exploiting dna replication stress for cancer treatment · review exploiting dna replication stress...

Review

Exploiting DNA Replication Stress for CancerTreatmentTajinder Ubhi1,2 and Grant W. Brown1,2

Abstract

Complete and accurate DNA replication is fundamental tocellular proliferation and genome stability. Obstacles thatdelay, prevent, or terminate DNA replication cause the phe-nomena termed DNA replication stress. Cancer cells exhibitchronic replication stress due to the loss of proteins thatprotect or repair stressed replication forks and due to thecontinuous proliferative signaling, providing an exploitabletherapeutic vulnerability in tumors. Here, we outline currentandpending therapeutic approaches leveraging tumor-specificreplication stress as a target, in addition to the challenges

associated with such therapies. We discuss how replicationstress modulates the cell-intrinsic innate immune responseand highlight the integration of replication stress with immu-notherapies. Together, exploiting replication stress for cancertreatment seems to be a promising strategy as it provides aselective means of eliminating tumors, and with continuousadvances in our knowledge of the replication stress responseand lessons learned from current therapies in use, we aremoving toward honing the potential of targeting replicationstress in the clinic.

IntroductionThe DNA replication machinery successfully carries out accu-

rate genome duplication in the face of numerous obstacles, manyof which cause DNA replication stress. Replication stress, definedas any hindrance to DNA replication that either stalls, blocks, orterminates DNA polymerization, activates a highly conservedreplication stress response pathway mediated by a network ofrepair proteins to resolve the damage. Efficient removal of obsta-cles to replication progression safeguards genome integrity byensuring timely completion of DNA replication and by limitingsusceptibility to mutagenesis. However, failure to remove repli-cation stressors due to the loss of replication stress response andrepair proteins and sustained proliferative signaling leads tochronic replication stress, and is a prominent feature of tumorcells. In particular, whole-genome sequencing efforts of tumorsamples have indicated the important role of replication stressin tumor progression and maintenance, complementing earlyfindings from the groups of Halazonetis and Bartek whodemonstrated that replication stress accumulates upon cellulartransformation, and is rarely observed in even the most prolifer-ative tissues (1, 2). Thus, due to the selective nature of replicationstress as a therapeutic target, the dependency of tumor cells onthe replication stress response for cell survival, and the ever-expanding network of replication stress response proteins thatcan be targeted, replication stress is beginning to be explored as anattractive target in the clinic. In contrast to normal cells, cancercells can be pushed toward cell death by introducing further DNAdamage in a catastrophic manner or by promoting their entranceinto cell cycle stages where unresolved replication stress is detri-

mental. In this review, we provide a summary of the therapiescentered on enhancing both endogenous and drug-induced rep-lication stress and discuss the rationales associated with them.Wealso highlight the potential of using replication stress to stimulatethe cell-intrinsic innate immune response to improve immuno-therapy effectiveness.

DNA Replication Stress: Causes,Consequences, and the Cellular Response

Eukaryotic DNA replication is a tightly regulated and complexprocess that requires the orchestrated functions of several hun-dred proteins during the S phase of each cell cycle (reviewed inrefs. 3, 4). Each time a cell divides, billions of nucleotides of DNAmust be accurately polymerized, and ensuring this process occursprior to the next cell cycle is essential for cellular homeostasis.However, there are numerous hindrances to DNA replication thatcan prevent progression of the replication machinery and causereplication forks to stall. Obstacles to replication fork progressioncan arise from several sources, either endogenous or exogenous,ranging from the depletion of nucleotide pools available forDNA synthesis to transcription–replication machinery collisions,RNA–DNA hybrids, oncogene-induced increases in replicationorigin firing, and inherently hard-to-replicate regions (Fig. 1A;reviewed in ref. 5). The type of stress challenging replicationfork progression dictates the cellular response and network ofrepair proteins activated in response to the replication stress.For example, ribonucleotides that are misincorporated intoreplicating DNA can cause replication stress, and are recognizedand removed by ribonucleotide excision repair, whereas inter-strand crosslinks are mainly repaired by the Fanconi anemiapathway, a specialized branch of the DNA damage response.Stalled replication forks are often transient, with cells able toresolve them and continue replication. However, when left unre-solved, replication stress can lead to small-scale and large-scalegenome alterations such as the mutations and chromosomalrearrangements found frequently in human cancers and devel-opmental disorders.

