging chemotherapies (Zaremba and Curtin, 2007).
Although PARP1 inhibitors are relatively nontoxic to most cancer cells
alone, it was subsequently discovered that tumor cells deficient for BRCA1
or BRCA2 were inherently and exquisitely sensitive to such agents even in
the absence of exogenous genotoxic stress (Bryant et al., 2005; Farmer et al.,
2005). The basis of this selective sensitization has been explained in terms of
synthetic lethality, a term applied to genetic functions or pathways whose
individual loss is tolerated but when combined result in lethality (Kaelin,
2005). In this context of course the synthetic lethal interaction occurs
between an inherent genetic deficiency for HRR resulting from BRCA1/
BRCA2 mutation and a phenocopy of functional BER deficiency that is
imposed through pharmacological inhibition of PARP1.
Thus, inhibition of PARP1 leads to accumulation of SSBs and other endogenous DNA damage lesions that would normally be corrected by BER (Jeggo,
1998). Such lesions are particularly problematical in S-phase, since when
encountered by replicative polymerases they lead to replication fork collapse
and formation of DSBs. HRR is thought to be the main mechanism through
which such DSBs can be repaired and replication restarted, enabling HRR
proficient cells to tolerate PARP inhibition (Li and Heyer, 2008). However,
because BRCA1- and BRCA2-deficient tumor cells are impaired both for
HRR and BER under conditions of PARP inhibition, replication-associated
DSBs cannot be repaired and thus escalate to lethal proportions (Bryant et al.,
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Joanne Smith et al.
2005; Farmer et al., 2005). As with resistance to platinum compounds,
acquired resistance to PARP inhibition can arise through reversion mutations
that 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 ovarian
cancers arising in BRCA1 and BRCA2 mutation carriers, and remarkably,
this activity is achieved without the toxicity associated with conventional
genotoxic chemotherapy (Fong et al., 2009, 2010). In addition, much preclinical evidence suggests that PARP inhibition may also synergize with
conventional genotoxic chemotherapies, including alkylating agents, platinum compounds, topoisomerase inhibitors, and radiation, both in BRCA1
and BRCA2 mutant and sporadic tumors (Ratnam and Low, 2007). As a
result, Olaparib and many other PARP inhibitors are currently undergoing
trials 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 widespread
this phenotype is in sporadic cancers arising in genetically normal individuals. Cell culture studies show that experimental inhibition of many diverse
gene functions involved either directly in HRR or in DNA damage signaling
more generally (including Rad51, RPA1, NBS1, ATM, ATR, Chk1, Chk2,
and certain Fanconi anemia gene products) all result in sensitization to PARP
inhibition (McCabe et al., 2006). In addition, human and mouse cells
genetically deficient for ATM are also sensitized to PARP inhibition (Loser
et al., 2010; Williamson et al., 2010). As already mentioned, inherited
mutations in several of these genes result in cancer predisposition; however,
there is little evidence that such genes are frequently mutated in sporadic
cancers.
The PTEN tumor suppressor, by contrast, is one of the most frequently
mutated or inactivated genes in human cancer and leads to upregulation of
the PI3-kinase-PKB/ Akt growth and survival signaling pathway (Chalhoub
and Baker, 2009). Loss of PTEN has been known for some time to compromise genome stability (Puc et al., 2005); however, recent data have revealed
unexpected connections with HR and DNA repair. Remarkably, human
HCT116 colon carcinoma cells genetically deleted for PTEN are both deficient for HR and sensitized to PARP inhibitors (Mendes-Pereira et al., 2009).
Similar effects have been documented in PTEN-deficient murine astrocytes
and human glioblastoma cell line (McEllin et al., 2010). The mechanism
through which PTEN affects HR is not yet fully understood; however, two
recent studies have shown that Akt, which is upregulated as a result of PTEN
loss, can inhibit both strand resection and recruitment of essential HR
factors such as BRCA1 and CtIP at radiation-induced DSBs (Tonic et al.,
DNA Damage and Cancer
95
2010; Xu et al., 2010). The prospect that frequently activated oncogenic
signaling pathways conventionally linked to cell proliferation and survival
may interact with DNA damage response and repair processes suggest that
impaired HRR may be widespread in sporadic cancer and that PARP inhibitors could have more generic application than currently thought.
