Cancer

Review Research

Exploiting DNA Replication Stress for Cancer

Treatment
Tajinder Ubhi1,2 and Grant W. Brown1,2

Abstract
Complete and accurate DNA replication is fundamental to
cellular proliferation and genome stability. Obstacles that
delay, prevent, or terminate DNA replication cause the phe-
nomena termed DNA replication stress. Cancer cells exhibit
chronic replication stress due to the loss of proteins that
protect or repair stressed replication forks and due to the
associated with such therapies. We discuss how replication
stress modulates the cell-intrinsic innate immune response
and highlight the integration of replication stress with immu-
notherapies. Together, exploiting replication stress for cancer
treatment seems to be a promising strategy as it provides a
selective means of eliminating tumors, and with continuous

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continuous proliferative signaling, providing an exploitable advances in our knowledge of the replication stress response
therapeutic vulnerability in tumors. Here, we outline current and lessons learned from current therapies in use, we are
and pending therapeutic approaches leveraging tumor-specific moving toward honing the potential of targeting replication
replication stress as a target, in addition to the challenges stress in the clinic.

Introduction mental. In this review, we provide a summary of the therapies

centered on enhancing both endogenous and drug-induced rep-
The DNA replication machinery successfully carries out accu-
lication stress and discuss the rationales associated with them. We
rate genome duplication in the face of numerous obstacles, many
also highlight the potential of using replication stress to stimulate
of which cause DNA replication stress. Replication stress, defined
the cell-intrinsic innate immune response to improve immuno-
as any hindrance to DNA replication that either stalls, blocks, or
therapy effectiveness.
terminates DNA polymerization, activates a highly conserved
replication stress response pathway mediated by a network of
repair proteins to resolve the damage. Efficient removal of obsta- DNA Replication Stress: Causes,
cles to replication progression safeguards genome integrity by Consequences, and the Cellular Response
ensuring timely completion of DNA replication and by limiting
Eukaryotic DNA replication is a tightly regulated and complex
susceptibility to mutagenesis. However, failure to remove repli-
process that requires the orchestrated functions of several hun-
cation stressors due to the loss of replication stress response and
dred proteins during the S phase of each cell cycle (reviewed in
repair proteins and sustained proliferative signaling leads to
refs. 3, 4). Each time a cell divides, billions of nucleotides of DNA
chronic replication stress, and is a prominent feature of tumor
must be accurately polymerized, and ensuring this process occurs
cells. In particular, whole-genome sequencing efforts of tumor
prior to the next cell cycle is essential for cellular homeostasis.
samples have indicated the important role of replication stress
However, there are numerous hindrances to DNA replication that
in tumor progression and maintenance, complementing early
can prevent progression of the replication machinery and cause
findings from the groups of Halazonetis and Bartek who
replication forks to stall. Obstacles to replication fork progression
demonstrated that replication stress accumulates upon cellular
can arise from several sources, either endogenous or exogenous,
transformation, and is rarely observed in even the most prolifer-
ranging from the depletion of nucleotide pools available for
ative tissues (1, 2). Thus, due to the selective nature of replication
DNA synthesis to transcription–replication machinery collisions,
stress as a therapeutic target, the dependency of tumor cells on
RNA–DNA hybrids, oncogene-induced increases in replication
the replication stress response for cell survival, and the ever-
origin firing, and inherently hard-to-replicate regions (Fig. 1A;
expanding network of replication stress response proteins that
reviewed in ref. 5). The type of stress challenging replication
can be targeted, replication stress is beginning to be explored as an
fork progression dictates the cellular response and network of
attractive target in the clinic. In contrast to normal cells, cancer
repair proteins activated in response to the replication stress.
cells can be pushed toward cell death by introducing further DNA
For example, ribonucleotides that are misincorporated into
damage in a catastrophic manner or by promoting their entrance
replicating DNA can cause replication stress, and are recognized
into cell cycle stages where unresolved replication stress is detri-
and removed by ribonucleotide excision repair, whereas inter-
1
Department of Biochemistry, University of Toronto, 1 King's College Circle, strand crosslinks are mainly repaired by the Fanconi anemia
Toronto, Ontario, Canada. 2Donnelly Centre for Cellular and Biomolecular pathway, a specialized branch of the DNA damage response.
Research, University of Toronto, Toronto, Ontario, Canada.
Stalled replication forks are often transient, with cells able to
Corresponding Author: Grant W. Brown, University of Toronto, 160 College resolve them and continue replication. However, when left unre-
Street, Toronto, ON M5S 3E1, Canada. Phone: 416-946-5733; Fax: 416-978-8548; solved, replication stress can lead to small-scale and large-scale
E-mail: grant.brown@utoronto.ca
genome alterations such as the mutations and chromosomal
doi: 10.1158/0008-5472.CAN-18-3631 rearrangements found frequently in human cancers and devel-

