achieving precision death with cell-cycle inhibitors that ......inevitable for cell-cycle...

Review

Achieving Precision Death with Cell-CycleInhibitors that Target DNA Replication andRepairAimee Bence Lin1, Samuel C. McNeely2, and Richard P. Beckmann3

Abstract

All cancers are characterized by defects in the systems thatensure strict control of the cell cycle in normal tissues. Theconsequent excess tissue growth can be countered by drugs thathalt cell division, and, indeed, the majority of chemotherapeu-tics developed during the last century work by disruptingprocesses essential for the cell cycle, particularly DNA synthesis,DNA replication, and chromatid segregation. In certain con-texts, the efficacy of these classes of drugs can be impressive, butbecause they indiscriminately block the cell cycle of all activelydividing cells, their side effects severely constrain the dose andduration with which they can be administered, allowing bothnormal and malignant cells to escape complete growth arrest.Recent progress in understanding how cancers lose control ofthe cell cycle, coupled with comprehensive genomic profiling of

human tumor biopsies, has shown that many cancers havemutations affecting various regulators and checkpoints thatimpinge on the core cell-cycle machinery. These defects intro-duce unique vulnerabilities that can be exploited by a nextgeneration of drugs that promise improved therapeutic win-dows in patients whose tumors bear particular genomic aberra-tions, permitting increased dose intensity and efficacy. Thesedevelopments, coupled with the success of new drugs targetingcell-cycle regulators, have led to a resurgence of interest in cell-cycle inhibitors. This review in particular focuses on the newerstrategies that may facilitate better therapeutic targeting ofdrugs that inhibit the various components that safeguard thefidelity of the fundamental processes of DNA replication andrepair. Clin Cancer Res; 23(13); 3232–40. �2017 AACR.

IntroductionTissue growth and homeostasis in multicellular organisms

necessitate a variety of systems to regulate and coordinate celldivision. When these systems fail, unabated and uncoordinatedgrowth can lead to cancer. The core cell-cycle machineryrequired for chromosome duplication, chromosome segrega-tion, and cytokinesis can be distinguished from cell-cycle reg-ulators that restrain the core engine via cell-cycle checkpoints.Many traditional cancer therapies act on the core machinery,either at S-phase, preventing nucleotide synthesis and DNAreplication (antimetabolites, DNA-damaging agents, and radio-therapy) or at mitosis (microtubule inhibitors). As they act onprocesses essential for all dividing cells, these classical therapiesalso affect normal proliferating tissues, and toxicities seemedinevitable for cell-cycle inhibitors. Hence, in the 1990s, cancerdrug discovery shifted focus from cell-cycle targets to mitogenicand oncogenic signaling pathways. Targeting somatic mutationsin genes encoding members of these signaling pathways offereda better therapeutic window in the cancers where they were

defective. This shift in focus was vindicated by success ofoncogene-targeting drugs such as trastuzumab, imatinib, erlo-tinib, vemurafenib, and crizotinib (1).

During this same period, next-generation sequencing usheredin efforts to comprehensively catalog all genomic aberrations thatcan drive cancer growth (2). Interestingly, these studies identifiedkey cell-cycle checkpoint genes that are commonly mutated incancer. In particular, checkpoints at three stages are targeted bymutations in established cancer genes: (i) G1 phase checkpoints,including the restrictionpoint; (ii) S-phase checkpoints, includingDNA damage and origin firing checkpoints; and (iii) M-phase,particularly the spindle assembly checkpoint.

Recent years have seen the introduction of inhibitors thatspecifically target kinases that facilitate DNA replication andrepair such as inhibitors of ataxia telangiectasia mutated kinase(ATM), ataxia telangiectasia and Rad3-related kinase (ATR),WEE1, checkpoint kinase 1 (CHK1), and checkpoint kinase 2(CHK2), collectively referred to herein as checkpoint kinaseinhibitors. Because their mechanisms of action inhibit crucialcomponents of DNA damage checkpoints, the initial interest inthis class of inhibitors was their use in combinationwith standardcytotoxic (genotoxic) therapies as chemopotentiators. However,preclinical investigations led to a next-generation strategy thatrelies on the identification of underlying genetic or expressionalterations that can potentially enhance the susceptibly of cancercells to checkpoint kinase inhibitor monotherapy or to combi-nation therapy with targeted drugs that do not induce generalizedDNA damage. Consequently, this article reviews the underlyinggenetic drivers in tumors that facilitate better selective targeting ofthese agents, with a focus on checkpoint kinase inhibitors that arecurrently in development (Table 1).

