Exploiting DNA Replication Stress for Cancer Treatment Tajinder Ubhi1,2 and Grant W

Home , Belotecan, DNA replication stress

Published OnlineFirst April 9, 2019; DOI: 10.1158/0008-5472.CAN-18-3631 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 associated with such therapies. We discuss how replication cellular proliferation and genome stability. Obstacles that stress modulates the cell-intrinsic innate immune response delay, prevent, or terminate DNA replication cause the phe- and highlight the integration of replication stress with immu- nomena termed DNA replication stress. Cancer cells exhibit notherapies. Together, exploiting replication stress for cancer chronic replication stress due to the loss of proteins that treatment seems to be a promising strategy as it provides a protect or repair stressed replication forks and due to the selective means of eliminating tumors, and with continuous 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- 1Department of Biochemistry, University of Toronto, 1 King's College Circle, strand crosslinks are mainly repaired by the Fanconi anemia 2 Toronto, Ontario, Canada. Donnelly 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: [email protected] 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.

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

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A Regulation of RNA processing enzymes, replication origin RNA-DNA helicases, ﬁring and topoisomerases Oncogene-induced replication stress RNA-DNA hybrids Nucleotide pool MYC and replication- DNA sliding clamp depletion transcription conﬂicts DNA polymerase C DNA helicase A H N Cl G 3 T Pt

H3N Cl

Inter-strand GCGG crosslinks

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

DNA helicases Ribonucleotide excision repair B

9-1-1 DNA clamp complex RPA DNA lesion

ATRIP ATRIP ETAA1 TOPBP1 ATR ATR P CHK1

Stabilization of Late origin ﬁring stalled replication Cell cycle forks progression C G : Gap phase I AZD1775 WEE1 1 S: Synthesis phase