2019 American Association for Cancer Research. opmental disorders.

1730 Cancer Res; 79(8) April 15, 2019


Replication stress triggers the activation of a signaling cascade
that promotes resolution of the stress and resumption of repli-
cation fork progression, collectively known as the S phase check-
point (Fig. 1B). Stalled replication forks are characterized by
stretches of single-stranded DNA (ssDNA), larger than those
found during lagging strand DNA synthesis, that activate the
S phase checkpoint. It is thought that the ssDNA gaps are caused
mainly by polymerase and helicase uncoupling (6), but ssDNA
can also be generated by nucleolytic processing of DNA at reversed
replication fork structures (7). The ssDNA is bound with high
affinity by replication protein A (RPA), which serves as a platform
for the recruitment of numerous sensor proteins, including
ataxia telangiectasia and Rad3-related (ATR)-interacting protein
(ATRIP), the 9-1-1 DNA clamp complex (RAD9-RAD1-HUS1),
topoisomerase II binding protein 1 (TOPBP1), and Ewing tumor-
associated antigen 1 (ETAA1), which recruit and activate the
central replication stress response kinase ATR (reviewed in 8).
Upon activation, ATR orchestrates a multifaceted response at
stalled replication forks by phosphorylating several downstream
targets, including its main effector kinase, checkpoint kinase 1
(CHK1). The ATR–CHK1 signaling pathway leads to cell cycle
arrest, stabilization of stalled replication forks, and the inhibition
of late origin firing to prevent excess ssDNA formation (9). In
doing so, the S phase checkpoint ultimately promotes replication
fork repair and restart, or resumption of replication from an
adjacent origin (10, 11), to ensure complete replication of the
affected genomic loci.
In instances where cells endure chronic replication stress, such
as in the early stages of tumorigenesis, or during extended periods
of antineoplastic drug treatment, replication forks can col-
lapse (12–14). It is thought that ssDNA stretches found at stressed
replication forks are converted to DNA double-strand breaks
during replication fork collapse and that the replication fork loses
its capacity to synthesize DNA. Although collapsed replication
forks can likely be rescued through homologous recombina-
tion (15) or mitotic DNA synthesis (16), inability to resume
DNA replication in these regions promotes mutagenesis and
genome instability. Cells recruit error-prone translesion DNA
polymerases that inaccurately synthesize DNA as a means to
complete bulk genome replication, failure of which leads to
incompletely replicated DNA persisting into mitosis. The presence
of under-replicated genomic loci in mitosis can result in chro-
mosomal entanglements between sister chromatids (17) or lag-
ging chromosomes that fail to segregate with the remaining
genetic material, leading to the formation of micronuclei (18).
These events not only increase the risk of genome instability
and promote transformation, but they also perpetuate the
existing genome instability through global missegregation of
chromosomes, thus providing a route of evolution for cancer
cells (19). Unresolved replication stress following mitosis leads
to the formation of nuclear bodies marked by the DNA damage
response protein p53 binding protein 1 (53BP1) in the subse-
quent G1 phase of the cell cycle in daughter cells, which may act
to protect or promote resolution of the replication stress (20).
Exploiting DNA Replication Stress–Centered
Vulnerabilities in Cancer
As it is well documented that tumor cells have persistent
replication stress (21), and with replication stress now acknowl-
edged as an enabling characteristic of cancer (22), it is not
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Exploiting Replication Stress
surprising that replication stress is beginning to be rationally
leveraged in cancer therapies. The elevated replication stress in
cancer cells has been largely attributed to the loss of cell cycle
checkpoint activators, often tumor suppressor genes, and to the
overexpression or constitutive expression of oncogenes (21, 23).
These alterations modify replication fork rates, increase replica-
tion initiation or origin firing, and promote premature entry into
S phase (2, 13, 24). Strategies aiming to exploit replication stress
fall within two major categories; they either function to increase
the endogenous replication stress present within tumor cells to
induce cell death or they produce tumor cell-specific replication
stress that can be further enhanced by additional therapeutics.
Traditional replication stress–inducing drugs largely resulted
from serendipitous observations of tumor cell death following
use of the agents (25), but increasingly drugs are designed with the
specific goal of exploiting replication stress (Table 1). The increase
in accessibility of deep sequencing approaches has provided
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remarkable insight into the catalogue of DNA repair genes mutat-
ed in cancers and offers another exciting means of targeting
endogenous replication stress by identifying mutation-specific
vulnerabilities (26).
Current Therapies That Harness
Replication Stress
Because a key feature that distinguishes tumor cells from
most normal cells is their sustained proliferation, DNA repli-
cation has been harnessed as a semi-selective target in cancer
therapies for decades (25). Many traditional anticancer drugs
act by increasing the replication stress within tumor cells, by
directly damaging the DNA, depleting cellular resources
required for successful cell division, or through a combination
of both. Nucleoside analogues, including 5-fluorouracil, cytar-
abine, and gemcitabine, were among the first chemotherapeutic
agents introduced to the clinic and act through diverse mechan-
isms to ultimately halt DNA replication. For instance, cytar-
abine and gemcitabine are deoxycytidine analogues that com-
pete directly with dCTP for incorporation into newly synthe-
sized DNA, leading to a delay in replication fork progression or
even a complete termination of some replication forks (27, 28).
In addition to their abilities to incorporate into DNA, these
agents also reduce the size of the intracellular nucleotide pools
available for DNA synthesis or limit the availability of a single
nucleotide. For example, gemcitabine potentiates its cytotoxic
effects by also inhibiting ribonucleotide reductase (29), where-
as 5-fluorouracil functions mainly by inhibiting thymidylate
synthetase, thereby reducing the amount of thymidine avail-
able for DNA replication and repair (30).
In contrast to nucleoside analogues, alkylating agents and
platinum-based compounds function by directly modifying DNA
through the formation of alkylation adducts on DNA bases, intra-
or interstrand crosslinks between DNA bases, or both (31, 32).
The generation of DNA adducts by alkylating agents such as
dacarbazine and temozolomide poses an obstacle for the
advancing DNA polymerase, leading to delayed replication fork
progression. Moreover, intra- and interstrand crosslinks generated
by platinum-based agents such as cisplatin and carboplatin are
physical barriers to DNA replication and block the replication
machinery. Intrastrand crosslinks present on the template DNA
strand during replication impair proper base pairing with the
nascent DNA strand, whereas interstrand crosslinks impede
Cancer Res; 79(8) April 15, 2019 1731
 Ubhi and Brown

A RNA processing enzymes,

Regulation of RNA-DNA helicases,
replication origin and topoisomerases
firing
Oncogene-induced
replication stress
RNA-DNA hybrids
Nucleotide pool MYC and replication- DNA sliding clamp
depletion transcription conflicts DNA polymerase
C DNA helicase
A H3N Cl
T G
Pt
H3N Cl

Inter-strand
GCGG crosslinks

OH Hard-to-replicate
regions Fanconi Anemia
Ribonucleotide pathway
incorporation

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DNA helicases
Ribonucleotide
excision repair

9-1-1 DNA clamp complex

RPA DNA lesion

ATRIP ATRIP
TOPBP1 ATR ATR ETAA1
P

CHK1

Stabilization of Late origin firing

stalled replication Cell cycle
forks progression
C
AZD1775 WEE1 G1: Gap phase I
S: Synthesis phase
G2: Gap phase II
M: Cell division phase
G2/M checkpoint

M
G1
G2
S
p53

Prexasertib,
GDC-0575,
SCH 900776, G1/S checkpoint
and SRA737

S phase
CHK1 ATR checkpoint

M6620, AZD6738,
and BAY1895344
© 2019 American Association for Cancer Research

1732 Cancer Res; 79(8) April 15, 2019 Cancer Research

 Exploiting Replication Stress

the very first step of DNA replication, that is, unwinding of the
double-strand helix (33). Another class of replication stress–
inducing agents that directly damage DNA is topoisomerase
inhibitors (34, 35). Topoisomerases generate transient DNA
breaks to relax DNA supercoiling that occurs during DNA
replication and transcription. The intermediate state where the
topoisomerases are bound to the cleaved DNA is trapped by
topoisomerase inhibitors and leads to the formation of stable
protein–DNA complexes that generate a physical barrier to ongo-
ing replication forks, often collapsing to double-strand breaks
when encountered by a replication fork. The action of topoisom-
erase inhibitors resembles that of another group of replication
stress–inducing therapeutics, PARP inhibitors. PARP inhibitors
generate an obstacle for the replication machinery by trapping
PARP on DNA (36), in addition to impairing single-strand break
repair (37) and accelerating replication fork progression (38),
amplifying the replication stress in tumor cells.
target cell cycle checkpoints (Fig. 1C) to ultimately promote the
premature entry of tumor cells into mitosis, where they undergo
cell death from abnormal mitosis, termed mitotic catastro-
phe (53). Most importantly, these approaches promise an
improved and practical therapeutic index relative to conventional
agents due to the intrinsically high levels of replication stress often
found within tumor cells.
Activation of the S phase checkpoint elicits a multifaceted
signaling response, where each pathway can provide additional
targets for therapeutic intervention (Fig. 1). The S phase check-
point response kinases ATR and CHK1 have been the leading
targets of the therapies currently under investigation (Table 1).
ATR delays the activation of dormant replication origins and
stabilizes stressed replication forks to prevent replication fork
collapse. Accordingly, ATR inhibition initiates widespread DNA
synthesis from normally dormant replication origins, generat-
ing enough ssDNA to exhaust the cellular pools of RPA and