VII. DNA DAMAGE SIGNALING AS A BARRIER TO
TUMORIGENESIS
Predisposition syndromes demonstrate that genomic instability can promote cancer; however, in recent years an alternative paradigm has emerged
in which DNA damage and the resulting downstream signaling processes act
as a tumor suppression mechanism (Halazonetis et al., 2008). This concept
originated with the observation that premalignant or early stage lesions in
several cancer types, including bladder, breast, lung, and colon, frequently
showed evidence of DNA damage as judged by the presence of multiple
surrogate or direct markers of damage signaling such as -H2AX, and
phosphorylated, active forms of ATM, Chk2, and p53 (Bartkova et al.,
2005). These markers were not present in proliferating normal tissue, and
strikingly, their prevalence diminished again in the more malignant, later
stages of disease (Bartkova et al., 2005). Crucially, the signs of DNA damage
in early stage lesions preceded the appearance of overt genomic instability or
of p53 mutations, both of which are frequent in fully malignant tumors
(Bartkova et al., 2005). Based on these observations it was proposed that
spontaneous DNA damage occurs early in the evolution of cancer and that
the resulting DNA damage signaling processes direct incipient cancer cells to
terminal, nonproductive fates such as senescence or apoptosis. It was furthermore postulated that this might provide a selective pressure for specific
secondary genetic alterations, such as p53 mutation, that would allow cells
to 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 drive
the proliferation of malignant tumor cells, expression of the same oncogenes
in naı̈ve, genetically normal cells in culture often results not in cell transformation but instead in growth inhibition through cell senescence or apoptosis
(Braig and Schmitt, 2006) . Oncogene-induced senescence (OIS) in cultured
cells is associated with physical DNA damage and a robust DNA damage
response (Di Micco et al., 2006), providing a tractable model to test the role
of downstream signaling processes in imposing this phenotype. Remarkably,
depletion or inhibition of several key DNA damage signaling components,
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Joanne Smith et al.
including ATM, Chk1, Chk2, and p53, relieved OIS to allow more vigorous
cell proliferation and efficient transformation of oncogene-expressing cells
(Bartkova et al., 2006; Di Micco et al., 2006). Although cell culture experiments obviously do not accurately recapitulate all features of tumorigenesis
in vivo, it is telling that markers of cell senescence and DNA damage
signaling are both present in early stage or premalignant lesions and both
are 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 or
epigenetic changes which enable tumor cells to survive and proliferate in
the face of such damage? With respect to the first question, telomere erosion,
reactive oxygen species, or acquired mutations in genes required for genome
stability could all plausibly generate DNA damage during tumorigenesis or
transformation; however, it has been argued that none of these occur sufficiently frequently to account for OIS either in vitro or in vivo (Negrini et al.,
2010). Instead, current evidence favors the view that dominant, growthpromoting oncogenes, such as Ras or EGFR, which are frequently and
recurrently mutated in sporadic cancers, disturb the DNA replication process directly in such a way as to generate DNA damage (Halazonetis et al.,
2008). Exactly how oncogene-induced replication stress occurs remains
unclear; however, Cdks are potential culprits. Ordered cycles of Cdk activation and deactivation are essential for the proper organization and activation of the replication program through the replication origin licensing
system (Diffley, 2004). Because Cdk activity is key to uncontrolled proliferation, it is deregulated by oncogene activation and tumor suppressor loss by
many different mechanisms. Amplification of Cdk activity as a result of
oncogenic signaling therefore may erode these ordered cycles, leading to
aberrant over- or under-replication and thus to DNA damage (Blow and
Gillespie, 2008). According to this view, uncontrolled proliferation inevitably leads to spontaneous DNA damage and genomic instability (Halazonetis
et al., 2008).