G2: Gap phase II M: Cell division phase

G2/M checkpoint

M G1 G2 S p53

Prexasertib, GDC-0575, G /S checkpoint SCH 900776, 1 and SRA737

S phase CHK1 ATR checkpoint

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the very first step of DNA replication, that is, unwinding of the target cell cycle checkpoints (Fig. 1C) to ultimately promote the double-strand helix (33). Another class of replication stress– premature entry of tumor cells into mitosis, where they undergo inducing agents that directly damage DNA is topoisomerase cell death from abnormal mitosis, termed mitotic catastro- inhibitors (34, 35). Topoisomerases generate transient DNA phe (53). Most importantly, these approaches promise an breaks to relax DNA supercoiling that occurs during DNA improved and practical therapeutic index relative to conventional replication and transcription. The intermediate state where the agents due to the intrinsically high levels of replication stress often topoisomerases are bound to the cleaved DNA is trapped by found within tumor cells. topoisomerase inhibitors and leads to the formation of stable Activation of the S phase checkpoint elicits a multifaceted protein–DNA complexes that generate a physical barrier to ongo- signaling response, where each pathway can provide additional ing replication forks, often collapsing to double-strand breaks targets for therapeutic intervention (Fig. 1). The S phase check- when encountered by a replication fork. The action of topoisom- point response kinases ATR and CHK1 have been the leading erase inhibitors resembles that of another group of replication targets of the therapies currently under investigation (Table 1). stress–inducing therapeutics, PARP inhibitors. PARP inhibitors ATR delays the activation of dormant replication origins and generate an obstacle for the replication machinery by trapping stabilizes stressed replication forks to prevent replication fork PARP on DNA (36), in addition to impairing single-strand break collapse. Accordingly, ATR inhibition initiates widespread DNA repair (37) and accelerating replication fork progression (38), synthesis from normally dormant replication origins, generat- amplifying the replication stress in tumor cells. ing enough ssDNA to exhaust the cellular pools of RPA and The replication stress–inducing effects of conventional thera- depleting the nucleotide pools necessary for DNA synthesis (9, pies have been directly visualized at the single-molecule level 11). ATR and CHK1 inhibition also promotes premature entry through DNA fiber analyses, where DNA synthesis rates of indi- into mitosis despite the presence of under-replicated DNA, vidual DNA replication forks can be measured following drug leading to chromosome fragmentation (54, 55). Given their treatment (39–41), and through DNA pull-down techniques mechanisms of action, ATR and CHK1 inhibitors synergize that identify proteins bound directly at replication forks with compounds that induce replication stress, and increase in vivo (42–46). Of particular interest, the interactions between the toxicity of nucleoside analogues in ovarian, lung, and pan- traditional replication stress–inducing drugs and cellular replica- creatic cancer (56–60), platinum-based agents in lung, gastric, tion stress response pathways are evident in preclinical studies, ovarian, bladder, and breast cancer (50, 61, 62) and topoisom- where inhibition or depletion of the key replication stress erase inhibitors in breast, colon, and brain tumors (63, 64). response kinases required for overcoming the replication stress Because ATR is activated following replication stress to ensure present within cells, ATR and/or CHK1, sensitizes tumor cells to successful resolution of the replication stress, ATR inhibitors 5-fluorouracil, gemcitabine, cisplatin, PARP inhibitors, and temo- should be sufficient as monotherapy in tumors that experience zolomide, with some combinations having advanced to clinical high levels of intrinsic replication stress, which is also a current trials (34, 35, 47–52). arm of investigation (NCT03188965). Early CHK1 inhibitors were not successful as monotherapies, largely due to their intol- Emerging Combination Therapies That erable side effects and pharmacokinetic properties, although Target Replication Stress: Cell Cycle clinical studies demonstrate potential for newer generation CHK1 inhibitors, as they have a tolerable toxicity profile (65). Checkpoints The high toxicity of CHK1 inhibitors is consistent with the central Although conventional replication stress–inducing agents have role of CHK1 in mediating the S phase checkpoint upon replica- been used for decades, their applicability and efficacy is limited by tion stress, both in the absence and presence of ATR, and their associated toxicities and the rapid emergence of resistant in preventing premature entry into mitosis (55, 66), but presents tumor cells. As noted, these therapies target DNA replication, and the possibility of targeting downstream effectors of CHK1 thus have an effect on highly proliferative cells, including normal therapeutically. fi cells within the gut epithelium and bone marrow, leading to As tumor cells are often de cient in the key G1/S transition undesirable and sometimes intolerable side effects. In light of this, regulator p53, the G2/M checkpoint becomes essential to prevent several promising rationally designed combination therapies that tumor cells with unreplicated or damaged DNA from entering increase the specificity of these traditional therapies toward tumor catastrophic mitoses. Promising therapies targeting regulators of cells are currently undergoing clinical trials. In addition to gen- mitotic entry to undermine the G2/M checkpoint are also being erating further replication stress, many of these therapies also 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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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 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). Precision Medicine Approaches That Drugs that increase replication stress should increase the impor- Leverage Replication Stress tance of the G2/M checkpoint for tumor cell survival, and accordingly there are currently greater than 30 clinical studies underway The identification of the underlying genetic alterations across for combination therapies with WEE1 inhibitors (clinicaltrials. all major subtypes of cancer has provided unprecedented insight gov, accessed January 2019). These include combinations of into DNA repair proteins that are altered in cancer (26) and has gemcitabine, temozolomide, cytarabine, irinotecan, carboplatin, laid the foundation for several precision medicine approaches. and cisplatin with the potent WEE1 inhibitor AZD1775 in Almost every tumor has at least one gene in the replication stress pancreatic,blood,brain,ovarian,andcolontumors,withan response altered, providing ample opportunities to exploit tumor- emphasis on p53 deficiency as the key selection criteria. In specific alterations without affecting normal cells. The concept of addition to driving tumor cells into unscheduled mitosis (68), synthetic lethality (73), whereby the presence of cancer-specific the cytotoxicity of AZD1775 could also be attributed to the mutations in specific genes renders other genes essential for cell effects WEE1 has on stabilizing stalled replication forks and proliferation and survival, has been fundamental for the discovery ensuring timely origin firing, further exacerbating the replica- of several of these gene-targeted strategies (74). Perhaps the best tion stress in tumor cells when WEE1 is inhibited (69, 70). example of this strategy is the successful use of PARP inhibitors for WEE1 inhibitors act synergistically with ATR and CHK1 inhi- the treatment of BRCA1/BRCA2-deficient breast and ovarian bitors in preclinical studies (71, 72), extending the catalogue of cancers, as these double-strand break repair defective tumors are possible combinations with other targeted inhibitors that dependent on PARP activity for cell survival (37, 75). Consider- could be implemented. Similar to ATR and CHK1 inhibitors, able effort is now being expended to identify additional synthetic although AZD1775 has shown single-agent activity in preclin- lethal interactions with DNA repair genes, including replication ical studies, its efficacy is potentiated when integrated into stress response genes. One of the most promising targets combination regimens. currently being explored is ATR. Tumors harboring mutations in

