VX-803 | Epoxide Hydrolase

Discovery of ATR kinase inhibitor berzosertib (VX-970, M6620): Clinical candidate for cancer therapy
Lukas Gorecki a, Martin Andrs a,b, Martina Rezacova c, Jan Korabecny a,⁎
a Biomedical Research Center, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech Republic
b Laboratory of Cancer Cell Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 142 20 Prague, Czech Republic
c Department of Medical Biochemistry, Faculty of Medicine in Hradec Kralove, Charles University, Simkova 870, 500 38 Hradec Kralove, Czech Republic

a r t i c l e i n f o

Available online xxxx

Keywords:
ATR kinase inhibitor Berzosertib
VX-970 M6620
DNA damage response replication stress cancer
drug development
a b s t r a c t

Chemoresistance, radioresistance, and the challenge of achieving complete resection are major driving forces in the search for more robust and targeted anticancer therapies. Targeting the DNA damage response has recently attracted research interest, as these processes are enhanced in tumour cells. The major repli- cation stress responder is ATM and Rad3-related (ATR) kinase, which is attracting attention worldwide with four drug candidates currently in phase I/II clinical trials. This review addresses a potent and selective small-molecule ATR inhibitor, which is known as VX-970 (also known as berzosertib or M6620), and sum- marizes the existing preclinical data to provide deep insight regarding its real potential. We also outline the transition from preclinical to clinical studies, as well as its relationships with other clinical candidates (AZD6738, VX-803 [M4344], and BAY1895344). The results suggest that VX-970 is indeed a promising anticancer drug that can be used both as monotherapy and in combination with either chemotherapy or radiotherapy strategies. Based on patient anamnesis and biomarker identification, VX-970 could become a valuable tool for oncologists in the fight against cancer.

Contents

⦁ Introduction 0
⦁ VE-821 and VX-970 in conventional cancer treatment 0
⦁ Simultaneous targeting of ATR-CHK1 downstream pathways 0
⦁ Synergism of VE-821 and VX-970 with other DNA damage-inducing agents 0
⦁ Synthetic lethal interactions and the potentiation of increased replication stress 0
⦁ Transition to clinical trials 0
⦁ VX-970 in clinical trials 0
⦁ Interconnectivity with AZD6738, BAY1895433, and VX-803 0
⦁ Conclusion 0
Funding 0

Abbreviations: ALT, alternative lengthening of telomeres; AML, acute myeloid leukemia; ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3-related; ATRIP, ATR interacting pro- tein; BER, base excision repair; BRCA, breast cancer susceptibility protein/gene; CED, convection-enhanced delivery; CHK1, checkpoint kinase 1; CQ, chloroquine; DDK, DBF4-dependent kinase; DSB, DNA double-strand break; DDR, DNA damage responder; DNA-PKcs, DNA dependent protein kinase catalytic subunit; dNTP, deoxyribonucleotide triphosphate; ETAA1, Ewing’s tumor-associated antigen 1; HR, homologous recombination; IR/IGF-1R, insulin/insulin-like growth factor-1 receptor; LET, linear energy transfer; MCM, minichromosome main- tenance complex; MMEJ, microhomology-mediated end joining; NHEJ, non-homologous end joining; NP, nanoparticle; PARP, poly(ADP-ribose) polymerase; PDAC, pancreatic ductal ad- enocarcinoma; PTEN, phosphate and tensin homolog; RFC2–5, replication factor C subunits 2–5; RHINO, RAD9-RAD1-HUS1-interacting nuclear orphan; ROS, reactive oxygen species; RPA, replication protein A; RS, replication stress; shRNA, short hairpin RNA; SCLC, small cell lung cancer; SSB, DNA single-strand break; ssDNA, single-stranded DNA; TNBC, triple negative breast cancer; Top, topoisomerase; TOPBP1, topoisomerase II binding protein 1; WEE1, Wee1-like protein kinase; XRCC, X-ray repair cross-complemented protein/gene.
⁎ Corresponding author.
E-mail address: [email protected] (J. Korabecny).

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Acknowledgment 0
References 0