With respect to mechanisms that may allow escape from oncogene-induced DNA damage, recent high-throughput sequencing studies have generally failed to find evidence for frequent mutations affecting DNA damage
response genes, such as ATM–Chk2 and ATR–Chk1, in sporadic cancers,
although clearly this does not rule out functional inactivation by epigenetic
or other means (Negrini et al., 2010). By contrast, missense mutations that
inactivate the p53 tumor suppressor protein are frequent in sporadic cancer
and some genetic evidence is consistent with the idea that these could be
selected to permit escape from DNA damage-induced OIS or apoptosis
(Negrini et al., 2010). One possible explanation for their prevalence is that
mutant p53 proteins can act dominantly, forming complexes with and
inhibiting normal p53 or the related p63 and p73 proteins (Brosh and
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97
Rotter, 2009). In contrast, biallelic mutations would presumably be required
to inactivate the function of most other DNA damage response genes. At all
events, frequent loss of p53 means that many tumor cells lack an effective
G1 checkpoint. As discussed below, this defect may render such tumors
inherently vulnerable to therapeutic strategies based on checkpoint
suppression.
Although inactivation of p53 could plausibly mitigate many of the negative effects of oncogene-induced DNA damage, it does not offer an obvious
explanation for why DNA damage signaling per se should be lost in the later
stages of tumorigenesis (Bartkova et al., 2005). Also, since malignant tumors
commonly exhibit high levels of genomic instability (Negrini et al., 2010), it
seems unlikely that the ongoing generation of oncogene-induced DNA
damage ceases as tumors progress, but more likely that its presence is simply
no longer detected. Why should this be? As previously mentioned, several
recent studies have revealed that upregulation of the PI3K-Akt pathway,
which is common in cancer as a result of PTEN inactivation and other
mechanisms (Chalhoub and Baker, 2009), inhibits both HRR and checkpoint activation by suppressing DNA damage processing (Tonic et al., 2010;
Xu et al., 2010). One prediction of these findings is that tumor cells with
high PI3K-Akt pathway activity will show a muted response to endogenous
DNA damage, potentially providing another mechanism through which
oncogene-induced DNA damage could be tolerated. However, a second
prediction is that this would inevitably be associated with genomic instability and an increased mutation rate, since ongoing oncogene-induced DNA
damage would continue undetected but unabated in cells with high PI3KAkt activity. Finally, it seems possible that tumors which have evolved
through such a route might show an altered response to conventional
genotoxic therapies, since presumably the recognition and signaling of
therapeutic DNA damage would also be suppressed.
VIII. CHECKPOINT SUPPRESSION AS A
THERAPEUTIC PRINCIPLE
Radiation and genotoxic chemotherapies remain the mainstays of cancer
treatment. Although new, molecularly targeted, drugs like imatinib have
revolutionized treatment of rare cancers such as chronic myeloid leukemia
(Agrawal et al., 2010), there seems little prospect that conventional therapies will be replaced in the treatment of other, more common malignancies
in the foreseeable future. Such treatments are, however, of limited efficacy
and toxic not only to tumor cells but also to normal tissues, leading to severe
side-effects. The realization that radiation and essentially all genotoxic
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Joanne Smith et al.
anticancer agents potently activate the ATM–Chk2 and ATR–Chk1 signaling pathways has stimulated much interest in whether these could be
manipulated pharmacologically to enhance the efficacy of conventional
therapies.
Several considerations argue that this may be the case. Firstly, defects in
DNA damage responses, whether inherent or experimentally imposed, can
result in sensitization to genotoxic stress. Examples already mentioned
include the acute radiosensitivity of ATM-deficient cells, the inherent vulnerability of BRCA1- and BRCA2-deficient cells to cross-linking agents, and
the synthetic lethality that results when cells with HRR deficiency are subject
to PARP inhibition. Secondly, enhanced DNA damage signaling has been
linked to radio- and chemo-resistance in leukemia and gliomas (Bao et al.,
2006; Nieborowska-Skorska et al., 2006), raising the possibility that this
might be reversed. Finally, it has been argued that the frequent functional
inactivation of p53 in human cancer creates a generic and exploitable
distinction between tumor and normal cells in terms of checkpoint proficiency. In essence this hypothesis holds that loss of p53-mediated G1 arrest
under conditions of genotoxic therapy will render tumor cells more dependent on checkpoints activated in S and G2 phases than their genetically
normal counterparts. Obviously this strategy is predicated on the
assumption that in tumor cells the primary function of these residual
p53-independent checkpoints is protective; that is, that their inhibition
will either escalate damage, promote the formation of more lethal lesions,
or trigger some mechanism of cell death that would not otherwise occur in
the 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 the
selective chemical inhibitor, UCN-01, has been shown to potentiate cell
killing by a wide range of genotoxic agents, including ionizing radiation,
alkylating agents, nucleoside analogs, cisplatin, and topoisomerase inhibitors (Carrassa et al., 2004; Cho et al., 2005; Ganzinelli et al., 2008; Hirose
et 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 inhibition
resulted in a greater degree of sensitization in tumor cells that were deficient
for p53 than in their proficient counterparts, consistent with the idea that
loss of G1 arrest indeed creates a therapeutic index. Sensitization has also
been observed as a consequence of inhibiting ATM, ATR, and other downstream components involved checkpoint regulation such as the Cdk1-regulating kinases, Wee1 and Myt1 (Karnitz et al., 2005; Mukhopadhyay et al.,
2005). In comparison, where tested, inhibition of Chk2 has in general been
found to have little or no effect on cell survival under conditions of damage,
emphasizing the fundamental functional distinction between Chk1 and
Chk2 (Carrassa et al., 2004; Karnitz et al., 2005; Pan et al., 2009).