1Early Phase Medical-Oncology, Eli Lilly and Company, Lilly Corporate Center,Indianapolis, Indiana. 2Oncology Business Unit-Patient Tailoring, Eli Lilly andCompany, Lilly Corporate Center, Indianapolis, Indiana. 3Oncology TranslationalResearch, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana.

Corresponding Author: Richard P. Beckmann, Eli Lilly and Company, LillyCorporate Center, Drop Code 0438, Indianapolis, IN 46285. Phone: 317-276-4285; Fax: 317-651-6346; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-0083

�2017 American Association for Cancer Research.

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ATM, ATR, WEE1, CHK1, and CHK2 are crucial components ofDNA damage response (DDR) signaling networks responsible forcontributing either to the detection and repair of damage or forcoordinating DNA replication (3–6). These DDR checkpointkinases function collectively to maintain genomic integrity byproviding cells time to repair any DNA damage prior to replica-tionormitosis, and to initiate an apoptotic response if thedamageis beyond repair. ATR and ATM act as sensors of single-strand anddouble-strand breaks (SSB and DSB), respectively, which activateCHK1 and CHK2 by phosphorylation (Fig. 1). CHK1 and CHK2suppress cell-cycle progression through downregulation of theCDC25A and CDC25C phosphatases, which promote progres-sion by removing inhibitory phosphates on CDK2 and CDK1,

respectively (7). WEE1 also prevents cell-cycle progression viainhibitory phosphorylation of CDK1 and CDK2 (8–11). TheATR–CHK1 response is also initiated in response to ssDNAgenerated by replication fork stalling (12, 13).

Strategies for Clinical Development: Pastand Present

The initial preclinical studies evaluating these inhibitors clearlydemonstrated that the chemical inhibition of checkpoint kinasesenhances the activity of cytotoxic therapies that damage DNAthrough diverse mechanisms. Good reviews have been publishedrecently describing this biology, and, therefore, it will not be

Table 1. Cell-cycle kinase inhibitors currently in development

Target Agent Phase Context Populations being evaluated in all clinical contexts

WEE1 AZD1775, MK1775 II MonotherapyChemotherapy combinations: cisplatin, carboplatin,docetaxel, gemcitabine, irinotecan, nab-paclitaxel,paclitaxel, peglyated liposomal doxorubicin,pemetrexedTargeted therapy combinations: olaparib, belinostatRadiation/chemoradiation combinations:monotherapy, cisplatin, gemcitabine, temozolomideImmunotherapy combinations: durvalumab

Cervical, CRC, gastric/GEJ, glioblastoma, gliomas, HNSCC,NSCLC, myeloid malignancies, ovarian, pancreatic,pediatric tumors, SCLC, squamous NSCLC, TNBCBiomarker focused:Basket trial: molecular profiling–based assignmentBladder: mutations in genes associated with cell-cycleregulation (RB1, CDKN2A, CCNE1 or MYC amp)CRC: KRAS/NRAS/BRAF mutationsGastric/GEJ: TP53 mutationsNSCLC: biomarker-directed basket trialOvarian: BRCA1 mutations, BRCA2 mutations, TP53 muta-tions, window trialPediatric: basket trial stratifying based on molecularanomalies

CHK1 Prexasertib,LY2606368

II MonotherapyChemotherapy combinations: cisplatin, 5-FU,pemetrexedTargeted therapy combinations: cetuximab,LY3023414 (PI3K/mTOR inhibitor), ralimetinib(p38 MAPK inhibitor), olaparibChemoradiation combinations: cetuximab, cisplatin

AML/high-risk MDS, CRC, HSNCC, NSCLC, ovarian,pediatric, prostate, SCC of anus, squamous NSCLC,SCLC, TNBCBiomarker focused:Basket trial: replication stress or homologous repairdeficiency (e.g., MYC or CCNE1 amp, Rb loss, FBXW7mutation, BRCA1, BRCA2, PALB2, RAD51C, RAD51D,ATR, ATM, CHK2, the Fanconi anemia pathway genes)Breast: BRCA1 mutations, BRCA2 mutationsCRC: KRAS/BRAF mutationsNSCLC: KRAS/BRAF mutationsOvarian: BRCA1 mutations, BRCA2 mutations