⦁ Introduction

Similar to the Three Musketeers, three kinases from the phos- phatidylinositol 3-kinase-related kinase family act as DNA damage re- sponders (DDRs) during the cell cycle: ATM (ataxia-telangiectasia mutated), ATR (ATM and Rad3-related), and DNA-PKcs (DNA-depen- dent protein kinase catalytic subunit) (Blackford & Jackson, 2017; Maréchal & Zou, 2013). The ATM and DNA-PKcs are preferably activated in response to double-strand breaks (DSBs) in DNA, while ATR is gener- ally activated for single strand breaks (SSBs) or in response to general replication stress (RS). During this process, ATR stabilizes the stalled replisome, regulates origin firing, and activates cell cycle checkpoints during the S/G2-phase (Foote, Lau, & Nissink, 2015). These “defenders” orchestrate a complex network of mediator and effector proteins to pro- tect human genome integrity against exogenous and endogenous insults, including ionizing or ultraviolet radiation, reactive oxygen spe- cies, or chemotherapeutic agents (e.g., topoisomerase inhibitors, anti- metabolites, and DNA crosslinkers) (Furgason & Bahassi, 2013; Mei, Zhang, He, & Zhang, 2019). In precancerous and cancer cells, DDRs ap- pear to be even more important because various oncogenes and tumour suppressors cause disruption of cell-cycle regulation, induce RS, cause defective DNA repair, and accelerate mutagenesis (Karnitz & Zou, 2015; Luis I. Toledo, Murga, & Fernandez-Capetillo, 2011).
More than one-half of human cancer cells have a mutation affecting either ATM or its downstream protein (p53), which makes it one of the most frequently mutated tumour suppressors (Gurpinar & Vousden, 2015; Jin & Oh, 2019). These cells exhibit accelerated mutagenesis that is associated with increased RS and resistance to conventional treat- ment. However, this also makes the cells substantially reliant on ATR and DNA-PKcs (Kantidze, Velichko, Luzhin, Petrova, & Razin, 2018; Reaper et al., 2011; Weber & Ryan, 2015). Furthermore, relative to ATM, mutations in ATR or DNA-PKcs are rare, which make them suitable targets for novel anticancer agents. Moreover, there is increasing inter- est in both types of inhibitors, as several drugs have reached clinical
trials (Kantidze et al., 2018; Mohiuddin & Kang, 2019). Although
N1,000 compounds have been evaluated as potential ATR inhibitors (ATRis), only a few have exhibited “drug-like” properties that merited further in vitro and in vivo studies (Andrs et al., 2016; Lecona &
Fernandez-Capetillo, 2018). The four ATRis currently in clinical studies are VX-970 (also known as VE-822, M6620, or berzosertib), VX-803 (M4344), BAY1895344, and AZD6738 (ceralasertib) (Fig. 1). Merck is currently evaluating VX-970 and VX-803, Bayer is currently evaluating BAY1895344, and AstraZeneca is currently evaluating AZD6738, with AZD6738 and VX-970 having undergone the most development. The structure of VX-970 is based on that of VE-821, which was identified by Vertex Pharmaceuticals during high-throughput screening for a po- tent ATRi (Charrier et al., 2011). Although VE-821 was effective at inhibiting ATR, it lacked the “drug-like” properties needed to advance into clinical trials. Ongoing research evaluated the biological activity of VX-970 (Fokas et al., 2012), with a more recent report describing its ra- tional design (Knegtel et al., 2019).
The ATR protein is activated in response to DNA replication pertur- bation or DNA damage involving the presence of single-stranded DNA (ssDNA). The presence of ssDNA can be caused by both endogenous events (e.g., transcription-replication interference or reactive oxygen species [ROS]) and exogenous agents (e.g., chemotherapy, ultraviolet radiation, and ionizing radiation). When ssDNA occurs, it is immediately coated by replication protein A (RPA), which directly binds to ATR interacting protein (ATRIP) and forms a complex with ATR. The still- inactive ATR-ATRIP complex is then activated through the ATR activat- ing domain and undergoes a conformational change (Mordes, Glick, Zhao, & Cortez, 2008; Saldivar, Cortez, & Cimprich, 2017). One possible pathway for activation is through Ewing’s tumour-associated antigen 1 (ETAA1), which is bound to RPA via a direct interaction, although its importance in ATR activation remains unclear (Bass et al., 2016; Feng et al., 2016; Haahr et al., 2016). A second alternate route for ATR activa- tion involves topoisomerase II binding protein 1 (TOPBP1), as mutations in its ATR activating domain are lethal in mice, unlike the non-lethal

Fig. 1. Structures of VE-821 and clinical candidates in phases I/II: VX-970, VX-803, AZD6738, BAY1895344, their enzymatic inhibitory constants and cellular median inhibitory concentration towards ATR. Data taken from (Charrier et al., 2011) for VE-821; from (Knegtel et al., 2019) for VX-970; from (Zenke et al., 2019) for VX-803; from (Foote et al., 2018) for AZD6738; and from (Luecking et al., 2017) for BAY1895344.

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mutations in the ATR activating domain of ETAA1 (Saldivar et al., 2017; Thada & Cortez, 2019; Zhou et al., 2013). The TOPBP1-ATR activation pathway is based on the presence of a single-strand/double-stranded DNA junction that serves as the loading point for the RAD9-RAD1- HUS1 (9–1-1) clamp complex, which is loaded onto DNA by RAD17- replication factor C subunits 2–5. The TOPBP1 protein is then recruited and activated through the MRN complex (MRE11-RAD50-NBS) and RHINO (RAD9-RAD1-HUS1-interacting nuclear orphan) (Fig. 2) (Delacroix, Wagner, Kobayashi, Yamamoto, & Karnitz, 2007; Joon Lee, Kumagai, & Dunphy, 2007).
Activated ATR phosphorylates a multitude of downstream media- tors and effectors (Fig. 3). The major regulatory pathway proceeds via phosphorylation of checkpoint kinase 1 (CHK1) through the mediator protein claspin (Qiu, Oleinick, & Zhang, 2018). The phosphorylation of CDC25A by CHK1 leads to proteasomal degradation that attenuates CDK2 activity, thus slowing down the S-phase (Petermann, Woodcock, & Helleday, 2010; Sørensen et al., 2003). Furthermore, the S-phase slowdown is related to CHK1-mediated phosphorylation of Treslin (also known as TOPBP1-interacting checkpoint and replication regula- tor [TICRR]), which negatively regulates the initiation of DNA replication (Guo et al., 2015). Replicon initiation is also abrogated by ATR-CHK1- mediated phosphorylation of DBF4-dependent kinase (DDK) (Heffernan et al., 2007).
In contrast, phosphorylation of CDC25B or CDC25C and their subse- quent degradation abrogates the activation of CDK1 and causes G2/M arrest (Peng et al., 1997). Similarly, CDK1 inactivation is also elicited by the activation of Wee1-like protein kinase (WEE1) after it is phos- phorylated by CHK1 (J. Lee, Kumagai, & Dunphy, 2001). The CHK1 pro- tein is exclusively phosphorylated at Ser345 by ATR, and serves as a potential biomarker for ATRi efficacy (Jazayeri et al., 2006; Reaper et al., 2011). Phosphorylation at Ser139 of histone protein H2AX may also serve as a marker for ATR activity, albeit only under experimental conditions because ATM and DNA-PKcs can also phosphorylate H2AX (Stiff et al., 2004; Ward & Chen, 2001). Phosphorylation of H2AX (γH2AX) serves as a more reliable biomarker of RS and is the standard marker for quantifying DNA damage, as it also accounts for DSBs (Buisson, Boisvert, Benes, & Zou, 2015; Luis I. Toledo et al., 2011).