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The replication stress–inducing effects of conventional thera-
pies have been directly visualized at the single-molecule level
through DNA fiber analyses, where DNA synthesis rates of indi-
vidual DNA replication forks can be measured following drug
treatment (39–41), and through DNA pull-down techniques
that identify proteins bound directly at replication forks
in vivo (42–46). Of particular interest, the interactions between
traditional replication stress–inducing drugs and cellular replica-
tion stress response pathways are evident in preclinical studies,
where inhibition or depletion of the key replication stress
response kinases required for overcoming the replication stress
present within cells, ATR and/or CHK1, sensitizes tumor cells to
5-fluorouracil, gemcitabine, cisplatin, PARP inhibitors, and temo-
zolomide, with some combinations having advanced to clinical
trials (34, 35, 47–52).
Emerging Combination Therapies That
Target Replication Stress: Cell Cycle
Checkpoints
Although conventional replication stress–inducing agents have
been used for decades, their applicability and efficacy is limited by
their associated toxicities and the rapid emergence of resistant
tumor cells. As noted, these therapies target DNA replication, and
thus have an effect on highly proliferative cells, including normal
cells within the gut epithelium and bone marrow, leading to
undesirable and sometimes intolerable side effects. In light of this,
several promising rationally designed combination therapies that
increase the specificity of these traditional therapies toward tumor
cells are currently undergoing clinical trials. In addition to gen-
erating further replication stress, many of these therapies also
depleting the nucleotide pools necessary for DNA synthesis (9,
11). ATR and CHK1 inhibition also promotes premature entry
into mitosis despite the presence of under-replicated DNA,
leading to chromosome fragmentation (54, 55). Given their
mechanisms of action, ATR and CHK1 inhibitors synergize
with compounds that induce replication stress, and increase
the toxicity of nucleoside analogues in ovarian, lung, and pan-
creatic cancer (56–60), platinum-based agents in lung, gastric,
ovarian, bladder, and breast cancer (50, 61, 62) and topoisom-
erase inhibitors in breast, colon, and brain tumors (63, 64).
Because ATR is activated following replication stress to ensure
successful resolution of the replication stress, ATR inhibitors
should be sufficient as monotherapy in tumors that experience
high levels of intrinsic replication stress, which is also a current
arm of investigation (NCT03188965). Early CHK1 inhibitors
were not successful as monotherapies, largely due to their intol-
erable side effects and pharmacokinetic properties, although
clinical studies demonstrate potential for newer generation
CHK1 inhibitors, as they have a tolerable toxicity profile (65).
The high toxicity of CHK1 inhibitors is consistent with the central
role of CHK1 in mediating the S phase checkpoint upon replica-
tion stress, both in the absence and presence of ATR, and
in preventing premature entry into mitosis (55, 66), but presents
the possibility of targeting downstream effectors of CHK1
therapeutically.
As tumor cells are often deficient in the key G1/S transition
regulator p53, the G2/M checkpoint becomes essential to prevent
tumor cells with unreplicated or damaged DNA from entering
catastrophic mitoses. Promising therapies targeting regulators of
mitotic entry to undermine the G2/M checkpoint are also being
explored. Inhibitors of the G2/M checkpoint kinase WEE1 have

Figure 1.
Generation and exploitation of DNA replication stress. A, Obstacles in replication fork progression can arise from exogenous and endogenous sources and
dictate the cellular response and network of repair proteins activated in response to the replication stress. Sources of replication stress include, but are not
limited to, the depletion of nucleotide pools available for DNA replication and repair, oncogene-induced stress, RNA–DNA hybrids, replication–transcription
conflicts, DNA crosslinks, inherently difficult-to-replicate regions, and the misincorporation of ribonucleotides into replicating DNA. B, Replication stress
activates a highly conserved signaling response called the S phase checkpoint. The S phase checkpoint is activated upon exposure of ssDNA from stressed
replication forks, which leads to the recruitment and activation of the central replication stress response kinase ATR and its main effector kinase, CHK1. The ATR–
CHK1 signaling pathway promotes cell cycle arrest, stabilization of stalled replication forks, and inhibition of late origin firing to ultimately ensure complete
replication of the affected genomic loci. C, Cancer cells become dependent on the S phase checkpoint for cell survival due to their high levels of intrinsic DNA
replication stress. In addition to S phase checkpoint response proteins being the leading targets for cancer therapies targeting replication stress, regulators of
mitotic entry are also being evaluated, as they drive cells with unresolved replication stress into catastrophic mitoses. Targets in the DNA replication stress
response are shown for each cell cycle checkpoint, with inhibitors targeting those proteins currently being evaluated in clinical trials in bold.

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 Ubhi and Brown

Table 1. Chemotherapeutics that increase DNA replication stress

Class of agents or target Function Compounds Clinical stage References
Nucleoside analogues Inhibition of DNA replication Azacitidine All approved for use 30, 109
Cytarabine
Decitabine
Gemcitabine
5-Fluorouracil
Alkylating agents and Direct modification of DNA Carboplatin All approved for use 31, 32
platinum compounds Cisplatin
Cyclophosphamide
Dacarbazine
Methotrexate
Mitomycin C
Oxaliplatin
Procarbazine
Temozolomide
Topoisomerase I and II Relax DNA supercoiling that occurs during Belotecan Approved 34, 35
DNA replication and transcription CRLX101 Phase II
Doxorubicin Approved
Epirubicin Approved

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Etoposide Approved
Idarubicin Approved
Irinotecan Approved
LMP400 Phase I
LMP776 Phase I
Mitoxantrone Approved
NKTR-102 Phase III
Teniposide Approved
Topotecan Approved
PARP Single-strand DNA break repair Olaparib Approved 110
Niraparib Approved
Rucaparib Approved
Talazoparib Approved
Veliparib Phase III
ATR Central replication stress response kinase AZD6738 Phase I/II 52
BAY1895344 Phase I/II
M6620 Phase II
CHK1 Main effector kinase of ATR in replication GDC-0575 Phase I 111
stress response SCH 900776 Phase I
SRA737 Phase I
Prexasertib Phase I/II
WEE1 G2/M checkpoint kinase AZD1775 Phase I/II 112

shown promising results with an acceptable toxicity profile (67).
Drugs that increase replication stress should increase the impor-
tance of the G2/M checkpoint for tumor cell survival, and accord-
ingly there are currently greater than 30 clinical studies underway
for combination therapies with WEE1 inhibitors (clinicaltrials.
gov, accessed January 2019). These include combinations of
gemcitabine, temozolomide, cytarabine, irinotecan, carboplatin,
and cisplatin with the potent WEE1 inhibitor AZD1775 in
pancreatic, blood, brain, ovarian, and colon tumors, with an
emphasis on p53 deficiency as the key selection criteria. In
addition to driving tumor cells into unscheduled mitosis (68),
the cytotoxicity of AZD1775 could also be attributed to the
effects WEE1 has on stabilizing stalled replication forks and
ensuring timely origin firing, further exacerbating the replica-
tion stress in tumor cells when WEE1 is inhibited (69, 70).
WEE1 inhibitors act synergistically with ATR and CHK1 inhi-
bitors in preclinical studies (71, 72), extending the catalogue of
possible combinations with other targeted inhibitors that
could be implemented. Similar to ATR and CHK1 inhibitors,
although AZD1775 has shown single-agent activity in preclin-
ical studies, its efficacy is potentiated when integrated into
combination regimens.
Precision Medicine Approaches That
Leverage Replication Stress
The identification of the underlying genetic alterations across
all major subtypes of cancer has provided unprecedented insight
into DNA repair proteins that are altered in cancer (26) and has
laid the foundation for several precision medicine approaches.
Almost every tumor has at least one gene in the replication stress
response altered, providing ample opportunities to exploit tumor-
specific alterations without affecting normal cells. The concept of
synthetic lethality (73), whereby the presence of cancer-specific
mutations in specific genes renders other genes essential for cell
proliferation and survival, has been fundamental for the discovery
of several of these gene-targeted strategies (74). Perhaps the best
example of this strategy is the successful use of PARP inhibitors for
the treatment of BRCA1/BRCA2-deficient breast and ovarian
cancers, as these double-strand break repair defective tumors are
dependent on PARP activity for cell survival (37, 75). Consider-
able effort is now being expended to identify additional synthetic
lethal interactions with DNA repair genes, including replication
stress response genes. One of the most promising targets
currently being explored is ATR. Tumors harboring mutations in