1Department of Biochemistry, University of Toronto, 1 King's College Circle,Toronto, Ontario, Canada. 2Donnelly Centre for Cellular and BiomolecularResearch, University of Toronto, Toronto, Ontario, Canada.

Corresponding Author: Grant W. Brown, University of Toronto, 160 CollegeStreet, Toronto, ONM5S 3E1, Canada. Phone: 416-946-5733; Fax: 416-978-8548;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-18-3631

�2019 American Association for Cancer Research.

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Replication stress triggers the activation of a signaling cascadethat 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 bystretches of single-stranded DNA (ssDNA), larger than thosefound during lagging strand DNA synthesis, that activate theS phase checkpoint. It is thought that the ssDNA gaps are causedmainly by polymerase and helicase uncoupling (6), but ssDNAcan also be generatedbynucleolytic processing ofDNAat reversedreplication fork structures (7). The ssDNA is bound with highaffinity by replication protein A (RPA), which serves as a platformfor the recruitment of numerous sensor proteins, includingataxia 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 thecentral replication stress response kinase ATR (reviewed in 8).Upon activation, ATR orchestrates a multifaceted response atstalled replication forks by phosphorylating several downstreamtargets, including its main effector kinase, checkpoint kinase 1(CHK1). The ATR–CHK1 signaling pathway leads to cell cyclearrest, stabilization of stalled replication forks, and the inhibitionof late origin firing to prevent excess ssDNA formation (9). Indoing so, the S phase checkpoint ultimately promotes replicationfork repair and restart, or resumption of replication from anadjacent origin (10, 11), to ensure complete replication of theaffected genomic loci.

In instances where cells endure chronic replication stress, suchas in the early stages of tumorigenesis, or during extended periodsof antineoplastic drug treatment, replication forks can col-lapse (12–14). It is thought that ssDNA stretches found at stressedreplication forks are converted to DNA double-strand breaksduring replication fork collapse and that the replication fork losesits capacity to synthesize DNA. Although collapsed replicationforks can likely be rescued through homologous recombina-tion (15) or mitotic DNA synthesis (16), inability to resumeDNA replication in these regions promotes mutagenesis andgenome instability. Cells recruit error-prone translesion DNApolymerases that inaccurately synthesize DNA as a means tocomplete bulk genome replication, failure of which leads toincompletely replicatedDNApersisting intomitosis. Thepresenceof 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 remaininggenetic material, leading to the formation of micronuclei (18).These events not only increase the risk of genome instabilityand promote transformation, but they also perpetuate theexisting genome instability through global missegregation ofchromosomes, thus providing a route of evolution for cancercells (19). Unresolved replication stress following mitosis leadsto the formation of nuclear bodies marked by the DNA damageresponse protein p53 binding protein 1 (53BP1) in the subse-quent G1 phase of the cell cycle in daughter cells, which may actto protect or promote resolution of the replication stress (20).

ExploitingDNAReplicationStress–CenteredVulnerabilities in Cancer

As it is well documented that tumor cells have persistentreplication stress (21), and with replication stress now acknowl-edged as an enabling characteristic of cancer (22), it is not

surprising that replication stress is beginning to be rationallyleveraged in cancer therapies. The elevated replication stress incancer cells has been largely attributed to the loss of cell cyclecheckpoint activators, often tumor suppressor genes, and to theoverexpression or constitutive expression of oncogenes (21, 23).These alterations modify replication fork rates, increase replica-tion initiation or origin firing, and promote premature entry intoS phase (2, 13, 24). Strategies aiming to exploit replication stressfall within two major categories; they either function to increasethe endogenous replication stress present within tumor cells toinduce cell death or they produce tumor cell-specific replicationstress that can be further enhanced by additional therapeutics.Traditional replication stress–inducing drugs largely resultedfrom serendipitous observations of tumor cell death followinguse of the agents (25), but increasingly drugs are designedwith thespecific goal of exploiting replication stress (Table 1). The increasein accessibility of deep sequencing approaches has providedremarkable insight into the catalogue of DNA repair genes mutat-ed in cancers and offers another exciting means of targetingendogenous replication stress by identifying mutation-specificvulnerabilities (26).