LY2880070, ESP-01 I MonotherapyChemotherapy combinations: gemcitabine

CRC, ovarian, TNBC

CCT245737, PNT-737 I MonotherapyChemotherapy combinations: gemcitabine, cisplatin

NSCLC, pancreatic

GDC-0575, ARRY-575 I MonotherapyChemotherapy combinations: gemcitabine

ATR VX970 II MonotherapyChemotherapy combinations: cisplatin, carboplatin,gemcitabine, etoposide, irinotecan, topotecanTargeted combinations: veliparibChemoradiation combinations: monotherapy, cisplatin

HNSCC (HPV negative), NSCLC, ovarian, SCLC, urothelial,squamous NSCLC, TNBC

AZD6738 I MonotherapyChemotherapy combinations: carboplatin, paclitaxelTargeted combinations: olaparibRadiationImmunotherapy combinations: durvalumab

B-cell malignancies, gastric/GEJ, HNSCC, NSCLCBiomarker focused:CLL: loss of chromosome 11q or ATM deficientGastric/GEJ: low ATM expressionHNSCC: window trialNSCLC: low ATM expression

VX803 I MonotherapyChemotherapy combinations: gemcitabine, carboplatin,cisplatin

Ovarian

ATM AZD0156 I MonotherapyChemotherapy combinations: irinotecan, olaparib

NOTE: Source: www.clinicaltrials.gov.Abbreviations: AML, acute myeloid leukemia; amp, amplification; CLL, chronic lymphocytic leukemia; CRC, colorectal cancer; GEJ, gastroesophageal junction; HPV,humanpapillomavirus; HNSCC, squamous cell head and neck cancer; MDS,myelodysplastic syndrome;NSCLC, non–small cell lung cancer; SCC, squamous cell cancer;SCLC, small-cell lung cancer; TNBC, triple-negative breast cancer.

Selective Targeting of Checkpoint Kinase Inhibitors

www.aacrjournals.org Clin Cancer Res; 23(13) July 1, 2017 3233



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summarized here (7, 10, 12–16). As a result of these data, clinicaltrials with early CHK1 inhibitors focused on the chemopotentia-tion of cytotoxic drugs. Although phase I trials demonstratedproof of concept that CHK1 inhibitors could be safely combinedwith chemotherapy (17–25), phase II studies failed to meet theirprimary endpoints (26, 27). Early CHK1 inhibitors were notsuccessful for a variety of reasons, including pharmacokineticproperties, unacceptable toxicities, and business considerations.

However, newer inhibitors, which aremore diverse and includenot only CHK1 inhibitors but also WEE1, ATR, and ATM inhibi-tors, continue to test this hypothesis with a variety of genotoxicagents and as monotherapies (Table 1). Although no phase IIstudies have been published with checkpoint inhibitors asmono-therapy, evidence of efficacy has been observed in phase I. A phaseI evaluation of AZD1775 as a monotherapy enrolled 24 patients,nine ofwhomhadBRCA1orBRCA2mutations. Twopatientswitha BRCA1mutation [squamous cell cancer (SCC) of the base of the

tongue and papillary serous ovarian cancer] had partial responses(28). Prexasertib, a CHK1 inhibitor, demonstrated objectiveresponses in patients with SCC of the anus and head and neckcancer in a phase I study with multiple expansion cohorts (29).These preliminary results suggest that the newer inhibitorsmay bemore successful than initial CHK1 inhibitors, which were onlydeveloped as chemopotentiators.