DNA Damage and Cancer
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Although these studies support the general principle that checkpoints are
protective and help tumor cells to survive exposure to genotoxic stress,
exactly how checkpoint suppression leads to cell death remains poorly
understood. Evidence for amplified levels of damage, mitotic catastrophe
with damaged or incompletely replicated DNA, and increased levels of
apoptosis have all been reported (Carrassa et al., 2004; Cho et al., 2005;
Ganzinelli et al., 2008; Hirose et al., 2001; Karnitz et al., 2005; Koniaras
et al., 2001; Yu et al., 2002). This is likely to be a complex issue; however,
since genotoxic agents with distinct mechanisms of action activate different
combinations of DNA damage and replication checkpoint responses, while
the effects of some genotoxins may also depend on cell cycle phase and thus
vary among individual cells in a population. The cellular consequences of
suppressing checkpoint responses and the mechanisms of induced cell death
that contribute to sensitization are therefore likely to be equally variable and
complex.
Some of this complexity is illustrated by studies in Chk1 knockout DT40
lymphoma cells examining the relationship between checkpoint deficiency
and cell survival under different conditions of genotoxic stress. Compared to
wild-type, Chk1-deficient DT40 cells are markedly sensitive to ionizing
radiation, the DNA polymerase inhibitor aphidicolin, and the nucleoside
analog 5-fluorouracil (5-FU); however, the mechanism of cell death arising
from checkpoint deficiency is different in each case. Thus, abrogation of the
Chk1-dependent G2 checkpoint leads to division with lethal damage after
irradiation, presumably because G2 arrest would normally provide a vital
opportunity to repair such damage prior to mitosis (Zachos et al., 2003).
By contrast, when DNA polymerase is blocked with aphidicolin, deficient
fork stabilization/origin suppression and S-M checkpoints result in massive
replication fork collapse and lethal premature entry to mitosis with unreplicated DNA (Zachos et al., 2003, 2005). 5-FU also inhibits DNA replication
through its inhibitory action on thymidylate synthase (TS) leading to nucleotide pool depletion (Longley et al., 2003); however, sensitization in this
case proved to stem from uncontrolled replication in the presence of drug
due to loss of Chk1-mediated slowing of DNA synthesis, rather than fork
collapse followed by premature entry to mitosis (Robinson et al., 2006).
Failure to slow replication resulted in enhanced incorporation of 5-FU into
cellular genomic DNA and a large increase in DSBs compared to Chk1
proficient cells (Robinson et al., 2006). Thus, the consequences of replication checkpoint suppression vary according to the nature of the genotoxic
agent 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 develop
drugs targeting the ATM–Chk2 and ATR–Chk1 pathways, some of which
have already entered clinical trials. Thus far these efforts have concentrated
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Joanne Smith et al.
mainly on Chk1, presumably in recognition of its role as the ultimate
effector of both DNA damage and replication checkpoints and the fact
that it is thought to be expressed and active in virtually all tumors (Dai
and Grant, 2010). Drugs specifically targeting ATM and Chk2 are, however,
also under development, although at an earlier stage, and also other checkpoint-regulating kinases such as Wee1 and Myt1 (Antoni et al., 2007;
Ashwell et al., 2008; Dai and Grant, 2010). Thus far no selective inhibitors
of ATR have been reported.