Current Therapies That HarnessReplication Stress

Because a key feature that distinguishes tumor cells frommost normal cells is their sustained proliferation, DNA repli-cation has been harnessed as a semi-selective target in cancertherapies for decades (25). Many traditional anticancer drugsact by increasing the replication stress within tumor cells, bydirectly damaging the DNA, depleting cellular resourcesrequired for successful cell division, or through a combinationof both. Nucleoside analogues, including 5-fluorouracil, cytar-abine, and gemcitabine, were among the first chemotherapeuticagents 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 oreven a complete termination of some replication forks (27, 28).In addition to their abilities to incorporate into DNA, theseagents also reduce the size of the intracellular nucleotide poolsavailable for DNA synthesis or limit the availability of a singlenucleotide. For example, gemcitabine potentiates its cytotoxiceffects by also inhibiting ribonucleotide reductase (29), where-as 5-fluorouracil functions mainly by inhibiting thymidylatesynthetase, thereby reducing the amount of thymidine avail-able for DNA replication and repair (30).

In contrast to nucleoside analogues, alkylating agents andplatinum-based compounds function by directlymodifying DNAthrough the formation of alkylation adducts onDNA bases, intra-or interstrand crosslinks between DNA bases, or both (31, 32).The generation of DNA adducts by alkylating agents such asdacarbazine and temozolomide poses an obstacle for theadvancing DNA polymerase, leading to delayed replication forkprogression.Moreover, intra- and interstrand crosslinks generatedby platinum-based agents such as cisplatin and carboplatin arephysical barriers to DNA replication and block the replicationmachinery. Intrastrand crosslinks present on the template DNAstrand during replication impair proper base pairing with thenascent DNA strand, whereas interstrand crosslinks impede

Exploiting Replication Stress

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© 2019 American Association for Cancer Research

P

OHRibonucleotideincorporation

Ribonucleotideexcision repair

Inter-strandcrosslinks

Fanconi Anemiapathway

RNA-DNA hybridsand replication-

transcription conflicts

RNA processing enzymes,RNA-DNA helicases,and topoisomerases

GCGG

Hard-to-replicateregions

DNA helicases

MYCNucleotide pooldepletion

Oncogene-inducedreplication stress

Regulation ofreplication origin

firing

AT G

C

PtCl

Cl

DNA sliding clampDNA polymerase

DNA helicase

B

A

C

M

SG2

G1

CHK1

WEE1AZD1775

p53

9-1-1 DNA clamp complex

RPA DNA lesion

Stabilization ofstalled replication

forksCell cycle

progression

Late origin firing

ATRIPATRIPATRATRTOPBP1

ETAA1

CHK1

ATR

M6620, AZD6738,and BAY1895344

Prexasertib,GDC-0575,SCH 900776,and SRA737

S phasecheckpoint

G1/S checkpoint

G2/M checkpoint

G1: Gap phase IS: Synthesis phaseG2: Gap phase IIM: Cell division phase

H3N

H3N

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the very first step of DNA replication, that is, unwinding of thedouble-strand helix (33). Another class of replication stress–inducing agents that directly damage DNA is topoisomeraseinhibitors (34, 35). Topoisomerases generate transient DNAbreaks to relax DNA supercoiling that occurs during DNAreplication and transcription. The intermediate state where thetopoisomerases are bound to the cleaved DNA is trapped bytopoisomerase inhibitors and leads to the formation of stableprotein–DNA complexes that generate a physical barrier to ongo-ing replication forks, often collapsing to double-strand breakswhen encountered by a replication fork. The action of topoisom-erase inhibitors resembles that of another group of replicationstress–inducing therapeutics, PARP inhibitors. PARP inhibitorsgenerate an obstacle for the replication machinery by trappingPARP on DNA (36), in addition to impairing single-strand breakrepair (37) and accelerating replication fork progression (38),amplifying the replication stress in tumor cells.