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genes encoding the chromatin remodeler ARID1A or the DNA or WEE1 activity is lethal in the context of cyclin E-, MYC-, and/or damage kinase ATM are synthetically sensitive to ATR inhibitors, RAS-overexpressing tumor cells (84–87), presumably because with a complete response observed in a patient with metastatic these tumors have a heightened requirement for the cell cycle ATM-deficient colorectal cancer (76–78). The dependency of checkpoints that respond to replication stress. Overexpression ARID1A-deficient tumors on ATR has been attributed to the of the G1/S checkpoint regulator, cyclin D and its effectors mitotic defects present in these cells, which causes an increased CDK4 and CDK6, are leveraged as biomarkers for patient selection reliance on ATR function. Both ARID1A and ATM are frequently in trials with DNA damaging agents and immunotherapies altered in tumors, particularly in ovarian and endometrial cancers (NCT03237390, NCT02290145, and NCT03088059). More for ARID1A and lymphoid malignancies for ATM, illustrating the recently, studies have also elucidated the role of apolipoprotein applicability of these findings. In addition to further interactions B mRNA editing enzyme catalytic polypeptide–like (APOBEC) with ATR, tumors containing mutations in other frequently proteins as an endogenous source of replication stress and have altered replication stress response genes including RPA1 found that overexpression of two APOBEC3 enzymes, APOBEC3A (NCT03207347 and NCT03377556) and the mutagenic DNA and APOBEC3B, confers sensitivity to ATR inhibition (88, 89). As polymerase POLQ (79) are currently being explored to identify APOBEC3A and APOBEC3B are overexpressed in several cancer druggable synthetic lethal interactions. Genetic interactions types (90, 91), this finding could have important implications in with replication stress–inducing agents including CHK1 (80, maximizing the potential of ATR inhibitors while sparing normal 81) and WEE1 (82) inhibitors are also being investigated for cells. The combination of the overexpression of the remaining five use in mutation-specific tumor backgrounds, such as the use of APOBEC3 paralogs and replication stress–inducing agents may, CHK1 inhibitors in EZH2-deficient T-cell acute lymphoblastic therefore, also be worth testing for possible synergies. leukemias. Patient-tailored therapies targeting vulnerabilities induced by The Innate Immune Response: An Emerging specific oncogenes are also being implemented. In particular, the genes encoding RAS, cyclin E, cyclin D, and MYC are frequently Marker of the Replication Stress Response amplified in many cancer types and are starting to be harnessed An exciting emerging concept is the link between replication as biomarkers of replication stress. These oncogenes stimulate stress and activation of the cell-intrinsic innate immune response premature entry into S phase, thereby disrupting the spatial (Fig. 2). Accumulating evidence indicates that replication stress– and temporal regulation of origin firing and replication fork inducing agents such as topoisomerase inhibitors and cells defi- speed, increase global transcription activity, and promote cient in replication stress response genes induce the expression RNA–DNA hybrid accumulation, ultimately causing replication of type I IFNs and proinflammatory cytokines (92–95). It is now stress (reviewed in ref. 83). Accordingly, inhibition of ATR, CHK1, apparent that this activation is largely mediated through the

Cytosol Replication stress conditions

TREX1 RPA and RAD51 Type I inteferon production cGAS and IFI16 STING Interferon-stimulated gene and pro-inﬂammatory cytokine SAMHD1 production

STING Cellular senescence or Cytosolic DNA activation elimination by adaptive sensing immune system