UCN-01 was the first Chk1 inhibitor approved for clinical trials; however,
undesirable pharmacological characteristics combined with lack of selectivity limited its utility and led to the development of several new and more
selective agents (Ashwell et al., 2008; Dai and Grant, 2010; Fuse et al.,
2005). Several of these have reached clinical trials while others remain under
preclinical development. Current Chk1 inhibitors in Phase I clinical trials
include AZD7762 (AstraZeneca), PF-00477736 (Pfizer), XL844 (Exelixis),
and SCH 900766 (Schering-Plough). It is important to note that although
each of these drugs was developed to inhibit Chk1, several also have significant, and in one case even greater, activity against Chk2. Whether the
potential for dual inhibition of both Chk1 and Chk2 is relevant to the
biological effects of these agents as reviewed below is currently unclear.
Each of these agents has shown promising preclinical activities that recapitulate many of the effects of experimental Chk1 inhibition on DNA damage and
replication checkpoint responses described above (Ashwell et al., 2008; Dai
and Grant, 2010). In addition to monitoring effects on cell survival, several of
these studies have sought evidence of drug efficacy in terms of Chk1 inhibition
and to gain insight into potential mechanisms of synergistic cell killing. Biochemical readouts of Chk1 activity include turnover of Cdc25A phosphatase,
phosphorylation of Cdc25 C on serine 216 (S216), and increased levels of
inhibitory T14/Y15 phosphorylation of Cdk1. To document the biological
consequences of Chk1 inhibition, nuclear foci of -H2AX and Rad51 provide
a means of quantifying DNA damage and proficiency for HRR respectively,
while premature entry to mitosis can be detected by flow cytometry when cells
with incompletely replicated DNA become positive for histone H3 phosphorylated 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 irinotecan
for solid malignancies (Morgan et al., 2010; Zabludoff et al., 2008).
In preclinical studies AZD7762 has been shown to synergize with ionizing
radiation, irinotecan, and gemcitabine in a variety of tumor cell lines and
xenografts with evidence of greater potency in p53-deficient cells (Morgan
et al., 2010; Zabludoff et al., 2008). In each case AZD7762 treatment
resulted in stabilization of Cdc25A, decreased levels of T14/Y15 phosphorylated Cdk1, and an increase in mitotic entry compared to DNA damaging
DNA Damage and Cancer
101
agent alone, indicating efficient Chk1 inhibition and checkpoint override
(Morgan et al., 2010; Zabludoff et al., 2008). Enhanced cell killing by
AZD7762 in the context of ionizing radiation was also associated with
increased and persistent -H2AX staining and a marked suppression of
Rad51 focus formation, indicating that drug treatment resulted in higher
levels 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 inhibits DNA replication via inhibition of both ribonucleotide reductase and
DNA polymerase, stalling replication forks and arresting cells in S-phase
(Ewald et al., 2008). A more detailed analysis of gemcitabine chemosensitization revealed that AZD7762 treatment caused collapse of stalled replication forks, ectopic replication origin firing, and ultimately premature entry
to mitosis with unreplicated DNA and apoptosis (McNeely et al., 2010).
Interestingly, siRNA depletion of Cdk1 mitigated cell death under conditions of AZD7762 and gemcitabine treatment, suggesting that premature
entry to mitosis as a result of S-M checkpoint failure was a direct cause of
cell death (McNeely et al., 2010). Replication fork collapse also resulted in
high levels of DSBs and ATM activation, and consistent with this, AZD7762
enhanced gemcitabine cytotoxicity particularly effectively in cells with DSB
repair 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 in
combination trials with gemcitabine for the treatment of advanced solid
tumors (Ashwell et al., 2008; Dai and Grant, 2010). In preclinical studies
PF-00477736 has been shown to abrogate both the G2 and intra-S-phase
checkpoints in cells treated with camptothecin and gemcitabine respectively,
and to enhance the cytotoxicity of gemcitabine and carboplatin in cell and
xenograft assays (Blasina et al., 2008). Unexpectedly, PF-00477736 also
synergizes strongly with docetaxel in cells and xenografts, releasing cells
from mitotic arrest and enhancing apoptosis (Zhang et al., 2009). The
mechanism of this synergy is not yet fully understood. High concentrations
of docetaxel can induce DNA damage which could be a factor; however,
other findings have shown that Chk1 is required for spindle checkpoint
function and mitotic progression, raising the possibility that Chk1 inhibitors
might enhance the effects of antimitotic drugs more generally (Carrassa
et 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 the
treatment of advanced solid tumors and lymphoma (Ashwell et al., 2008;
Dai and Grant, 2010). In PANC-1 cells treated with gemcitabine XL-844
has been shown to override the S-phase checkpoint leading to increased
levels of Chk1 phosphorylation (S317) and -H2AX, indicative of increased
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Joanne Smith et al.