The replication stress–inducing effects of conventional thera-pies have been directly visualized at the single-molecule levelthrough DNA fiber analyses, where DNA synthesis rates of indi-vidual DNA replication forks can be measured following drugtreatment (39–41), and through DNA pull-down techniquesthat identify proteins bound directly at replication forksin vivo (42–46). Of particular interest, the interactions betweentraditional replication stress–inducing drugs and cellular replica-tion stress response pathways are evident in preclinical studies,where inhibition or depletion of the key replication stressresponse kinases required for overcoming the replication stresspresent within cells, ATR and/or CHK1, sensitizes tumor cells to5-fluorouracil, gemcitabine, cisplatin, PARP inhibitors, and temo-zolomide, with some combinations having advanced to clinicaltrials (34, 35, 47–52).

Emerging Combination Therapies ThatTarget Replication Stress: Cell CycleCheckpoints

Although conventional replication stress–inducing agents havebeen used for decades, their applicability and efficacy is limited bytheir associated toxicities and the rapid emergence of resistanttumor cells. As noted, these therapies target DNA replication, andthus have an effect on highly proliferative cells, including normalcells within the gut epithelium and bone marrow, leading toundesirable and sometimes intolerable side effects. In light of this,several promising rationally designed combination therapies thatincrease the specificity of these traditional therapies toward tumorcells are currently undergoing clinical trials. In addition to gen-erating further replication stress, many of these therapies also

target cell cycle checkpoints (Fig. 1C) to ultimately promote thepremature entry of tumor cells into mitosis, where they undergocell death from abnormal mitosis, termed mitotic catastro-phe (53). Most importantly, these approaches promise animproved and practical therapeutic index relative to conventionalagents due to the intrinsically high levels of replication stress oftenfound within tumor cells.

Activation of the S phase checkpoint elicits a multifacetedsignaling response, where each pathway can provide additionaltargets for therapeutic intervention (Fig. 1). The S phase check-point response kinases ATR and CHK1 have been the leadingtargets of the therapies currently under investigation (Table 1).ATR delays the activation of dormant replication origins andstabilizes stressed replication forks to prevent replication forkcollapse. Accordingly, ATR inhibition initiates widespread DNAsynthesis from normally dormant replication origins, generat-ing enough ssDNA to exhaust the cellular pools of RPA anddepleting the nucleotide pools necessary for DNA synthesis (9,11). ATR and CHK1 inhibition also promotes premature entryinto mitosis despite the presence of under-replicated DNA,leading to chromosome fragmentation (54, 55). Given theirmechanisms of action, ATR and CHK1 inhibitors synergizewith compounds that induce replication stress, and increasethe 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 ensuresuccessful resolution of the replication stress, ATR inhibitorsshould be sufficient as monotherapy in tumors that experiencehigh levels of intrinsic replication stress, which is also a currentarm of investigation (NCT03188965). Early CHK1 inhibitorswere not successful as monotherapies, largely due to their intol-erable side effects and pharmacokinetic properties, althoughclinical studies demonstrate potential for newer generationCHK1 inhibitors, as they have a tolerable toxicity profile (65).The high toxicity of CHK1 inhibitors is consistent with the centralrole of CHK1 in mediating the S phase checkpoint upon replica-tion stress, both in the absence and presence of ATR, andin preventing premature entry into mitosis (55, 66), but presentsthe possibility of targeting downstream effectors of CHK1therapeutically.