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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presence of cytosolic DNA derived from the nucleus or mitochon- replication stress. Knowledge of the replication stress response dria, which engages the pattern recognition receptors that nor- continues to develop as we discover novel proteins and reveal mally scan for viral infections. The mechanisms through which intricate response networks, providing greater opportunities to replication stress induces DNA release into the cytoplasm appear target these proteins in therapy. In particular, advances in molec- to be specific to the perturbation. Recent work has found micro- ular biology tools continue to facilitate identification of novel nuclei to be a key source of immunostimulatory DNA following molecular targets in the replication stress response and the inter- mitotic progression in cells lacking RNase H2 (96, 97), whereas play between the replication stress response and different cellular the release of ssDNA directly from stalled replication forks has pathways such as metabolism (104). In addition to RNA inter- also been demonstrated in TREX1-, SAMHD1-, RPA-, and RAD51- ference screens, the advent of genome-wide screening using the deficient backgrounds and following cytarabine treatment CRISPR-Cas9 technology will provide a powerful tool moving (93, 98–101). Whether these conditions could result in cyto- forward to rapidly identify novel clinically relevant synthetic plasmic DNA accumulation through both observed mechanisms lethal interactions in cancer cells, and is already yielding prom- has yet to be ruled out, and several aspects of these mechanisms, ising results (105–108). As these technologies begin to be adapted including the interplay of proteins at replication forks that pro- in other systems, similar screens performed in more biologically mote ssDNA release, have yet to be defined. appropriate settings, such as in 3D organoid cultures or in vivo Central to the innate immune response is the adaptor protein mouse models, will likely yield more clinically relevant synthetic "stimulator of IFN genes" (STING), which couples signals from lethal interactions. Furthermore, advances in whole-genome cytosolic DNA sensors to a transcriptional response from activa- sequencing of tumors will also assist precision medicine tion of both the NF-kB and type I IFN signaling axes, ultimately approaches by identifying key actionable genetic alterations for promoting cellular senescence or elimination by the adaptive therapeutic targeting in the clinic. immune system (reviewed in ref. 102). Interestingly, STING Despitetheunprecedentedrateatwhichweareidentifying signaling is suppressed in several tumors (103) and multiple novel replication stress response targets, there remain several cancer cell types contain genome-derived cytosolic ssDNA (100), obstacles to overcome for optimal treatment outcomes. One further affirming the presence and importance of persistent rep- limitation of all replication stress–targeting therapies that will lication stress in tumors. As type I IFN production from the innate be difficult to diminish is achieving an acceptable therapeutic response is critical in priming the adaptive immune system, window, due to the essentiality of the replication stress robust STING signaling has been attributed to an increased response in normal cells. In contrast to conventional therapies immunotherapy response. In fact, blocking T-cell inhibitory path- that target all dividing cells, targeted inhibitors have shown ways using immune checkpoint inhibitors is ineffective in mice greater precision in selectively affecting tumors, yet they con- lacking TMEM173 (STING; ref. 96). However, despite current tinuetopresentadversesideeffectsthataresometimesintol- efforts to combine STING agonists with checkpoint inhibitors as erable. Another important limitation is the current technolo- antitumor therapy, therapeutic delivery of the agonists has yet to gies available to predict tumor backgrounds that would benefit be improved for clinical settings. Combination approaches inte- most from certain therapies. Markers that can be effectively grating replication stress–inducing agents, such as carboplatin detected by immunohistochemistry, such as the overexpres- or gemcitabine, with immunotherapies like the immune check- sion of certain oncogenes, are starting to be implemented as a point inhibitor nivolumab, have advanced to clinical trials means to stratify patients for targeted treatment strategies in (NCT02944396, NCT03662074, NCT03061188, NCT02734004, clinical trials. However, the dependence on immunohistolo- NCT02849496, NCT02657889, and NCT02571725). It is tempt- gical approaches for biomarker identification restricts the use ing to speculate that the efficacy observed could be due to the of important replication stress markers such as RPA foci and engagement of STING by replication stress, and would support ssDNA and the use of functional DNA repair assays as proxies the use of replication stress as a predictive marker for immu- for therapy response. notherapy efficacy. Further work is required to better under- The notion of exploiting intrinsic replication stress has driven stand the interplay between the host immune systems and considerable excitement in the medical oncology field as it pro- replication stress and would provide insight into how these vides a selective means of eliminating tumors. Several approaches responses can be modulated optimally. Interestingly, somatic undergoing clinical investigation are already showing promise, mutations in the genes encoding the replicative polymerases and with the advent of powerful new technologies, both in POLD1 and POLE are also being used as indicators for success- academic and clinical settings, many more are likely to be revealed ful immunotherapy outcomes due to the high mutation for therapeutic development. burden present in these genetic backgrounds (NCT03375307, NCT03207347, NCT03428802, and NCT03491345). The role Disclosure of Potential Conflicts of Interest of the innate response in this interaction has yet to be revealed No potential conflicts of interest were disclosed. and will likely also provide additional exploitable therapeutic vulnerabilities. Acknowledgments Work in the authors' lab is supported by the Canadian Cancer Society, the Canadian Institutes of Health Research, and an Ontario Graduate Scholarship Future Directions (to T. Ubhi). We thank Haley Wyatt and Dan Durocher for helpful discussions and careful reading of the manuscript. Our knowledge of replication stress–centered vulnerabilities in tumor cells has grown dramatically in recent years and is leading Received November 19, 2018; revised January 8, 2019; accepted January 28, to an increasingly complex view of how tumor cells deal with 2019; published first April 9, 2019.

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OF10 Cancer Res; 79(8) April 15, 2019 Cancer Research

Exploiting DNA Replication Stress for Cancer Treatment

Tajinder Ubhi and Grant W. Brown

Cancer Res Published OnlineFirst April 9, 2019.

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