1734 Cancer Res; 79(8) April 15, 2019 Cancer Research


genes encoding the chromatin remodeler ARID1A or the DNA
damage kinase ATM are synthetically sensitive to ATR inhibitors,
with a complete response observed in a patient with metastatic
ATM-deficient colorectal cancer (76–78). The dependency of
ARID1A-deficient tumors on ATR has been attributed to the
mitotic defects present in these cells, which causes an increased
reliance on ATR function. Both ARID1A and ATM are frequently
altered in tumors, particularly in ovarian and endometrial cancers
for ARID1A and lymphoid malignancies for ATM, illustrating the
applicability of these findings. In addition to further interactions
with ATR, tumors containing mutations in other frequently
altered replication stress response genes including RPA1
(NCT03207347 and NCT03377556) and the mutagenic DNA
polymerase POLQ (79) are currently being explored to identify
druggable synthetic lethal interactions. Genetic interactions
with replication stress–inducing agents including CHK1 (80,
81) and WEE1 (82) inhibitors are also being investigated for
use in mutation-specific tumor backgrounds, such as the use of
CHK1 inhibitors in EZH2-deficient T-cell acute lymphoblastic
leukemias.
Patient-tailored therapies targeting vulnerabilities induced by
specific oncogenes are also being implemented. In particular,
the genes encoding RAS, cyclin E, cyclin D, and MYC are frequently
amplified in many cancer types and are starting to be harnessed
as biomarkers of replication stress. These oncogenes stimulate
premature entry into S phase, thereby disrupting the spatial
and temporal regulation of origin firing and replication fork
speed, increase global transcription activity, and promote
RNA–DNA hybrid accumulation, ultimately causing replication
stress (reviewed in ref. 83). Accordingly, inhibition of ATR, CHK1,
Cytosol
Replication stress conditions
TREX1
RPA and
RAD51
SAMHD1
Nucleus
Nuclear envelope
Micronucleus
Figure 2.
Activation of the cell-intrinsic innate immune response by DNA replication stress. In replication stress conditions, nuclear DNA is released into the cytosol, either
directly from stalled replication forks in three prime repair exonuclease 1 (TREX1)-, sterile alpha motif and HD domain–containing protein 1 (SAMHD1)-, RPA- or
RAD51-deficient backgrounds, or from micronuclei, which are small nuclei formed predominantly following mitotic progression in cells experiencing genome
instability. The cytosolic DNA fragments are sensed by the pattern recognition receptors, cyclic GMP-AMP synthase (cGAS) and IFNg-inducible factor 16 (IFI16),
which activate the central adaptor protein STING, inducing a transcriptional response to ultimately promote cellular senescence or elimination by the adaptive
immune system.
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Exploiting Replication Stress
or WEE1 activity is lethal in the context of cyclin E-, MYC-, and/or
RAS-overexpressing tumor cells (84–87), presumably because
these tumors have a heightened requirement for the cell cycle
checkpoints that respond to replication stress. Overexpression
of the G1/S checkpoint regulator, cyclin D and its effectors
CDK4 and CDK6, are leveraged as biomarkers for patient selection
in trials with DNA damaging agents and immunotherapies
(NCT03237390, NCT02290145, and NCT03088059). More
recently, studies have also elucidated the role of apolipoprotein
B mRNA editing enzyme catalytic polypeptide–like (APOBEC)
proteins as an endogenous source of replication stress and have
found that overexpression of two APOBEC3 enzymes, APOBEC3A
and APOBEC3B, confers sensitivity to ATR inhibition (88, 89). As
APOBEC3A and APOBEC3B are overexpressed in several cancer
types (90, 91), this finding could have important implications in
maximizing the potential of ATR inhibitors while sparing normal
cells. The combination of the overexpression of the remaining five
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APOBEC3 paralogs and replication stress–inducing agents may,
therefore, also be worth testing for possible synergies.
The Innate Immune Response: An Emerging
Marker of the Replication Stress Response
An exciting emerging concept is the link between replication
stress and activation of the cell-intrinsic innate immune response
(Fig. 2). Accumulating evidence indicates that replication stress–
inducing agents such as topoisomerase inhibitors and cells defi-
cient in replication stress response genes induce the expression
of type I IFNs and proinflammatory cytokines (92–95). It is now
apparent that this activation is largely mediated through the
Type I inteferon production
cGAS and
IFI16 STING
Interferon-stimulated gene
and pro-inflammatory cytokine
production
STING Cellular senescence or
Cytosolic DNA activation elimination by adaptive
sensing immune system
© 2019 American Association for Cancer Research
Cancer Res; 79(8) April 15, 2019 1735
 Ubhi and Brown
presence of cytosolic DNA derived from the nucleus or mitochon-
dria, which engages the pattern recognition receptors that nor-
mally scan for viral infections. The mechanisms through which
replication stress induces DNA release into the cytoplasm appear
to be specific to the perturbation. Recent work has found micro-
nuclei to be a key source of immunostimulatory DNA following
mitotic progression in cells lacking RNase H2 (96, 97), whereas
the release of ssDNA directly from stalled replication forks has
also been demonstrated in TREX1-, SAMHD1-, RPA-, and RAD51-
deficient backgrounds and following cytarabine treatment
(93, 98–101). Whether these conditions could result in cyto-
plasmic DNA accumulation through both observed mechanisms
has yet to be ruled out, and several aspects of these mechanisms,
including the interplay of proteins at replication forks that pro-
mote ssDNA release, have yet to be defined.
Central to the innate immune response is the adaptor protein
"stimulator of IFN genes" (STING), which couples signals from
cytosolic DNA sensors to a transcriptional response from activa-
tion of both the NF-kB and type I IFN signaling axes, ultimately
promoting cellular senescence or elimination by the adaptive
immune system (reviewed in ref. 102). Interestingly, STING
signaling is suppressed in several tumors (103) and multiple
cancer cell types contain genome-derived cytosolic ssDNA (100),
further affirming the presence and importance of persistent rep-
lication stress in tumors. As type I IFN production from the innate
response is critical in priming the adaptive immune system,
robust STING signaling has been attributed to an increased
immunotherapy response. In fact, blocking T-cell inhibitory path-
ways using immune checkpoint inhibitors is ineffective in mice
lacking TMEM173 (STING; ref. 96). However, despite current
efforts to combine STING agonists with checkpoint inhibitors as
antitumor therapy, therapeutic delivery of the agonists has yet to
be improved for clinical settings. Combination approaches inte-
grating replication stress–inducing agents, such as carboplatin
or gemcitabine, with immunotherapies like the immune check-
point inhibitor nivolumab, have advanced to clinical trials
(NCT02944396, NCT03662074, NCT03061188, NCT02734004,
NCT02849496, NCT02657889, and NCT02571725). It is tempt-
ing to speculate that the efficacy observed could be due to the
engagement of STING by replication stress, and would support
the use of replication stress as a predictive marker for immu-
notherapy efficacy. Further work is required to better under-
stand the interplay between the host immune systems and
replication stress and would provide insight into how these
responses can be modulated optimally. Interestingly, somatic
mutations in the genes encoding the replicative polymerases
POLD1 and POLE are also being used as indicators for success-
ful immunotherapy outcomes due to the high mutation
burden present in these genetic backgrounds (NCT03375307,
NCT03207347, NCT03428802, and NCT03491345). The role
of the innate response in this interaction has yet to be revealed
and will likely also provide additional exploitable therapeutic
vulnerabilities.
Future Directions
Our knowledge of replication stress–centered vulnerabilities in
tumor cells has grown dramatically in recent years and is leading
to an increasingly complex view of how tumor cells deal with
1736 Cancer Res; 79(8) April 15, 2019
replication stress. Knowledge of the replication stress response
continues to develop as we discover novel proteins and reveal
intricate response networks, providing greater opportunities to
target these proteins in therapy. In particular, advances in molec-
ular biology tools continue to facilitate identification of novel
molecular targets in the replication stress response and the inter-
play between the replication stress response and different cellular
pathways such as metabolism (104). In addition to RNA inter-
ference screens, the advent of genome-wide screening using the
CRISPR-Cas9 technology will provide a powerful tool moving
forward to rapidly identify novel clinically relevant synthetic
lethal interactions in cancer cells, and is already yielding prom-
ising results (105–108). As these technologies begin to be adapted
in other systems, similar screens performed in more biologically
appropriate settings, such as in 3D organoid cultures or in vivo
mouse models, will likely yield more clinically relevant synthetic
lethal interactions. Furthermore, advances in whole-genome
Downloaded from http://aacrjournals.org/cancerres/article-pdf/79/8/1730/2791709/1730.pdf by guest on 05 March 2023
sequencing of tumors will also assist precision medicine
approaches by identifying key actionable genetic alterations for
therapeutic targeting in the clinic.
Despite the unprecedented rate at which we are identifying
novel replication stress response targets, there remain several
obstacles to overcome for optimal treatment outcomes. One
limitation of all replication stress–targeting therapies that will
be difficult to diminish is achieving an acceptable therapeutic
window, due to the essentiality of the replication stress
response in normal cells. In contrast to conventional therapies
that target all dividing cells, targeted inhibitors have shown
greater precision in selectively affecting tumors, yet they con-
tinue to present adverse side effects that are sometimes intol-
erable. Another important limitation is the current technolo-
gies available to predict tumor backgrounds that would benefit
most from certain therapies. Markers that can be effectively
detected by immunohistochemistry, such as the overexpres-
sion of certain oncogenes, are starting to be implemented as a
means to stratify patients for targeted treatment strategies in
clinical trials. However, the dependence on immunohistolo-
gical approaches for biomarker identification restricts the use
of important replication stress markers such as RPA foci and
ssDNA and the use of functional DNA repair assays as proxies
for therapy response.
The notion of exploiting intrinsic replication stress has driven
considerable excitement in the medical oncology field as it pro-
vides a selective means of eliminating tumors. Several approaches
undergoing clinical investigation are already showing promise,
and with the advent of powerful new technologies, both in
academic and clinical settings, many more are likely to be revealed
for therapeutic development.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Work in the authors' lab is supported by the Canadian Cancer Society, the
Canadian Institutes of Health Research, and an Ontario Graduate Scholarship
(to T. Ubhi). We thank Haley Wyatt and Dan Durocher for helpful discussions
and careful reading of the manuscript.
Received November 19, 2018; revised January 8, 2019; accepted January 28,
2019; published first April 9, 2019.
Cancer Research