In addition, these data suggest the optimal use of these inhi-bitors may require the identification of contexts where tumor-specific vulnerabilities are leveraged to achieve selective cytotox-icity in cancer cells. p53 is a critical mediator of the DDR, which isactivated through phosphorylation by CHK2 and ATM, andparticipates in aparallel pathway toATR,CHK1, andWEE1 (Fig. 1;ref. 7). For this reason, cancer cells that have lost either p53function or ATM may show greater reliance on ATR, CHK1, andWEE1 for efficient repair and, hence, greater sensitivity to treat-ments that inhibit these kinases (30–35). This hypothesis has

© 2017 American Association for Cancer Research

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ATR, ATM, CHK1, CHK2, and WEE1 inhibit cell-cycle progression into S-phase and mitosis following DNA damage. This regulation occurs ultimately through thecontrol of the cyclin-dependent kinases (CDK) that facilitate entry into the S- and M-phases of the cell cycle, namely CDK2 and CDK1, respectively. Theactivity of CDK1 and CDK2 are controlled by phosphorylation as well as by the partnership of these kinases with their regulatory cyclin subunits,whereby activation of CDK2 requires association with cyclin E and CDK1 activation requires association with either cyclin A or B. Phosphorylation of eitherCDK1 or CDK2 is regulated in part by the opposing actions of the WEE1 kinase and the CDC25A/CDC25C phosphatases. ATR and ATM are sensors ofSSBs and DSBs, respectively, which activate CHK1 and CHK2, respectively, by phosphorylation. The inhibition of cell-cycle progression by activated(phosphorylated) CHK1 and CHK2 results from the inhibition of CDK1 and CDK2, which is mediated by preventing the removal of inhibitory phosphates placedon CDK1 and CDK2, by WEE1. CHK1 and CHK2 prevent the removal of these phosphates by suppressing the CDC25A and CDC25C phosphatasesthrough phosphorylation, whereby this phosphorylation facilitates proteolytic degradation of CDC25A and the sequestration of CDC25C by 14-3-3. p53 isactivated upon DNA damage by phosphorylation by ATM and CHK2 and serves as an additional pathway to inhibit cell-cycle progression bypromoting transcription of p21, which in turn inhibits the kinase activity of the cyclinE–CDK2 complex.

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Clin Cancer Res; 23(13) July 1, 2017 Clinical Cancer Research3234




been tested in a phase II trial where patients with TP53-mutatedovarian cancer who were refractory or resistant (<3 months) tofirst-line therapy received AZD1775 in combination with carbo-platin. The combination demonstrated manageable toxicity, andin 21 evaluable patients, the objective response rate was 43%.Median progression-free and overall survival times were 5.3months and 12.6 months, respectively. In addition to TP53mutations, patients with an objective response had alterationsin BRCA1, KRAS, MYC, or CCNE (cyclin E; ref. 36). Similarly, in arandomized, phase II trial in platinum-sensitive, TP53-mutantovarian cancer, the WEE1 inhibitor AZD1775 combined withpaclitaxel and carboplatin met the primary and secondary end-points and significantly prolonged progression-free survival com-pared with the combination of paclitaxel and carboplatin (37). Ina phase I, multi-arm combination study assessing AZD1775 witheither gemcitabine, cisplatin, or carboplatin, 176 patients wereevaluable for response. Responses were observed in patients withovarian cancer (n ¼ 7), melanoma (n¼ 3), breast cancer (n¼ 2),head and neck cancer (n¼ 3), colorectal cancer (n¼ 1), and SCCof the skin (n¼ 1). Patientswith TP53mutations (4/19, 23%)hada partial response compared with 4/33 (12%) of patients withTP53 wild-type tumors (38).

Ongoing studies with AZD1775 continue to evaluate thishypothesis in TP53-mutated gastric cancer (with paclitaxel,NCT02448329), TP53- or KRAS-mutated solid tumors (witholaparib, NCT02576444), and TP53-mutated (with either MYCamplification or CDKN2A mutation) relapsed small-cell lungcancer (SCLC; monotherapy, NCT02688907). Similarly, theATR inhibitor AZD6738 has an ongoing clinical study(NCT02264678) in ATM-low/deficient NSCLC (with carbopla-tin) or gastric cancer (with olaparib). These studies will furthercharacterize the impact of TP53 mutations and ATM deficiencynot only in the context of cytotoxic chemotherapy but also withtargeted agents and monotherapy.