During RS, ATR ensures genomic stability by preserving the fork’s in- tegrity after stalling (Tercero & Difﬂey, 2001), as the stalled fork con- tains two vulnerable ssDNAs that could lead to genomic instability. The ATR protein facilitates remodelling of the replication fork via the translocases SMARCAL1 and/or ZRANB3, which creates a four-way structure known as a reversed fork or “chicken foot”. This structure pre- vents formation of DSBs resulting from the breaking of ssDNA. Thus, ATR helps stabilizes the replication fork until the cause of the stalled fork (e.g., DNA nicks or crosslinking) can be resolved (Neelsen & Lopes, 2015). In addition, ATR coordinates the restarting of a reversed fork via Bloom and Werner helicases (Couch et al., 2013; Davies, North, & Hickson, 2007; Pichierri, Rosselli, & Franchitto, 2003). The ATR protein can also help repair a collapsed fork by promoting the recruitment of homologous recombination factors (HRs, such as PALB2 or FANCD2). While PALB2 is recruited through ATR-mediated phosphorylation of RPA, FANCD2 is directly phosphorylated by ATR (Lossaint et al., 2013; Murphy et al., 2014). Noteworthy, FANCD2 can accurately inter- act with minichromosome maintenance complex (MCM) (Lossaint et al., 2013).
Apart from stalled forks, ATR also regulates the initiation of origin firing at dormant origins through MCM or Fanconi anaemia group I (FANCI) (Y.-H. Chen et al., 2015; Cortez, Glick, & Elledge, 2004; Ibarra, Schwob, & Méndez, 2008). In contrast, ATR inhibition leads to massive deregulation of origin firing, which results in global depletion of RPA, DNA breakage, and ultimately replication catastrophe (L. Toledo, Neelsen, & Lukas, 2017; Luis Ignacio Toledo et al., 2013).
Finally, genome integrity is also preserved via two mechanisms that secure a sufficient stock of deoxyribonucleotide triphosphate (dNTP). First, ATR increases RRM2 transcription and downregulates its cyclin F-mediated degradation, thus stimulating de novo dNTP biosynthesis via ribonucleotide reductase (RNR), the rate-limiting enzyme (Buisson et al., 2015; D’Angiolella et al., 2012). Second, ATR controls the basal activity of the key nucleotide salvage enzyme deoxycytidine kinase (dCK), as well as its response to RS, which also ensures that there is a sufficient pool of dNTP (Beyaert, Starczewska, Van Den Neste, & Bontemps, 2016; D’Angiolella et al., 2012; Le et al., 2017; Poczta, Rogalska, Łukawska, & Marczak, 2019). Fig. 3 shows a simplified version

Fig. 2. Activation of the ATR-CHK1 pathway. At a stalled replication fork or after DNA end resection during HR, ssDNA is detected and coated by RPA. ATRIP is directly bound to RPA calling ATR up to this site. The ATR-ATRIP complex could be directly stimulated through ETAA1 bound to RPA or through TOPBP1 activation. Initially, 9–1-1 clamp is loaded on RAD17-RFC2–5, and this complex recruits MRN and RHINO together with TOPBP1 which can thereafter activate ATR. Finally, ATR phosphorylates CHK1 through claspin adaptor and initiates the downstream pathways. MCM – minichromosome maintenance complex – DNA helicase; DNA pol – DNA polymerase. Figure was modified from several review articles (Blackford & Jackson, 2017; Foote et al., 2015; Forment & O’Connor, 2018; Karnitz & Zou, 2015; Lecona & Fernandez-Capetillo, 2018; Saldivar et al., 2017; Yazinski & Zou, 2016).

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Fig. 3. ATR-CHK1 downstream after replication stress: Checkpoint management through CDC25 phosphatases (B or C) together with WEE1 results to attenuation of CDK1 activity and thus promotion of G2-M arrest, or through CDC25A abrogation of CDK2 activity and slowing down of S-phase; Regulation of origin firing is mediated through MCM or FANCI by ATR or through Treslin and DDK by CHK1. Responses to fork instability is mediated via fork stabilization and repaired by activation of helicases (SMARCAL1; BLM; WRN) and HR factors (PALB2; FANCD2). Preservation of sufficient dNTP pools is managed through their biosynthesis (induction of RRM2 transcription) or salvage (dCK stimulation).