As tumor cells are often deficient in the key G1/S transitionregulator p53, the G2/M checkpoint becomes essential to preventtumor cells with unreplicated or damaged DNA from enteringcatastrophic mitoses. Promising therapies targeting regulators ofmitotic entry to undermine the G2/M checkpoint are also beingexplored. 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 anddictate the cellular response and network of repair proteins activated in response to the replication stress. Sources of replication stress include, but are notlimited to, the depletion of nucleotide pools available for DNA replication and repair, oncogene-induced stress, RNA–DNA hybrids, replication–transcriptionconflicts, DNA crosslinks, inherently difficult-to-replicate regions, and the misincorporation of ribonucleotides into replicating DNA. B, Replication stressactivates a highly conserved signaling response called the S phase checkpoint. The S phase checkpoint is activated upon exposure of ssDNA from stressedreplication 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 completereplication 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 DNAreplication stress. In addition to S phase checkpoint response proteins being the leading targets for cancer therapies targeting replication stress, regulators ofmitotic entry are also being evaluated, as they drive cells with unresolved replication stress into catastrophic mitoses. Targets in the DNA replication stressresponse are shown for each cell cycle checkpoint, with inhibitors targeting those proteins currently being evaluated in clinical trials in bold.






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 underwayfor combination therapies with WEE1 inhibitors (clinicaltrials.gov, accessed January 2019). These include combinations ofgemcitabine, temozolomide, cytarabine, irinotecan, carboplatin,and cisplatin with the potent WEE1 inhibitor AZD1775 inpancreatic, blood, brain, ovarian, and colon tumors, with anemphasis on p53 deficiency as the key selection criteria. Inaddition to driving tumor cells into unscheduled mitosis (68),the cytotoxicity of AZD1775 could also be attributed to theeffects WEE1 has on stabilizing stalled replication forks andensuring 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 ofpossible combinations with other targeted inhibitors thatcould 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 intocombination regimens.

Precision Medicine Approaches ThatLeverage Replication Stress

The identification of the underlying genetic alterations acrossall major subtypes of cancer has provided unprecedented insightinto DNA repair proteins that are altered in cancer (26) and haslaid the foundation for several precision medicine approaches.Almost every tumor has at least one gene in the replication stressresponse altered, providing ampleopportunities to exploit tumor-specific alterations without affecting normal cells. The concept ofsynthetic lethality (73), whereby the presence of cancer-specificmutations in specific genes renders other genes essential for cellproliferation and survival, has been fundamental for the discoveryof several of these gene-targeted strategies (74). Perhaps the bestexample of this strategy is the successful use of PARP inhibitors forthe treatment of BRCA1/BRCA2-deficient breast and ovariancancers, as these double-strand break repair defective tumors aredependent on PARP activity for cell survival (37, 75). Consider-able effort is now being expended to identify additional syntheticlethal interactions with DNA repair genes, including replicationstress response genes. One of the most promising targetscurrently being explored is ATR. Tumors harboring mutations in

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, 109CytarabineDecitabineGemcitabine5-Fluorouracil

Alkylating agents and Direct modification of DNA Carboplatin All approved for use 31, 32platinum compounds Cisplatin

CyclophosphamideDacarbazineMethotrexateMitomycin COxaliplatinProcarbazineTemozolomide

Topoisomerase I and II Relax DNA supercoiling that occurs during Belotecan Approved 34, 35DNA replication and transcription CRLX101 Phase II

Doxorubicin ApprovedEpirubicin ApprovedEtoposide ApprovedIdarubicin ApprovedIrinotecan ApprovedLMP400 Phase ILMP776 Phase IMitoxantrone ApprovedNKTR-102 Phase IIITeniposide ApprovedTopotecan Approved

PARP Single-strand DNA break repair Olaparib Approved 110Niraparib ApprovedRucaparib ApprovedTalazoparib ApprovedVeliparib Phase III

ATR Central replication stress response kinase AZD6738 Phase I/II 52BAY1895344 Phase I/IIM6620 Phase II

CHK1 Main effector kinase of ATR in replication GDC-0575 Phase I 111stress response SCH 900776 Phase I