The emerging clinical data with TP53mutations are one exam-ple of potential synthetic lethality (SL), where functional altera-tions in one pathway lead to enhanced sensitivity to inhibition ofanother pathway. This is the strategy guiding the use of PARPinhibitors as therapies for BRCA1- or BRCA2-deficient breast orovarian cancers (39–42). Leveraging SL for checkpoint kinaseinhibitors requires knowledge about functional alterations incancer cells that make them more vulnerable to the inhibitionof these kinases than normal cells. Although TP53 mutations arethe most well-characterized example of SL with the checkpointinhibitors, the concept of replication stress (RS) is emerging as apotential SL mechanism (43, 44). RS can arise in any situationthat leads to inappropriate replication origin licensing or firing(45). This results in stalled replication forks and DNA breaks ifthe stalled forks are not adequately resolved (46–48). In cancercells, oncogene-driven events can contribute to higher degrees ofRS by promoting growth to a point that strains the replicativecapacity of the cell (45, 49). As a result, inhibitors of checkpointkinases may induce SL with tumors that have high RS, as all ofthese kinases contribute to sensing and reducing RS (Fig. 2). Inparticular, WEE1 and CHK1 play complementary roles in restrict-ing the initiation of replication origins by inhibiting CDK2,whichwhen activated, promotes replication (6, 50, 51). Thus, treatmentwith an inhibitor of CHK1 or WEE1 augments ongoing RS byeffectively neutralizing one of the mechanisms available forsuppressing replication origin firing, resulting in more stallingand DNA breaks. As both CHK1 and WEE1 are important

signaling components in the response to DNA damage, damagethat results from the additional RS is potentially exacerbatedbecause it cannot be repaired effectively. Likewise, ATR plays animportant role as a sensor for RS as it is recruited to regions ofssDNA that become exposed by replication fork stalling. Subse-quently, ATR is responsible for activating CHK1, which in turnsuppresses replication, as indicated above (15). An ongoingchallenge is to identify tumors that have reached near-criticallevels of RS and are likely the most susceptible to treatment withcheckpoint kinase inhibitors.

Validation of therapeutic strategies to exploit RS has recentlyemerged from several studies (52–63). For example, an SL rela-tionship was described between oncogene-induced RS by MYCactivation and ATR loss in lymphomas (49). Specifically in thecontext of ATR-deficient cells, MYC expression induced higher RSand apoptosis and contributed to greater sensitivity to ATR andCHK1 inhibitors. Analogous observations were derived fromstudies in neuroblastoma models wherein sensitivity to CHK1inhibition was correlated with MYCN expression (52). Studies inother model systems for hematologic malignancies such as MYC-driven diffuse large B-cell lymphoma, cyclin D1–driven mantlecell lymphomas, T-cell acute lymphoblastic leukemia, and acutemyeloid leukemia suggest that oncogene-induced RS in thesecancers maymake them particularly vulnerable to treatment withcheckpoint kinase inhibitors (53–55, 58, 61, 63). The hypothesisthat MYC may drive RS and increase sensitivity to checkpointkinase inhibitors is being tested in several ongoing clinical trials,including a biomarker-directed study with AZD1775 and durva-lumab in muscle-invasive bladder cancer. To be eligible, patientsmust have mutations in CDKN2A or RB1 genes and/or amplifi-cation of CCNE1, MYC, MYCL, or MYCN genes, all of whichpresumably contribute to heightened RS (Fig. 2). Other agentsare focusing on SCLC, a tumor associated with RS and MYCamplifications: AZD1775 (NCT02593019) and prexasertib(NCT02735980) are being assessed as monotherapy, whereasVX970 is being evaluated with topotecan (NCT02487095).

Another opportunity for SL may be inhibition of ATR in thecontext of ATM deficiency. In preclinical models of hematologicmalignancies, cells that lack ATM (e.g., due to 11q deletions) orhave defects in p53 signaling were particularly vulnerable to theATR inhibitor AZD6738. Treatment with AZD6738 increasedreplication initiation and fork stalling, resulting in RS and DNAdamage that likely contributed to selective cytotoxicity in cellswith p53 and/or ATM defects (64). In parallel, in a phase I trialwith the ATR inhibitor VX970 and carboplatin, a patient withcolorectal cancer and ATM loss achieved a complete response(65). This hypothesis is being further tested in a trial of AZD6738in patients with relapsed/refractory B-cell malignancies withprospectively identified 11q-deleted or ATM-deficient relapsed/refractory CLL (NCT01955668).