of ATR’s activation of downstream effectors, and a detailed description of their pathways can be found elsewhere (Forment & O’Connor, 2018; Lecona & Fernandez-Capetillo, 2018; Saldivar et al., 2017). Never- theless, the specific ATR-related mechanisms are a fundamental concept in the search for potent synthetic lethal interactions, and several of these mechanisms are described below.
Exploiting synthetic lethal interactions has attracted considerable attention as an anticancer strategy, as it enables a selective targeting of defective cancer cells while sparing healthy cells (Jackson & Chen, 2016; O’Neil, Bailey, & Hieter, 2017). However, this approach requires identification and characterisation of precise synthetic lethal interac- tions, as well as the development of screening tools and biomarkers that can identify suitable patients. Compounds affecting DNA repair have prominent status in this new anticancer strategy, with one poly (ADP-ribose) polymerase inhibitor (PARPi, olaparib) already approved for the treatment of breast cancer susceptibility gene 2 (BRCA2)- deficient breast and ovarian cancers (Lord & Ashworth, 2017; Robson et al., 2017). Another example of synthetic lethality would be the simul- taneous inactivation of co-acting ATM and ATR (Fig. 4). In this scenario, loss of ATM and downstream p53 would allow a cell with damaged DNA to proceed through the G1 checkpoint, resulting in increased reliance on intra-S and G2/M checkpoints for cell survival (Kwok et al., 2015; Nghiem, Park, Kim, Vaziri, & Schreiber, 2001; Luis I. Toledo, Murga, Zur, et al., 2011). Furthermore, combined inactivation of ATR and ATM/p53 would lead to cell death through replication or mitotic catas- trophes (Denisenko, Sorokina, Gogvadze, & Zhivotovsky, 2016; L. Toledo et al., 2017). Thus, ATR inhibition has great potential because it inter- venes in various DNA damage response pathways.
This review focuses on our knowledge of ATR kinase activity in anticancer treatment, which has led to the evaluation of VX-970 in numerous preclinical and clinical studies. Although VX-970 has replaced VE-821 (its predecessor), VE-821 also still serves as a valid template for designing new ATRis and as a good standard for in vitro biological activity assays and cell cultures (Middleton, Pollard, & Curtin, 2018). The following chapters describe the pre- clinical and limited clinical studies that have evaluated VE-821 and VX-970, which have been investigated as monotherapies or in combination with chemotherapy or radiotherapy strategies. Fi- nally, we evaluate the current status of other ATRis (AZD6738, BAY1895344, and VX-803) and brieﬂy discuss their differences in relation to VX-970.

⦁ VE-821 and VX-970 in conventional cancer treatment

⦁ Combination with cisplatin, oxaliplatin, or carboplatin

The main mechanism for the antitumor effects of cisplatin and its derivatives involves the interaction with DNA and the covalent link between cisplatin and purine bases (mainly guanine). This causes intra-strand and inter-strand crosslinks, leading to cytotoxicity that mainly occurs in the S-phase. Cisplatin-induced DNA damage triggers DNA repair pathways (mostly through the nucleotide excision and mis- match repair systems), cell cycle arrest, and apoptosis. Activation of ATR is one of the earliest events in the response to cisplatin and its deriva- tives (Galluzzi et al., 2012).

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Fig. 4. Schematic representation of synthetic lethal interaction. ATR inhibition in a normal cell will not significantly affect DNA repair as it could be compensated through activation of ATM. In cancer cells, spontaneous mutation of ATM results in reliance on ATR DDR; however, if ATR is also inhibited, unrepaired DNA promotes cell death.

Preliminary studies evaluated the effects of VE-821 plus cisplatin in 14 cancer cell lines and 6 normal cell lines (Reaper et al., 2011). The results revealed that p53-deficient cancer cell lines were particularly sensitive to VE-821 plus cisplatin, while the cell lines with normal p53 (H460, A549, MCF7, and RKO) exhibited the weakest response. Further- more, a p53-mutant cancer cell line (H23) and normal cells (HFL1) were compared, which revealed a distinct synergistic effect in H23 cells, even at a low concentrations of cisplatin, which was not observed in the nor- mal HFL1 cells. The dependence of VE-821 sensitivity on ATM activity was also confirmed in ATM-null cells (AT1BR) and cells expressing ATM (161BR). In the absence of ATR activity, ATM phosphorylation was clearly escalated as a compensatory response after the combination treatment, relative to cisplatin treatment alone. This finding proved that ATM and ATR may help support each other in the DDR, which suggest a potentially direct synthetic lethal interaction between the ATM-p53 ab- rogated pathway and ATR (Reaper et al., 2011). Similarly, VE-821 inhi- bition of ATR was increased after cisplatin treatment of SKOV3, PEO1, and OVCAR ovarian cancer cells, while this sensitization was not ob- served after treatment using MK-8776 (a CHK1 inhibitor) (Huntoon et al., 2013). This may be because cisplatin-induced DNA damage in the form of DSBs could be repaired via HR (Huehls, Wagner, Huntoon, & Karnitz, 2012; W. Wang, 2007). These findings indicate that CHK1- independent events are regulated by ATR during HR.
In 2012, one year after the synergic effect of VE-821 and cisplatin
was reported, VX-970 was introduced by Vertex Pharmaceuticals Inc. (Fokas et al., 2012). Subsequent in vivo proof-of-concept experiments were performed to evaluate VX-970 plus cisplatin using lung xenografts (Hall et al., 2014). In these experiments, 35 lung cancer cell lines were treated using DNA-damaging drugs (cisplatin and oxaliplatin) and VX-970 or a CHK1 inhibitor (AZD7762) as a sensitizing agent. The clearest synergistic effect was observed after treatment using cisplatin plus VX-970, which sensitized N80% of the cell lines. While loss of p53
function apparently enables better sensitization by VX-970, the TP53
status was not a statistically significant factor. Moreover, in all seven non-small cell lung cancer xenograft models, markedly improved cis- platin response was observed using VX-970 doses that did not produce any clear effect as monotherapy, even in cases where cisplatin mono- therapy had no effect (Hall et al., 2014). Further studies examined how ATM/p53 alterations in KRAS-mutant murine lung adenocarcinoma might affect chemotherapy response, which surprisingly revealed that bi-allelic ATM deletion had no effect on the response to cisplatin (Schmitt et al., 2017).
Oesophageal tumours have also been analysed for sensitivity to the combination of VX-970 plus cisplatin (Leszczynska et al., 2016). After VX-970 treatment, two oesophageal squamous cell carcinoma lines (OE21 and OE33) and one adenocarcinoma cell line (FLO-1) exhibited sensitivity to cisplatin and carboplatin, and were also responsive in hyp- oxic conditions. Interestingly, ATR activation has been identified as a po- tential biomarker of chemoresistance in oesophageal squamous cell carcinoma (Shi et al., 2018). In that study, samples were obtained from 144 oesophageal squamous cell carcinoma patients, which re- vealed that ATM/ATR signalling activation was the major responder for overcoming RS that was induced by cisplatin treatment. In particu- lar, ATM-deficient cells were significantly sensitized by the combination of VX-970 plus cisplatin. Similar results were also obtained in mice xe- nograft models using both ATM-null KYSE70 and KYSE450 cells, which exhibited good ability to tolerate ATR inhibition (Shi et al., 2018). In vitro evaluation during a paediatric preclinical testing program also revealed modest sensitization to the combinations of VX-970 plus cisplatin (1.48-fold) and VX-970 plus melphalan (an alkylating cyto- static agent, 1.96-fold) (Kurmasheva et al., 2018). In vivo testing of various paediatric solid tumour xenograft revealed that VX-970 mono- therapy only provided event-free survival in 5 of 24 cases. However, the combination of VX-970 plus cisplatin provided outstanding potenti- ation (event-free survival for 21 of 24 xenografts), relative to cisplatin alone. That study also failed to detect a significant relationship with TP53 status (Kurmasheva et al., 2018).
In the context of treatment-resistant SCLC, a CRISPR-Cas9 screen also favoured the ATR-CHK1 pathway for a potential synthetic lethality target (Nagel et al., 2019). Six human SCLC cell lines (DMS-273, NCI-H69, NCI-H82, NCI-H187, NCI-H446, NCI-H1304)
and primary human lung fibroblasts were treated using VX-970 plus cisplatin, which provided greater lethality than a cisplatin/ etoposide mixture. This synergy was not detected in non-cancer cells, and the same results were observed using DMS-273 and NCI- H187 xenografts, where VX-970 plus cisplatin substantially de- creased tumour growth, relative to treatment using single agent or the cisplatin/etoposide mixture (Nagel et al., 2019). A similar ap- proach using kinome library screening also favoured the ATR-CHK1 pathway for restoring sensitivity in oxaliplatin-resistant colorectal cancer (Combès et al., 2019). In that study, VX-970 plus oxaliplatin provided a synergistic effect on all four colorectal cancer cell lines (HCT116, SW48, SW620, and HT29) and on three oxaliplatin- resistant colorectal cancer cells (HCT116-R1, HCT116-R2, and