References
1. Gorgoulis VG, Vassiliou LV, Karakaidos P, Zacharatos P, Kotsinas A,
Liloglou T, et al. Activation of the DNA damage checkpoint and genomic
instability in human precancerous lesions. Nature 2005;434:907–13.
2. Bartkova J, Ho
r ej
sí Z, Koed K, Kr€amer A, Tort F, Zleger K, et al. DNA
damage response as a candidate anti-cancer barrier in early human
tumorigenesis. Nature 2005;434:864–70.
3. Fragkos M, Ganier O, Coulombe P, Mechali M. DNA replication origin
activation in space and time. Nat Rev Mol Cell Biol 2015;16:360–74.
4. Burgers PMJ, Kunkel TA. Eukaryotic DNA replication fork. Annu Rev
Biochem 2017;86:417–38.
5. Zeman M, Cimprich K. Causes and consequences of replication stress.
Nat Cell Biol 2014;16:2–9.
6. Byun TS, Pacek M, Yee MC, Walter JC, Cimprich KA. Functional uncou-
pling of MCM helicase and DNA polymerase activities activates the
ATR-dependent checkpoint. Genes Dev 2005;19:1040–52.
7. Zellweger R, Dalcher D, Mutreja K, Berti M, Schmid JA, Herrador R, et al.
Rad51-mediated replication fork reversal is a global response to genotoxic
treatments in human cells. J Cell Biol 2015;208:563–79.
8. Saldivar JC, Cortez D, Cimprich KA. The essential kinase ATR: Ensuring
faithful duplication of a challenging genome. Nat Rev Mol Cell Biol 2017;
18:622–36.
9. Toledo LI, Altmeyer M, Rask MB, Lukas C, Larsen DH, Povlsen LK, et al.
ATR prohibits replication catastrophe by preventing global exhaustion of
RPA. Cell 2013;155:1088–103.
10. Ge XQ, Blow JJ. Chk1 inhibits replication factory activation but allows
dormant origin firing in existing factories. J Cell Biol 2010;191:1285–97.
11. Chen YH, Jones MJK, Yin Y, Crist SB, Colnaghi L, Sims RJ, et al. ATR-
mediated phosphorylation of FANCI regulates dormant origin firing in
response to replication stress. Mol Cell 2015;58:323–38.
12. Petermann E, Orta ML, Issaeva N, Schultz N, Helleday T. Hydroxyurea-
stalled replication forks become progressively inactivated and require two
different RAD51-mediated pathways for restart and repair. Mol Cell 2010;
37:492–502.
13. Macheret M, Halazonetis TD. Intragenic origins due to short G1 phases
underlie oncogene-induced DNA replication stress. Nature 2018;555:
112–6.
14. Cortez D. Preventing replication fork collapse to maintain genome
integrity. DNA Repair 2015;32:149–57.
15. Costantino L, Sotiriou SK, Rantala JK, Magin S, Mladenov E, Helleday T,
et al. Break-induced replication repair of damaged forks induces genomic
duplications in human cells. Science 2014;343:88–91.
16. Minocherhomji S, Ying S, Bjerregaard VA, Bursomanno S, Aleliunaite A,
Wu W, et al. Replication stress activates DNA repair synthesis in mitosis.
Nature 2015;528:286–90.
17. Gisselsson D, Pettersson L, Hoglund M, Heidenblad M, Gorunova L,
Wiegant J, et al. Chromosomal breakage-fusion-bridge events cause
genetic intratumor heterogeneity. Proc Natl Acad Sci U S A 2000;97:
5357–62.
18. Crasta K, Ganem NJ, Dagher R, Lantermann AB, Ivanova EV, Pan Y, et al.
DNA breaks and chromosome pulverization from errors in mitosis.
Nature 2012;482:53–8.
19. Zhang CZ, Spektor A, Cornils H, Francis JM, Jackson EK, Liu S, et al.
Chromothripsis from DNA damage in micronuclei. Nature 2015;522:
179–84.
20. Lukas C, Savic V, Bekker-Jensen S, Doil C, Neumann B, Pedersen RS, et al.
53BP1 nuclear bodies form around DNA lesions generated by mitotic
transmission of chromosomes under replication stress. Nat Cell Biol
2011;13:243–53.
21. Macheret M, Halazonetis TD. DNA replication stress as a hallmark of
cancer. Annu Rev Pathol 2015;10:425–48.
22. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell
2011;144:646–74.
23. Halazonetis TD, Gorgoulis VG, Bartek J. An oncogene-induced DNA
damage model for cancer development. Science 2008;319:1352–5.
24. Bester AC, Roniger M, Oren YS, Im MM, Sarni D, Chaoat M, et al.
Nucleotide deficiency promotes genomic instability in early stages of
cancer development. Cell 2011;145:435–46.
25. Cheung-Ong K, Giaever G, Nislow C. DNA-damaging agents in cancer
chemotherapy: serendipity and chemical biology. Chem Biol 2013;20:
648–59.
www.aacrjournals.org
Exploiting Replication Stress
26. Knijnenburg TA, Wang L, Zimmermann MT, Chambwe N, Gao GF,
Cherniack AD, et al. Genomic and molecular landscape of DNA damage
repair deficiency across The Cancer Genome Atlas. Cell Rep 2018;23:
239–54.
27. Richardson KA, Vega TP, Richardson FC, Moore CL, Rohloff JC, Tomkinson
B, et al. Polymerization of the triphosphates of AraC, 20 ,20 -difluorodeox-
ycytidine (dFdC) and OSI-7836 (T-araC) by human DNA polymerase a
and DNA primase. Biochem Pharmacol 2004;68:2337–46.
28. Huang P, Chubb S, Hertel LW, Grindey GB, Plunkett W. Action of 20 ,20 -
difluorodeoxycytidine on DNA synthesis. Cancer Res 1991;51:6110–7.
29. Cerqueira NM, Fernandes PA, Ramos MJ. Understanding ribonucleotide
reductase inactivation by gemcitabine. Chemistry 2007;13:8507–15.
30. Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms of
action and clinical strategies. Nat Rev Cancer 2003;3:330–8.
31. Wang D, Lippard SJ. Cellular processing of platinum anticancer drugs.
Nat Rev Drug Discov 2005;4:307–20.
32. Fu D, Calvo JA, Samson LD. Balancing repair and tolerance of DNA
damage caused by alkylating agents. Nat Rev Cancer 2012;12:104–20.
33. Deans AJ, West SC. DNA interstrand crosslink repair and cancer. Nat Rev
Downloaded from http://aacrjournals.org/cancerres/article-pdf/79/8/1730/2791709/1730.pdf by guest on 05 March 2023
Cancer 2011;11:467–80.
34. Pommier Y. Topoisomerase I inhibitors: Camptothecins and beyond.
Nat Rev Cancer 2006;6:789–802.
35. Nitiss JL. Targeting DNA topoisomerase II in cancer chemotherapy.
Nat Rev Cancer 2009;9:338–50.
36. Str€
om CE, Johansson F, Uhlen M, Szigyarto CAK, Erixon K, Helleday T.