In addition to neuroblastomas, as mentioned above, otherpediatric cancers, particularly sarcomas, may represent anotheropportunity for exploiting oncogenic drivers of RS, as demon-strated by recent studies in models for Ewing sarcoma (ES)wherein the oncogenic drivers were fusion proteins unique tothis tumor (EWS/FLI1 or EWS/ERG; ref. 59). In these studies, EScell lines were more sensitive to inhibition of ATR compared withsarcoma cell lines that lacked the EWS fusions, and ATR inhibitormonotherapy resulted in near-complete to complete growthinhibition of humanES xenografts. Related studieswith theCHK1inhibitor prexasertib (56) showed that monotherapy resulted in






complete regressions in xenograft models for desmoplastic smallround cell tumor, which also expresses an oncogenic EWS fusionprotein known as EWS–WT1, or alveolar rhabdomyosarcoma, amalignancy driven by another unique oncogenic fusion protein(66, 67). These nonclinical studies with prexasertib have led to aninterest in exploring prexasertib in pediatric solid tumors(NCT02808650). In addition, WEE1 was identified as a target inmedulloblastoma, the most common pediatric malignant braintumor (68). AZD1775 induced DNA damage and suppressed thegrowth of medulloblastoma cells both in vitro and in vivo (68),potentially by exploiting MYC-induced RS (69). AZD1775 hasalso demonstrated in vitro activity in rhabdomyosarcoma asmonotherapy and in combination with conventional therapies(70) and improved the efficacy of radiation in mouse models of

pediatric high-grade glioma (71). Aphase I study of AZD1775 andradiation in children with diffuse intrinsic pontine gliomas isongoing (NCT01922076). AZD1775 is also being assessed in theEuropean Proof-of-Concept Therapeutic Stratification Trial ofMolecular Anomalies in Relapsed or Refractory Tumors(ESMART). In this basket study, molecular profiling of pediatrictumors is used to direct patients to individual treatmentarms, including a combination of AZD1775 and carboplatin(NCT02813135).

Strategies exploiting RS also include combining inhibitorstargeting different checkpoint kinases. Accordingly, RNA inter-ference (RNAi) studies exploring SL with the CHK1 inhibitorPF-00477736 identified WEE1 as the most significant hit (72).In addition, synergistic growth inhibition was observed with

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The ATR–CHK1 pathway reduces RS. The kinase function of CDK2 requires activation by association with cyclin E. When activated by cyclin E, CDK2 facilitatesthe progression of cells from G1 into S-phase and subsequently promotes replication origin firing. ATR and CHK1 suppress replication origin firing in part bysuppressing the activity of CDK2 through regulation of downstream effectors that coordinate the phosphorylation of CDK2. Specifically, the presence of longstretches of single-stranded DNA, which can occur in situations where the DNA polymerases lag behind the unwinding activity of the helicases, trigger ATR, which isrecruited by a complex of proteins including replication protein A (RPA), topoisomerase-binding protein 1 (TopBP1), and ATR-interacting protein (ATRIP).ATR then activates CHK1 (through phosphorylation), which subsequently through phosphorylation of the CDC25A phosphatase, negatively regulates CDK2 bypreventing the removal of inhibitory phosphates by this phosphatase. The actions of CDC25A are also opposed by WEE1, which contributes to the inhibitionof CDK2 by catalyzing the inhibitory phosphates that are removed by CDC25A. As shown at the bottom half of the figure, RS can result from endogenous orexogenous DNA damage as well as through the action of growth-promoting oncogenes, such as MYC, which serve to drive progression into S-phase. Inaddition, RS can also arise through other alterations that disrupt control at the G1–S interface, such as changes that lead to enhanced expression of cyclinE or loss of inhibition of CDK2. Alterations that may enhance cyclin E expression or CDK2 activity possibly include (i) losses in FBXW7, which facilitatesproteasomal degradation of cyclin E or (ii) alterations that disrupt regulation of the primary restriction point controlling progression from G1 into S includingactivation of CDK4, CDK6, and D-type cyclins or loss of RB1 that serves to activate E2F, thereby promoting the transcription of the genes that encode cyclin Eand CDK2 as well as other gene products that promote DNA replication. Notably, as indicated by the red Xs, the inhibition or loss of function of ATR, CHK1,or WEE1 can elevate ongoing RS by effectively removing the control over CDK2. The result of this inhibition or loss is replication fork collapse, with eventualDNA DSBs leading to the activation of DNA damage and repair responses such as homologous recombination repair.