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SW48-R), based on results from both two-dimensional and spheroid three-dimensional cultures. The combination of VX-970 plus cis- platin also suppressed tumour growth and extended survival in a mouse model with HCT116-R1 xenografts (Combès et al., 2019).

⦁ Combination with gemcitabine

Gemcitabine is an antimetabolite nucleoside analogue that stalls replication after integration into the growing strand created by DNA polymerases. Gemcitabine also inhibits crucial enzymes involved in nu- cleotide synthesis (thymidylate synthase and ribonucleotide reduc- tase), which limits the supply for DNA synthesis (Schelhaas et al., 2016). This is relevant because stalled replication forks are a major trig- ger for ATR activation.
In 2012, Vertex Pharmaceuticals, Inc. evaluated whether VX-970 could be used to induce chemosensitivity or radiosensitivity in in vitro and in vivo models of pancreatic ductal adenocarcinoma (PDAC) (Fokas et al., 2012). Those experiments revealed that VX-970 improved the cytotoxicity of gemcitabine in p53-mutant PDAC cell lines (MiaPaCa-2, PSN-1, and PancM) and in PDAC xenografts (PSN-1 and MiaPaCa-2). Furthermore, VX-970 provided sensitization even at doses of gemcitabine that had no effect as monotherapy. Interestingly, VX-970 plus gemcitabine had no detrimental effects on normal cells, and the combination was also well tolerated in animals (Fokas et al., 2012). Lung xenografts were also used as a proof-of-concept model for the combination of gemcitabine plus VX-970 (Hall et al., 2014). Moreover, a mouse model using MV4–11 xenografts (acute myeloid leukaemia cells) revealed substantial susceptibility to VX-970 plus gemcitabine, which resulted in a significant reduction in graft growth and a higher survival rate (Fordham et al., 2018).
Similar results have been reported for VE-821. Prevo et al. treated PSN-1, MiaPaCa-2, and primary PanCM pancreatic cancer cell lines (p53 mutants) with gemcitabine (100 nM), and noted substantially increased sensitivity when VE-821 (1 μM) was added under both normoxic and hypoxic conditions. Gemcitabine-induced phosphoryla- tion of CHK1 is blocked by ATR inhibition, which results in lethal syner- gism and G2/M arrest in cancer cells (Prevo et al., 2012). The use of VE-821 also increased sensitivity to gemcitabine in SKOV3, PEO1, and OVCAR ovarian cancer cells, similar to the use of a CHK1 inhibitor (MK-8776) (Huntoon et al., 2013).
Another study evaluated whether VE-821 could sensitize AML cell lines to gemcitabine or hydroxyurea, which also inhibits ribonucle- otide reductase. Noticeable synergy was observed in all cases: in chemotherapy-resistant AML samples from patients with de novo AML, relapsed AML, paediatric t-AML, and adult t-AML, although no synergy was observed in primary bone marrow cells from healthy donors (Fordham et al., 2018).
The relationship between VE-821-induced sensitization and TP53 status was also explored, although the potency of ATR inhibition was not related to TP53 status in three p53-deficient and four p53 wild-type (p53-wt) cell lines (Middleton et al., 2018). Furthermore, p53 dysfunction (loss of G1 checkpoint control) was not sufficient to evoke sensitivity to VE-821 as monotherapy, although gemcitabine ad- ministration increased the sensitivity of p53-null cells. Moreover, gemcitabine (100 nM) plus VE-821 (1 μM) reduced the clonogenic sur- vival of p53-dysfunctional cells (U2OS: 35-fold, HCT116: 60-fold, and MDA-MB-231: 16-fold), while the reductions were substantially lower for p53-wt cells (U2OS: 2.3-fold, HCT116: 2-fold, and MCF7: 4-fold). Importantly, there was no significant chemosensitization in non- tumorigenic MCF10A breast cells (Middleton et al., 2018).