SRA737 Phase IPrexasertib Phase I/II

WEE1 G2/M checkpoint kinase AZD1775 Phase I/II 112

Ubhi and Brown





genes encoding the chromatin remodeler ARID1A or the DNAdamage kinase ATM are synthetically sensitive to ATR inhibitors,with a complete response observed in a patient with metastaticATM-deficient colorectal cancer (76–78). The dependency ofARID1A-deficient tumors on ATR has been attributed to themitotic defects present in these cells, which causes an increasedreliance on ATR function. Both ARID1A and ATM are frequentlyaltered in tumors, particularly in ovarian and endometrial cancersfor ARID1A and lymphoid malignancies for ATM, illustrating theapplicability of these findings. In addition to further interactionswith ATR, tumors containing mutations in other frequentlyaltered replication stress response genes including RPA1(NCT03207347 and NCT03377556) and the mutagenic DNApolymerase POLQ (79) are currently being explored to identifydruggable synthetic lethal interactions. Genetic interactionswith replication stress–inducing agents including CHK1 (80,81) and WEE1 (82) inhibitors are also being investigated foruse in mutation-specific tumor backgrounds, such as the use ofCHK1 inhibitors in EZH2-deficient T-cell acute lymphoblasticleukemias.

Patient-tailored therapies targeting vulnerabilities induced byspecific oncogenes are also being implemented. In particular,the genes encodingRAS, cyclin E, cyclinD, andMYCare frequentlyamplified in many cancer types and are starting to be harnessedas biomarkers of replication stress. These oncogenes stimulatepremature entry into S phase, thereby disrupting the spatialand temporal regulation of origin firing and replication forkspeed, increase global transcription activity, and promoteRNA–DNA hybrid accumulation, ultimately causing replicationstress (reviewed in ref. 83). Accordingly, inhibition of ATR, CHK1,

or WEE1 activity is lethal in the context of cyclin E-, MYC-, and/orRAS-overexpressing tumor cells (84–87), presumably becausethese tumors have a heightened requirement for the cell cyclecheckpoints that respond to replication stress. Overexpressionof the G1/S checkpoint regulator, cyclin D and its effectorsCDK4andCDK6, are leveraged as biomarkers for patient selectionin trials with DNA damaging agents and immunotherapies(NCT03237390, NCT02290145, and NCT03088059). Morerecently, studies have also elucidated the role of apolipoproteinB mRNA editing enzyme catalytic polypeptide–like (APOBEC)proteins as an endogenous source of replication stress and havefound that overexpressionof twoAPOBEC3enzymes,APOBEC3Aand APOBEC3B, confers sensitivity to ATR inhibition (88, 89). AsAPOBEC3A and APOBEC3B are overexpressed in several cancertypes (90, 91), this finding could have important implications inmaximizing the potential of ATR inhibitors while sparing normalcells. The combination of the overexpression of the remaining fiveAPOBEC3 paralogs and replication stress–inducing agents may,therefore, also be worth testing for possible synergies.

The Innate ImmuneResponse:AnEmergingMarker of the Replication Stress Response

An exciting emerging concept is the link between replicationstress 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 expressionof type I IFNs and proinflammatory cytokines (92–95). It is nowapparent that this activation is largely mediated through the

© 2019 American Association for Cancer Research

Micronucleus

Nuclear envelope

TREX1

Nucleus

Cytosol

SAMHD1

Cytosolic DNAsensing

Replication stress conditions

cGAS andIFI16 STING

STINGactivation

Type I inteferon production

Interferon-stimulated geneand pro-inflammatory cytokineproduction

Cellular senescence orelimination by adaptiveimmune system

RPA andRAD51

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, eitherdirectly from stalled replication forks in three prime repair exonuclease 1 (TREX1)-, sterile alpha motif and HD domain–containing protein 1 (SAMHD1)-, RPA- orRAD51-deficient backgrounds, or frommicronuclei, which are small nuclei formed predominantly followingmitotic progression in cells experiencing genomeinstability. 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 adaptiveimmune system.