Lin et al.





PF-00477736 and AZD1775 in cell lines and in a xenograftmodel for ovarian cancer (72). Synergy was also observed withAZD1775 and the CHK1 inhibitor MK-8776 (73). Analogousstudies showed that treatment of cancer cells in culture with theCHK1 inhibitor AZD7762 and the ATR inhibitor VE-821 ele-vated RS, leading to replication catastrophe and death byapoptosis. The combination was associated with a significantinhibition of tumor growth and an increase in overall survivalin mice bearing human NCI-H460 NSCLC xenografts whencompared with the monotherapy treatments (74).

In addition to RS, deficiencies in homologous recombinationrepair (HRR) may also contribute to greater sensitivity to check-point kinase inhibition, as homologous recombination deficien-cies (HRD) reduce the efficiency of repair of DSBs that result fromRS generated by these inhibitors (Fig. 2). Deficiencies in theFanconi anemia (FA) genes (FANC), such as defects in BRCA1or BRCA2, also disrupt HRR (75), and studies with the CHK1inhibitor G€o6976 showed that FA-deficient cell lines were highlysensitive to G€o6976 and to RNAi silencing of CHK1 mRNAexpression when compared with isogenic lines that were FAproficient (76). An RNAi-based screen that targeted 230 DNArepair genes identified many FANC genes as SL with G€o6976treatment, including FANCA, FANCC, FANCD1, FANCD2,FANCE, FANCF, and FANCG.

A similar approach using siRNA to target 240 DNA replicationand repair genes explored SL with ATR inhibitors (57). Thesestudies uncovered that loss of expression of ATR pathway geneshad the strongest association for SL including losses of ATR itself,ATRIP, RPA, and CHK1. As the ATR pathway is crucial forresponding to RS, the observations from this study as well asothers provide support regarding the therapeutic benefits thatmay be derived from the combined inhibition of targets withinthis pathway to elevate RS further (62, 72–74). In addition, theresults confirm the benefit that may be attained through mono-therapy by selective targeting of tumors with preexisting muta-tions in this pathway. Of note, these studies demonstrated thatloss of excision repair cross-complementation group 1 protein(ERCC1), which functions in the repair of bulky DNA adducts,DSBs, and interstrand cross-links (77), was a significant SL inter-action with ATR and CHK1 inhibition. Similar SL interactionswere observed between ATR inhibition and loss of another repairgene known as XRCC1, which plays a role base excision repair(78) and with ATM deficiency (34).

These studies suggest that therapeutic strategies aimed at dis-rupting HRR may facilitate SL with checkpoint kinase inhibitors.In this regard, CHK1, in addition to its role in limiting RS, isrequired for HRR by facilitating the essential recruitment ofRAD51 to sites of repair (79). Thus, CHK1 inhibition would beexpected to promote further deficiencies in HRR in addition toexacerbating RS. In support of this concept, augmented growthinhibition by the PARP inhibitor olaparib was observed upon theRNAi silencing of genes encoding CHK1, CHK2, ATR, ATM, andRPA1 as well as genes encoding proteins that contribute to HRRsuch as RAD51, NBS1, FANCA, FANCD2, and FANCC (40). Inaddition, studies in breast carcinoma cell lines showed that thecombination of PARP inhibitors (olaparib, veliparib, NU1025, orrucaparib) with CHK1 inhibitors (UCN-01, AZD7762, orLY2603618) increased DNA damage and cytotoxicity, as com-pared with the single-agent treatments (80). More recent studieshave shown in BRCA1- or BRCA2-mutated high-grade serousovarian cancer (HGSOC) models that combination therapy with

olaparib and either MK-8776 (CHK1) or AZD6738 (ATR) actedsynergistically to decrease survival and colony formation in vitroand inhibit tumor xenograft growth in vivo (81). Clinical trialsevaluating PARP inhibition with AZD1775 (NCT02511795),AZD0156 (NCT02588105), VX970 (NCT02723864), orAZD6738 (NCT02264678) are ongoing. In addition, a studywithAZD1775 and olaparib (OLAPCO) is a molecularly directed trialthat requires mutations in TP53 or KRAS (NCT02576444).