Poly (ADP-ribose) polymerase (PARP) is not involved in base excision
repair but PARP inhibition traps a single-strand intermediate.
Nucleic Acids Res 2011;39:3166–75.
37. Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, et al.
Targeting the DNA repair defect in BRCA mutant cells as a therapeutic
strategy. Nature 2005;434:917–21.
38. Maya-Mendoza A, Moudry P, Merchut-Maya JM, Lee M, Strauss R, Bartek J.
High speed of fork progression induces DNA replication stress and
genomic instability. Nature 2018;559:279–84.
39. Merrick CJ, Jackson D, Diffley JF. Visualization of altered replication
dynamics after DNA damage in human cells. J Biol Chem 2004;279:
20067–75.
40. Kopper F, Bierwirth C, Schon M, Kunze M, Elvers I, Kranz D, et al. Damage-
induced DNA replication stalling relies on MAPK-activated protein kinase
2 activity. Proc Natl Acad Sci U S A 2013;110:16856–61.
41. Hagenkort A, Paulin CBJ, Desroses M, Sarno A, Wiita E, Mortusewicz O,
et al. dUTPase inhibition augments replication defects of 5-Fluorouracil.
Oncotarget 2017;8:23713–26.
42. Ribeyre C, Zellweger R, Chauvin M, Bec N, Larroque C, Lopes M, et al.
Nascent DNA proteomics reveals a chromatin remodeler required for
topoisomerase I loading at replication forks. Cell Rep 2016;15:300–9.
43. Sirbu BM, Couch FB, Cortez D. Monitoring the spatiotemporal dynamics
of proteins at replication forks and in assembled chromatin using isola-
tion of proteins on nascent DNA. Nat Protoc 2012;7:594–605.
44. Lossaint G, Larroque M, Ribeyre C, Bec N, Larroque C, Decaillet C, et al.
FANCD2 binds MCM proteins and controls replisome function upon
activation of S phase checkpoint signaling. Mol Cell 2013;51:678–90.
45. Dungrawala H, Rose KL, Bhat KP, Mohni KN, Glick GG, Couch FB, et al.
The replication checkpoint prevents two types of fork collapse without
regulating replisome stability. Mol Cell 2015;59:998–1010.
46. Alabert C, Bukowski-Wills JC, Lee SB, Kustatscher G, Nakamura K, De
Lima Alves F, et al. Nascent chromatin capture proteomics determines
chromatin dynamics during DNA replication and identifies unknown
fork components. Nat Cell Biol 2014;16:281–93.
47. Fujinaka Y, Matsuoka K, Iimori M, Tuul M, Sakasai R, Yoshinaga K, et al.
ATR-Chk1 signaling pathway and homologous recombinational repair
protect cells from 5-fluorouracil cytotoxicity. DNA Repair 2012;11:
247–58.
48. Calvo E, Braiteh F, Von Hoff D, McWilliams R, Becerra C, Galsky MD, et al.
Phase I study of CHK1 inhibitor LY2603618 in combination with
gemcitabine in patients with solid tumors. Oncology 2016;91:251–60.
49. Kim H, George E, Ragland RL, Rafail S, Zhang R, Krepler C, et al. Targeting
the ATR/CHK1 axis with PARP inhibition results in tumor regression
in BRCA-mutant ovarian cancer models. Clin Cancer Res 2017;23:
3097–108.
Cancer Res; 79(8) April 15, 2019 1737
 Ubhi and Brown
50. Mohni KN, Thompson PS, Luzwick JW, Glick GG, Pendleton CS,
Lehmann BD, et al. A synthetic lethal screen identifies DNA repair
pathways that sensitize cancer cells to combined ATR inhibition and
cisplatin treatments. PLoS One 2015;10:e0125482.
51. Eich M, Roos WP, Nikolova T, Kaina B. Contribution of ATM and ATR to
the resistance of glioblastoma and malignant melanoma cells to the
methylating anticancer drug temozolomide. Mol Cancer Ther 2013;12:
2529–40.
52. Lecona E, Fernandez-Capetillo O. Targeting ATR in cancer. Nat Rev Cancer
2018;18:586–95.
53. Mansilla S, Bataller M, Portugal J. Mitotic catastrophe as a consequence of
chemotherapy. Anticancer Agents Med Chem 2006;6:589–602.
54. Eykelenboom JK, Harte EC, Canavan L, Pastor-Peidro A, Calvo-Asensio I,
Llorens-Agost M, et al. ATR activates the S-M checkpoint during unper-
turbed growth to ensure sufficient replication prior to mitotic onset.
Cell Rep 2013;5:1095–107.
55. Suzuki M, Yamamori T, Bo T, Sakai Y, Inanami O. MK-8776, a novel Chk1
inhibitor, exhibits an improved radiosensitizing effect compared to
UCN-01 by exacerbating radiation-induced aberrant mitosis. Transl Oncol
2017;10:491–500.
56. Hall AB, Newsome D, Wang Y, Boucher DM, Eustace B, Gu Y, et al.
Potentiation of tumor responses to DNA damaging therapy by the
selective ATR inhibitor VX-970. Oncotarget 2014;5:5674–85.
57. Liu S, Ge Y, Wang T, Edwards H, Ren Q, Jiang Y, et al. Inhibition of ATR
potentiates the cytotoxic effect of gemcitabine on pancreatic cancer cells
through enhancement of DNA damage and abrogation of ribonucleotide
reductase induction by gemcitabine. Oncol Rep 2017;37:3377–86.
58. Wallez Y, Dunlop CR, Johnson TI, Koh S-B, Fornari C, Yates JWT, et al. The
ATR inhibitor AZD6738 synergizes with gemcitabine in vitro and in vivo to
induce pancreatic ductal adenocarcinoma regression. Mol Cancer Ther
2018;17:1670–82.
59. Koh SB, Courtin A, Boyce RJ, Boyle RG, Richards FM, Jodrell DI. CHK1
inhibition synergizes with gemcitabine initially by destabilizing the DNA
replication apparatus. Cancer Res 2015;75:3583–95.
60. Liu Y, Li Y, Wang X, Liu F, Gao P, Quinn MM, et al. Gemcitabine and Chk1
inhibitor AZD7762 synergistically suppress the growth of Lkb1-deficient
lung adenocarcinoma. Cancer Res 2017;77:5068–76.
61. Vendetti FP, Lau A, Schamus S, Conrads TP, O'Connor MJ, Bakkenist CJ.
The orally active and bioavailable ATR kinase inhibitor AZD6738 potenti-
ates the anti-tumor effects of cisplatin to resolve ATM-deficient non-small
cell lung cancer in vivo. Oncotarget 2015;6:44289–305.
62. Li CC, Yang JC, Lu MC, Lee CL, Peng CY, Hsu WY, et al. ATR-Chk1
signaling inhibition as a therapeutic strategy to enhance cisplatin che-
mosensitivity in urothelial bladder cancer. Oncotarget 2016;7:1947–59.
63. Josse R, Martin SE, Guha R, Ormanoglu P, Pfister TD, Reaper PM, et al. ATR
inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase I
inhibitors by disabling DNA replication initiation and fork elongation