levels of damage, followed by subsequent premature entry into mitosis and
decreased clonogenic survival (Matthews et al., 2007). Similarly, SCH900766 is a selective Chk1 inhibitor that is in combination trials with
gemcitabine for the treatment of solid tumor and lymphoma and cytarabine
for the treatment of acute leukemia. SCH-900766 has been shown to abrogate both the intra-S and G2 checkpoints resulting in sensitization of tumor
cells to IR and alkylating agents, although full details of these effects are not
yet in the public domain (Dai and Grant, 2010).
Taken together, these studies validate Chk1 as a target whose pharmacological inhibition can potentiate tumor cell killing by a wide range of
genotoxic agents in vitro. As depicted in Fig. 4, much remains to be learned
about the detailed mechanisms involved in chemosensitization, however,
Chk1 inhibition can clearly both amplify the extent of damage inflicted by
a given agent and promote the formation of more lethal lesions, for example
by triggering stalled replication fork collapse to form DSBs. In addition,
evidence suggests that damage escalation as a result of Chk1 inhibition can
enhance tumor cell killing both by conventional routes, for example by
increasing apoptosis, but also by triggering novel mechanisms such as premature entry to mitosis with unreplicated DNA. Clearly, the challenge now
will be to determine whether these preclinical principles established in the
laboratory using novel Chk1 inhibitor drugs will have utility in the clinic.
IX. FUTURE PERSPECTIVES
Genomic instability has long been recognized as a cardinal feature, and
arguably an important cause of, cancer, however, in recent years it has also
emerged as a potential Achilles heel that offers new therapeutic opportunities.
Thus far this prospect has been most evident in cancers that arise in predisposed individuals, for example in BRCA1 and BRCA2 mutation carriers,
where impairment of one particular form of DNA repair specifically in
tumor cells creates sensitivity to both existing and novel treatments.
Although similar loss-of-function mutations do not appear to be common in
sporadic cancers, genomic instability is, perhaps because DNA damage response genes are inactivated epigenetically, or the proteins they encode are
inhibited by oncogenic signaling processes. This raises the possibility that
individual sporadic cancers might also be selectively targeted if the functional
basis and consequences of genomic instability in individual tumors could be
understood. Alternatively, it may be possible to improve the efficacy, or mitigate the undesirable side-effects, of existing genotoxic therapies by developing
drugs that inhibit DNA damage responses controlled by the ATM–Chk2 and
ATR–Chk1 pathways. Evidence suggests that inhibition of the Chk1 protein
103
DNA Damage and Cancer
IR/topoisomerase inhibitors
(DSBs)
ATM
Fork collapse
leading to DSBs
DNA stand
resection
ATR
Gemcitabine, 5-FU,
Platinum compounds
(stalled replication forks)
Chk1
Damage escalation,
Suppression of HRR,
Division with damage,
Premature mitosis,
Increased apoptosis
G2 arrest,
Intra-S checkpoint,
S-M checkpoint,
Fork stabilization,
Origin suppression,
Chk1 inhibition
Fig. 4 Chk1 inhibition potentiates cell killing by genotoxic agents by multiple mechansims
Activation 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 ionizing
radiation (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 the
text for further details and explanation.
kinase in particular may have therapeutic potential, particularly in tumors that
have suffered loss of p53 function during their evolution. Such efforts are,
however, in their infancy, and as understanding of the biological and molecular
functions of these pathways deepens, additional rational therapeutic strategies
based 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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Joanne Smith et al.
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