⦁ Combination with topoisomerase I and II inhibitors

Topoisomerases (Top) relax DNA supercoils during replication, and their inhibition creates SSBs and DSBs because their activity is interrupted before the DNA strands can be ligated together. Top I
inhibitors include camptothecin and its derivatives (e.g., topotecan and irinotecan), as well as indenoisoquinoline derivatives, such as indotecan, and are mainly responsible for DNA SSBs (Hevener, Verstak, Lutat, Riggsbee, & Mooney, 2018). Top II inhibitors (namely etoposide) prevent the re-ligation of DNA and create DSBs (i.e., the most deleterious DNA lesions) (Stanulla, Wang, Chervinsky, & Aplan, 1997). These damages obviously trigger the full DDR, including ATR activity.
Large genome screening using MDA-MB-231 cells supported the ATR-CHK1 pathway as a synthetic lethal partner for Top I inhibitors (Jossé et al., 2014). The effect of VE-821 was investigated using three colon cancer cell lines (HT29, HCT116 [p53-wt], and HCT116 [p53-knockout]) and one breast cell line (MDA-231 [p53 knockout]). As expected, VE-821 had minimal effects as monotherapy, although substantial synergy was observed for the combinations with campto- thecin and indotecan (Jossé et al., 2014). Furthermore, VE-821 induced topotecan sensitivity in SKOV3, PEO1, and OVCAR ovarian cancer cells, although CHK1 inhibitor (MK-8776) did not (Huntoon et al., 2013).
Synergy between Top inhibitors and VX-970 has been evaluated in two xenograft studies. In one study, VX-970 plus irinotecan caused dis- tinct tumour regression in a COLO205 xenograft model, relative to the single-agent treatments (Jossé et al., 2014). Similarly, a lung xenograft model revealed synergy for the combinations of VX-970 with etoposide or SN38 (the active metabolite of irinotecan). While the most pro- nounced synergy was observed for VX-970 plus cisplatin, co-action was also detected with etoposide and for the combination of SN38 with VX-970 or a CHK1 inhibitor (Hall et al., 2014). While bi-allelic ATM deletion in murine lung adenocarcinoma did not inﬂuence the re- sponse to cisplatin, these deletions significantly increased the sensitivity to Top I and II inhibitors with VX-970 (Schmitt et al., 2017).

⦁ Combination with PARP inhibitors

The PARP proteins play an important role in the initiation of DNA repair, where they are critical for the SSB repair and base excision repair (BER) pathways. Inhibitors of PARP provide excellent synergies in HR- deficient cells, and several of these inhibitors have been approved for treating BRCA1/2-mutated breast or ovarian cancer. Thus, PARPis are an interesting tool for combination with ATRis, and this topic has al- ready been covered previously (Zheng et al., 2020). A study of KRAS- mutant murine lung adenocarcinoma (Schmitt et al., 2017) revealed significant reliance on the PARP1 and ATR DNA damage repair path- ways. Allograft model studies using a combination of olaparib plus VX-970 also confirmed the dependence on p53 expression, with a lack of p53 and ATM being associated with a remarkable loss of cell viability after treatment using olaparib plus VX-970 (Schmitt et al., 2017).
Similarly, VE-821 induced veliparib sensitivity in SKOV3, PEO1, and OVCAR ovarian cancer cells, which was not achieved via CHK1 inhibi- tion (Huntoon et al., 2013). However, DNA damage caused by veliparib could be repaired via HR if it led to DSBs (Huehls et al., 2012; W. Wang, 2007), which indicates that this synergy was also related to CHK1- independent ATR-regulated events during HR. Thus, an ATRi could be combined with a PARPi in BRCA1/2-deficient cells (Huntoon et al., 2013), and similar results were observed in colon cancer cells using a combination of a PARPi, VE-821, and irinotecan (Abu-Sanad et al., 2015). Furthermore, the three-agent combination was more potent in the HCT-116, HT-29, and LoVo cell lines, relative to irinotecan with ei- ther a PARPi or VE-821. The HCT-116 cells were the most sensitive be- cause they have significantly decreased ATM levels, although the authors also noted that increased DNA-PKcs levels might help compen- sate for the ATR inhibition (Abu-Sanad et al., 2015). A recent study also evaluated olaparib in 61 lung adenocarcinoma cell lines, which revealed a better response in the presence of ATM deficiency, relative to ATM- proficient cells. A single olaparib treatment combined with loss of ATM caused only transient G2 arrest and not apoptosis, while the

L. Gorecki et al. / Pharmacology & Therapeutics xxx (2020) xxx 7

combination of olaparib plus VE-821 resulted in cells proceeding into mitosis, which resulted in increased apoptotic cell death (Jette et al., 2019).