responses. Cancer Res 2014;74:6968–79.
64. Flatten K, Dai NT, Vroman BT, Loegering D, Erlichman C, Karnitz LM, et al.
The role of checkpoint kinase 1 in sensitivity to topoisomerase I poisons.
J Biol Chem 2005;280:14349–55.
65. Hong D, Infante J, Janku F, Jones S, Nguyen LM, Burris H, et al. Phase I
study of LY2606368, a checkpoint kinase 1 inhibitor, in patients with
advanced cancer. J Clin Oncol 2016;34:1764–71.
66. Buisson R, Boisvert JL, Benes CH, Zou L. Distinct but concerted roles of
ATR, DNA-PK, and Chk1 in countering replication stress during S phase.
Mol Cell 2015;59:1011–24.
67. Leijen S, van Geel RM, Pavlick AC, Tibes R, Rosen L, Razak AR, et al. Phase I
study evaluating WEE1 inhibitor AZD1775 as monotherapy and in
combination with gemcitabine, cisplatin, or carboplatin in patients with
advanced solid tumors. J Clin Oncol 2016;34:4371–80.
68. De Witt Hamer PC, Mir SE, Noske D, Van Noorden CJ, W€ urdinger T. WEE1
kinase targeting combined with DNA-damaging cancer therapy catalyzes
mitotic catastrophe. Clin Cancer Res 2011;17:4200–7.
69. Domínguez-Kelly R, Martín Y, Koundrioukoff S, Tanenbaum ME, Smits
VA, Medema RH, et al. Wee1 controls genomic stability during replication
by regulating the Mus81-Eme1 endonuclease. J Cell Biol 2011;194:
567–79.
70. Beck H, Nahse-Kumpf V, Larsen MS, O'Hanlon KA, Patzke S, Holmberg C,
et al. Cyclin-dependent kinase suppression by WEE1 kinase protects the
1738 Cancer Res; 79(8) April 15, 2019
genome through control of replication initiation and nucleotide con-
sumption. Mol Cell Biol 2012;32:4226–36.
71. Jin J, Fang H, Yang F, Ji W, Guan N, Sun Z, et al. Combined inhibition of
ATR and WEE1 as a novel therapeutic strategy in triple-negative breast
cancer. Neoplasia 2018;20:478–88.
72. Hauge S, Naucke C, Hasvold G, Joel M, Rødland GE, Juzenas P, et al.
Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in
S-phase due to distinct regulation of CDK activity and CDC45 loading.
Oncotarget 2017;8:10966–79.
73. Dobzhansky T. Genetics of natural populations; recombination and
variability in populations of Drosophila pseudoobscura. Genetics
1946;31:269–90.
74. O'Neil NJ, Bailey ML, Hieter P. Synthetic lethality and cancer. Nat Rev
Genet 2017;18:613–23.
75. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, et al.
Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-
ribose) polymerase. Nature 2005;434:913–7.
76. Williamson CT, Miller R, Pemberton HN, Jones SE, Campbell J, Konde A,
et al. ATR inhibitors as a synthetic lethal therapy for tumours deficient in
ARID1A. Nat Commun 2016;7:13837.
Downloaded from http://aacrjournals.org/cancerres/article-pdf/79/8/1730/2791709/1730.pdf by guest on 05 March 2023
77. Brown JS, O'Carrigan B, Jackson SP, Yap TA. Targeting DNA repair in
cancer: beyond PARP inhibitors. Cancer Discov 2017;7:20–37.
78. Kwok M, Davies N, Agathanggelou A, Smith E, Oldreive C, Petermann E,
et al. ATR inhibition induces synthetic lethality and overcomes chemore-
sistance in TP53- or ATM-defective chronic lymphocytic leukemia cells.
Blood 2016;127:582–95.
79. Higgins GS, Boulton SJ. Beyond PARP—POLq as an anticancer target.
Science 2018;359:1217–8.
80. Leon TE, Rapoz-D'Silva T, Rahman S, Magnussen M, Farah N, Guerra-
Assunç~ao JA, et al. Synthetic lethal screening identifies CHK1 inhibition as
an exploitable vulnerability in EZH2 deficient T-ALL. Blood 2017;130:
1325.
81. Dietlein F, Kalb B, Jokic M, Noll EM, Strong A, Tharun L, et al. A synergistic
interaction between Chk1- and MK2 Inhibitors in KRAS-mutant cancer.
Cell 2015;162:146–59.
82. Pfister SX, Markkanen E, Jiang Y, Sarkar S, Woodcock M, Orlando G, et al.
Inhibiting WEE1 selectively kills histone H3K36me3-deficient cancers by
dNTP starvation. Cancer Cell 2015;28:557–68.
83. Kotsantis P, Petermann E, Boulton SJ. Mechanisms of oncogene-
induced replication stress: Jigsaw falling into place. Cancer Discov
2018;8:537–55.
84. Toledo LI, Murga M, Zur R, Soria R, Rodriguez A, Martinez S, et al. A cell-
based screen identifies ATR inhibitors with synthetic lethal properties for
cancer-associated mutations. Nat Struct Mol Biol 2011;18:721–7.
85. Schoppy DW, Ragland RL, Gilad O, Shastri N, Peters AA, Murga M, et al.
Oncogenic stress sensitizes murine cancers to hypomorphic suppression
of ATR. J Clin Invest 2012;122:241–52.
86. Gilad O, Nabet BY, Ragland RL, Schoppy DW, Smith KD, Durham AC,
et al. Combining ATR suppression with oncogenic Ras synergistically
increases genomic instability, causing synthetic lethality or tumorigenesis
in a dosage-dependent manner. Cancer Res 2010;70:9693–702.
87. Sanjiv K, Hagenkort A, Calder on-Monta~ no JM, Koolmeister T, Reaper PM,
Mortusewicz O, et al. Cancer-specific synthetic lethality between ATR and
CHK1 kinase activities. Cell Rep 2016;14:298–309.
88. Nikkil€a J, Kumar R, Campbell J, Brandsma I, Pemberton HN, Wallberg F,
et al. Elevated APOBEC3B expression drives a kataegic-like mutation
signature and replication stress-related therapeutic vulnerabilities in
p53-defective cells. Br J Cancer 2017;117:113–23.
89. Buisson R, Lawrence MS, Benes CH, Zou L. APOBEC3A and APOBEC3B
activities render cancer cells susceptible to ATR inhibition. Cancer Res
2017;77:4567–78.
90. Burns MB, Temiz NA, Harris RS. Evidence for APOBEC3B mutagenesis in
multiple human cancers. Nat Genet 2013;45:977–83.
91. Roberts SA, Lawrence MS, Klimczak LJ, Grimm SA, Fargo D, Stojanov P,
et al. An APOBEC cytidine deaminase mutagenesis pattern is widespread
in human cancers. Nat Genet 2013;45:970–6.
92. Luthra P, Aguirre S, Yen BC, Pietzsch CA, Sanchez-Aparicio MT, Tigabu B,
et al. Topoisomerase II inhibitors induce DNA damage-dependent
interferon responses circumventing ebola virus immune evasion. MBio
2017;8:pii: e00368–17.
Cancer Research