presence of cytosolic DNA derived from the nucleus ormitochon-dria, which engages the pattern recognition receptors that nor-mally scan for viral infections. The mechanisms through whichreplication stress induces DNA release into the cytoplasm appearto be specific to the perturbation. Recent work has found micro-nuclei to be a key source of immunostimulatory DNA followingmitotic progression in cells lacking RNase H2 (96, 97), whereasthe release of ssDNA directly from stalled replication forks hasalso 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 mechanismshas 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 fromcytosolic DNA sensors to a transcriptional response from activa-tion of both the NF-kB and type I IFN signaling axes, ultimatelypromoting cellular senescence or elimination by the adaptiveimmune system (reviewed in ref. 102). Interestingly, STINGsignaling is suppressed in several tumors (103) and multiplecancer 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 innateresponse is critical in priming the adaptive immune system,robust STING signaling has been attributed to an increasedimmunotherapy response. In fact, blocking T-cell inhibitory path-ways using immune checkpoint inhibitors is ineffective in micelacking TMEM173 (STING; ref. 96). However, despite currentefforts to combine STING agonists with checkpoint inhibitors asantitumor therapy, therapeutic delivery of the agonists has yet tobe improved for clinical settings. Combination approaches inte-grating replication stress–inducing agents, such as carboplatinor 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 theengagement of STING by replication stress, and would supportthe 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 andreplication stress and would provide insight into how theseresponses can be modulated optimally. Interestingly, somaticmutations in the genes encoding the replicative polymerasesPOLD1 and POLE are also being used as indicators for success-ful immunotherapy outcomes due to the high mutationburden present in these genetic backgrounds (NCT03375307,NCT03207347, NCT03428802, and NCT03491345). The roleof the innate response in this interaction has yet to be revealedand will likely also provide additional exploitable therapeuticvulnerabilities.

Future DirectionsOur knowledge of replication stress–centered vulnerabilities in

tumor cells has grown dramatically in recent years and is leadingto an increasingly complex view of how tumor cells deal with

replication stress. Knowledge of the replication stress responsecontinues to develop as we discover novel proteins and revealintricate response networks, providing greater opportunities totarget these proteins in therapy. In particular, advances in molec-ular biology tools continue to facilitate identification of novelmolecular targets in the replication stress response and the inter-play between the replication stress response and different cellularpathways such as metabolism (104). In addition to RNA inter-ference screens, the advent of genome-wide screening using theCRISPR-Cas9 technology will provide a powerful tool movingforward to rapidly identify novel clinically relevant syntheticlethal interactions in cancer cells, and is already yielding prom-ising results (105–108). As these technologies begin to be adaptedin other systems, similar screens performed in more biologicallyappropriate settings, such as in 3D organoid cultures or in vivomouse models, will likely yield more clinically relevant syntheticlethal interactions. Furthermore, advances in whole-genomesequencing of tumors will also assist precision medicineapproaches by identifying key actionable genetic alterations fortherapeutic targeting in the clinic.

Despite the unprecedented rate at which we are identifyingnovel replication stress response targets, there remain severalobstacles to overcome for optimal treatment outcomes. Onelimitation of all replication stress–targeting therapies that willbe difficult to diminish is achieving an acceptable therapeuticwindow, due to the essentiality of the replication stressresponse in normal cells. In contrast to conventional therapiesthat target all dividing cells, targeted inhibitors have showngreater 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 benefitmost from certain therapies. Markers that can be effectivelydetected by immunohistochemistry, such as the overexpres-sion of certain oncogenes, are starting to be implemented as ameans to stratify patients for targeted treatment strategies inclinical trials. However, the dependence on immunohistolo-gical approaches for biomarker identification restricts the useof important replication stress markers such as RPA foci andssDNA and the use of functional DNA repair assays as proxiesfor therapy response.

The notion of exploiting intrinsic replication stress has drivenconsiderable excitement in the medical oncology field as it pro-vides a selectivemeans of eliminating tumors. Several approachesundergoing clinical investigation are already showing promise,and with the advent of powerful new technologies, both inacademic and clinical settings,manymore are likely to be revealedfor therapeutic development.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsWork 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 discussionsand careful reading of the manuscript.

Received November 19, 2018; revised January 8, 2019; accepted January 28,2019; published first April 9, 2019.

Ubhi and Brown





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2019;79:1730-1739. Published OnlineFirst April 9, 2019.Cancer Res Tajinder Ubhi and Grant W. Brown Exploiting DNA Replication Stress for Cancer Treatment

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