⦁ Combination with radiotherapy

Irradiation of mammalian cells using a 1-Gy dose causes approxi- mately 1,000–2,000 damaged bases, 800–1,600 damaged deoxyribose bases, 500–1,000 SSBs, 200–300 alkaline labile sites, 20–40 DSBs, 30 DNA–DNA covalent bonds, and 150 DNA-protein covalent bonds (Prasad, 1995). Thus, targeting the DDR in combination with radiother- apy should be an excellent strategy, especially in cases involving radioresistance. Researchers at Oxford University and Vertex Pharma- ceuticals have evaluated whether VE-821 could be used to sensitize radiotherapy-resistant hypoxic cancer cells (Pires et al., 2012), and the in vitro results indicated that VE-821 (1 μM) caused radiosensitization (at doses of ≤6 Gy) in 12 cancer cell lines, independent of their p53 sta- tus. Furthermore, VE-821 caused a decreased hypoxic response through HIF-1-mediated signalling, which is relevant because prevention of ATR-mediated HIF-1 phosphorylation (potentially at Ser696) plays a significant role in hypoxia-induced angiogenesis (Economopoulou et al., 2009). Although VE-821 increases hypoxia-induced ATM activa- tion (both ATM and DNA-PKcs are able to phosphorylate HIF-1), this compensation may be redundant in ATM-deficient tumours (Cam, Easton, High, & Houghton, 2010; Pires et al., 2012). Interestingly, acti- vated ATM, but not ATR, was mainly detected in growing hypoxic spher- oids (A673, Ewing sarcoma), although ATR inhibition by VE-821 was sufficient to alter spheroid growth (Rifﬂe, Pandey, Albert, & Hegde, 2017). Other studies revealed that VE-821 induced radiosensitivity at a dose of 3 Gy in human p53-deficient promyelocytic leukaemia cells (HL-60) and T-lymphocyte leukemic cells (MOLT-4) (Šalovská et al., 2014, 2018; Vávrová et al., 2013). Moreover, irradiation increased the sensitivity of p53-null cells to ATR inhibition by VE-821 (Middleton et al., 2018). Interestingly, higher radiation doses were the most potent, which suggests that hypofractionated radiotherapy may be useful in this setting. Importantly, no radiosensitization was observed in non- cancerous MCF10A breast cells (Middleton et al., 2018).
The radiosensitizing properties of VX-970 were first reported in 2012, based on preclinical data generated by Vertex Pharmaceuticals Inc. using PDAC cells and xenografts (Fokas et al., 2012). The results in- dicated that VX-970 improved the cytotoxicity of radiotherapy in all PDAC cell lines (p53-mutants: MiaPaCa-2, PSN-1, and PanCM) as well as in PDAC xenografts (PSN-1 and MiaPaCa-2), even when fractionated radiation doses were used. Moreover, VX-970 exerted no detrimental effects on normal cells when it was combined with radiotherapy (Fokas et al., 2012). Prevo et al. also tested PSN-1, MiaPaCa-2, and pri- mary PanCM pancreatic cancer cell lines (p53 mutants), and reported significant sensitization using a combination of radiotherapy (6 Gy) and VE-821 (1 μM) under both normoxic and hypoxic conditions. The radiation-induced phosphorylation of CHK1 was blocked by ATR inhibi- tion, resulting in lethal synergism and G2/M arrest in cancer cells (Prevo et al., 2012). Co-activity with radiotherapy was observed for VE-821- mediated inhibition of HR, based on the persistence of the γH2AX and 53BP1 foci, and inhibition of the RAD51 foci (Prevo et al., 2012). Oesophageal squamous cell carcinomas (OE21 and OE33) and an ade- nocarcinoma cell line (FLO-1) have also exhibited VX-970-induced radiosensitization under hypoxic conditions, with substantially inhibited growth of an OE21 xenograft containing broad hypoxic re- gions (Leszczynska et al., 2016).
Brain tumours usually have a poor prognosis, and the blood–brain barrier is an obstacle to the intracranial delivery of therapeutic agents, complicating the treatment of central nervous system diseases. Convection-enhanced delivery (CED) drug-loaded nanoparticles (NPs) are a potential solution, as they provide easier and prolonged CNS distri- bution of the compounds. For example, rats with intracranial RG2 tu- mours were treated using fractioned radiotherapy (5 doses of 3 Gy

each) plus free VX-970 or VX-970 in CED-NPs, which revealed prolonged survival after treatment using the VX-970-loaded NPs (drug persistence of approximately 5 days for the CED-NPs versus 10 h for free VX-970) (E. M. Chen et al., 2018).
The ATR-CHK1 network in breast cancers has been evaluated in pre- vious studies (Abdel-Fatah et al., 2015; Alsubhi et al., 2016; Al-Subhi et al., 2018). One study evaluated the levels of ATR, CHK1, and phos- phorylated CHK1Ser345 (pCHK1) in 1,712 breast cancers (Abdel-Fatah et al., 2015), which revealed that high ATR levels were linked to aggres- sive tumours (based on size, high grade, pleomorphism, and a higher mitotic index) and poor survival. Two cancer cell lines (MCF7 [p53- wt] and MDA-MB-231 [p53 mutant]) and non-tumorigenic MCF10A cells were evaluated to determine their LC50 values for VE-821, which was poorly selective, regardless of TP53 status (LC50 of 1.89 μM for MCF7, 1.93 μM for MDA-MB-231, and N10 μM for MCF10A) (Abdel-
Fatah et al., 2015). This finding highlighted the potential need to person-
alize ATRi treatment based on ATR levels. An ongoing clinical study has also revealed an association between high levels of cytoplasmic pCHK1 and early local recurrence (Alsubhi et al., 2016), although high nuclear expression of p53 was also related to this phenomenon. Radiotherapy was also combined with VE-821, which surprisingly revealed better radiosensitization in MCF7 cells than in MDA-MB-231 cells (3-fold in MCF7 cells and 2-fold in MDA-MB-231 cells, relative to radiotherapy alone) (Alsubhi et al., 2016). A recent study has also suggested that ATR signalling was related to the poor survival associated with phos- phate and tensin homolog deficient (PTEN)-deficient breast cancer, which exhibited aggressive phenotypes and generally upregulated ATR activity. The pre-clinical data revealed that VE-821 was more toxic to PTEN-deficient cells (MDA-MB-468 and BT-549) than to PTEN-proficient cells (MDA-MB-231) (Al-Subhi et al., 2018). Triple- negative breast cancer (TNBC) is a highly aggressive and treatment- resistant cancer where radiosensitizing agents (such as VX-970) might have therapeutic value. One study revealed successful VX-970-induced radiosensitization of TNBC cell lines (MDA-MB-231, HCC1806, and BT- 549), with the most robust effect observed in MDA-MB-231 cells and the least synergy observed in non-cancerous MCD10A breast cells. Four TNBC patient-derived xenografts were also evaluated for radiosen- sitivity and chemosensitivity, which exhibited a good response to VX- 970 plus radiotherapy and improved sensitivity in the chemoresistant xenografts. Enhanced synergy was observed during combination therapy for cases involving TP53 mutations or increased MYC expres- sion, as well as in cells with HR dysregulation (BRCA1 mutated) (Tu et al., 2018).