As outlined above, the optimal context for a checkpoint kinaseinhibitor might be tumors that have both high RS and HRD. Ofnotable interest are ovarian cancers, particularly HGSOCs, asapproximately half harbor mutations in genes that modulateHRR, including mutations that impact BRCA1 and BRCA2(39, 82). In addition, nearly all HGSOCs have mutations in TP53(83). Mutations in ATM and ATR have been observed in 2% ofHGSOCs, and 5% have mutations in genes of the FA DNA repairpathway (39). Mutually exclusive with mutations in BRCA1 andBRCA2,CCNE1 is amplified in 15% to 20% of HGSOCs (82, 84).RB1 loss is also observed in about 15% of these cancers (83). Ascyclin E is required for activation of CDK2, its overexpressioninduces RS andDNAdamage that activates HRR andmay increasesensitivity to single-agent CHK1, ATR1, and/or WEE1 inhibition(85). Likewise loss of RB1 can contribute to RS by promoting theprogression of cells into S-phase. Therefore, HGSOCs present aprovocative context for therapy with the checkpoint kinase inhi-bitors by providing an opportunity to leverage the dual presenceof higher RS and HRD (28, 86). Accordingly, AZD1775, prexa-sertib, LY2880070, VX970, VX803, and AZD0156 are beingassessed in a variety of subsets of ovarian cancer (Table 1), andit may be notable that AZD1775 has demonstrated clinicalactivity in both platinum-sensitive and platinum-resistant ovar-ian cancer (36, 37).

As our understanding of SL interactions and the underlyingmechanisms of the checkpoint kinase inhibitors grows, theoptimal context may shift from a focus on a particular histologysuch as ovarian cancer, to the genetic attributes of the tumorregardless of histology. This approach is already being imple-mented in multiple clinical trials. In a prexasertib basket trial(NCT02873975), patients whose tumors show alterations con-sistent with RS (MYC amplification, CCNE1 amplification, Rbloss, or FBXW7 mutation) or HRD (BRCA1, BRCA2, PALB2,RAD51C, RAD51D, ATR, ATM, CHK2, or the FA pathway genes)are eligible. Similarly, AZD1775 is being assessed in the Molec-ular Profiling–Based Targeted Therapy (MPACT) trial, whichassigns patients with solid tumors to a treatment regimenbased on their mutation/amplification category. One of thesearms assesses AZD1775 in combination with carboplatin(NCT01827384). One obvious goal from these types of studies,besides improved efficacy, is to identify tumor-specific biomar-kers that can be used to predict response to monotherapy orcombination therapy with one or more checkpoint kinaseinhibitors. Preclinical studies indicate that alterations inducingRS or HRD contribute to greater sensitivity to these inhibitors.However, the crucial challenge from a clinical perspective isgoing beyond just identifying which of these alterations indi-cate the presence of RS or HRD to identifying situations wheretumors are at near-unstainable levels of RS or HRD and,therefore, are most susceptible to interventions that furtheraugment RS or compromise HRR.

The identification of expression or genetic alterations in a smallsubset of stand-alone biomarkers for predicting response is






certainly desirable and may be achievable in certain cancers, butfor other cancers, additional methodologies may be required toadequately measure the extent to which RS and HRD are nearcatastrophic. However, these challenges should not dampen ourenthusiasm for the ongoing clinical trials with the various check-point kinase inhibitors, as someof these trialsmayprovidepatientdata that will allow us to validate and refine our biomarkerhypotheses. The preclinical studies have provided a solid foun-dation leading to the concept of leveraging RS andHRD to achieveSL in human tumors. The clinical datawill allowus to return to thebench to explore additional concepts that will further enhancethe therapeutic benefit of the checkpoint kinase inhibitors.

Disclosure of Potential Conflicts of InterestA.B. Lin, S.C. McNeely, and R.P. Beckmann hold ownership interest in

Eli Lilly and Company. No other potential conflicts of interest weredisclosed.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received October 17, 2016; revised November 29, 2016; accepted March 15,2017; published OnlineFirst March 22, 2017.

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