93. Coquel F, Silva MJ, Techer H, Zadorozhny K, Sharma S, Nieminuszczy J,
et al. SAMHD1 acts at stalled replication forks to prevent interferon
induction. Nature 2018;557:57–61.
94. Dunphy G, Flannery SM, Almine JF, Connolly DJ, Paulus C, Jønsson KL,
et al. Non-canonical activation of the DNA sensing adaptor STING by
ATM and IFI16 mediates NF-kB signaling after nuclear DNA damage.
Mol Cell 2018;71:745–60.
95. Shen J, Zhao W, Ju Z, Wang L, Peng G. PARPi triggers STING-dependent
immune response and enhances therapeutic efficacy of immune check-
point blockade independent of BRCAness. Cancer Res 2018;79:311–9.
96. Harding SM, Benci JL, Irianto J, Discher DE, Minn AJ, Greenberg RA.
Mitotic progression following DNA damage enables pattern recognition
within micronuclei. Nature 2017;548:466–70.
97. Mackenzie KJ, Carroll P, Martin CA, Murina O, Fluteau A, Simpson DJ,
et al. cGAS surveillance of micronuclei links genome instability to innate
immunity. Nature 2017;548:461–5.
98. Bhattacharya S, Srinivasan K, Abdisalaam S, Su F, Raj P, Dozmorov I, et al.
RAD51 interconnects between DNA replication, DNA repair and immu-
nity. Nucleic Acids Res 2017;45:4590–605.
99. Erdal E, Haider S, Rehwinkel J, Harris AL, McHugh PJ. A prosurvival DNA
damage-induced cytoplasmic interferon response is mediated by end
resection factors and is limited by Trex1. Genes Dev 2017;31:353–69.
100. Shen YJ, Le Bert N, Chitre AA, Koo CX, Nga XH, Ho SS, et al. Genome-
derived cytosolic DNA mediates type I interferon-dependent rejection of
B cell lymphoma cells. Cell Rep 2015;11:460–73.
101. Wolf C, Rapp A, Berndt N, Staroske W, Schuster M, Dobrick-Mattheuer M,
et al. RPA and Rad51 constitute a cell intrinsic mechanism to protect the
cytosol from self DNA. Nat Commun 2016;7:11752.
102. Li T, Chen ZJ. The cGAS–cGAMP–STING pathway connects DNA damage
to inflammation, senescence, and cancer. J Exp Med 2018;215:1287–99.
www.aacrjournals.org
Exploiting Replication Stress
103. Xia T, Konno H, Ahn J, Barber GN. Deregulation of STING signaling in
colorectal carcinoma constrains DNA damage responses and correlates
with tumorigenesis. Cell Rep 2016;14:282–97.
104. Somyajit K, Gupta R, Sedlackova H, Neelsen KJ, Ochs F, Rask MB, et al.
Redox-sensitive alteration of replisome architecture safeguards genome
integrity. Science 2017;358:797–802.
105. Toledo CM, Ding Y, Hoellerbauer P, Davis RJ, Basom R, Girard EJ, et al.
Genome-wide CRISPR-Cas9 screens reveal loss of redundancy between
PKMYT1 and WEE1 in glioblastoma stem-like cells. Cell Rep 2015;13:
2425–39.
106. Wang T, Yu H, Hughes NW, Liu B, Kendirli A, Klein K, et al. Gene
essentiality profiling reveals gene networks and synthetic lethal interac-
tions with oncogenic Ras. Cell 2017;168:890–903.
107. Kanarek N, Keys HR, Cantor JR, Lewis CA, Chan SH, Kunchok T, et al.
Histidine catabolism is a major determinant of methotrexate sensitivity.
Nature 2018;559:632–6.
108. Zimmermann M, Murina O, Reijns MAM, Agathanggelou A, Challis
R, Tarnauskaite Z,
et al. CRISPR screens identify genomic ribonu-
cleotides as a source of PARP-trapping lesions. Nature 2018;559:
285–9.
Downloaded from http://aacrjournals.org/cancerres/article-pdf/79/8/1730/2791709/1730.pdf by guest on 05 March 2023
109. Jordheim LP, Durantel D, Zoulim F, Dumontet C. Advances in the
development of nucleoside and nucleotide analogues for cancer and
viral diseases. Nat Rev Drug Discov 2013;12:447–64.
110. Lord CJ, Ashworth A. PARP inhibitors: synthetic lethality in the clinic.
Science 2017;355:1152–8.
111. Ma CX, Janetka JW, Piwnica-Worms H. Death by releasing the breaks:
CHK1 inhibitors as cancer therapeutics. Trends Mol Med 2011;17:
88–96.
112. Matheson CJ, Backos DS, Reigan P. Targeting WEE1 kinase in cancer.
Trends Pharmacol Sci 2016;37:872–81.
Cancer Res; 79(8) April 15, 2019 1739