⦁ Simultaneous targeting of ATR-CHK1 downstream pathways

In addition to classic chemotherapy or radiotherapy strategies, several distinct DDR-related pathways have been evaluated for poten- tial synergism in cancer therapy. One promising strategy involves si- multaneous inhibition of ATR and CHK1, as double blockade of the two downstream partners should produce a synergistic effect. Using cancer cell lines (U2OS and MCF-7), a coactive effect was observed using a CHK1 inhibitor (AZD7762) and VE-821, which was not observed in normal VH-10 fibroblasts. Moreover, the combination provided sub- stantial tumour growth reduction in mouse xenograft model using H460 lung cancer, while the single-agent treatments had limited effects (Sanjiv et al., 2016). Another study evaluated the relationship between CHK1 and ATR inhibition (Massey, 2016), which revealed that a CHK1 inhibitor (V158411) plus VX-970 provided a greater reduction in cell viability than combinations of V158411 with an ATM inhibitor (KU- 60019) or with a DNA-PKcs inhibitor (NU7441). These results were independent of TP53 status (Massey, 2016).
There are limitations to the simultaneous inhibition of ATR and CHK1, as CHK1 inhibition leads to ATR activation in a feedback loop, and potential vulnerability is mediated by additional ATR silencing in cancer cells. Thus, any synergistic effect might be limited by the

8 L. Gorecki et al. / Pharmacology & Therapeutics xxx (2020) xxx

available levels of CHK1 (Massey, 2016), and the combination with at- tenuated toxicity in non-cancerous cells might be less effective than ei- ther monotherapy (Sanjiv et al., 2016). It is also possible that the synthetic lethal interaction of VX-970 might be enhanced if another ATR/CHK1 downstream target is selected, and WEE1 inhibition has re- cently attracted attention in this setting. Some studies have revealed that VX-970 plus WEE1 inhibitor (AZD1775) provided a substantial antimetastatic effect (Bukhari et al., 2019; Chaudhuri et al., 2014; Qi et al., 2019), and better growth inhibition than VX-970 alone in AML cell lines (MV4–11, HL-60, MOLM-3 THP-1, OCL-AML3, and U937). In
this context, VX-970 was able to abrogate the G2/M checkpoint through increased DSB levels that were related to the WEE1 inhibitor treatment (Qi et al., 2019). Similarly, the combination of a WEE1 inhibitor with VE- 821 provided dose-dependent activity in AML cell lines with impaired p53 function (TF-1, HEL and THP-1) (Chaudhuri et al., 2014).
Phase I/II studies are currently investigating CHK1 and WEE1 inhib- itors (Bukhari et al., 2019; Otto & Sicinski, 2017; Qiu et al., 2018). The preclinical data clearly indicate that ATR inhibition provides broader chemosensitization (relative to CHK1 inhibition), as ATR controls addi- tional cell responses (Hall et al., 2014; Huntoon et al., 2013; Karnitz & Zou, 2015; Qiu et al., 2018). Moreover, CHK1 inhibition is lethal at low levels of RS, which may affect healthy cells. In contrast, ATR inhibition can activate CHK1 through a pathway involving DNA-PKcs, so ATR inhi- bition is more preferably lethal at high levels of RS (Buisson et al., 2015; Yazinski & Zou, 2016).

⦁ Synergism of VE-821 and VX-970 with other DNA damage- inducing agents

An unbiased kinome inhibition screen identified dependence on the RS response (ATR-CHK1 pathway) in PDAC cell lines after treatment with chloroquine (CQ) (Elliott et al., 2019). Furthermore, CQ can deplete aspartate in PDAC cells, thereby restricting de novo dNTP biosynthesis, with the antiproliferative effects of CQ and VX-970 being confirmed in 23 human PDAC cell lines, primary cultures, three-dimensional spheroid cultures (MiaPaca2; Panc03.27; CFPAC1; and DANG), and one co-culture (Miapaca2/CAF). The combination of CQ and VX-970 also inhibited growth of MiaPaca2 xenografts, although increased levels of aspartate provided protective effects (induced via supplemen- tation or overexpression of the aspartate transporter SLC1A3) (Elliott et al., 2019).
Breast cancer cells can also be sensitized using an insulin/insulin-like growth factor-1 receptor (IR/IGF-1R) inhibitor combined with VE-821 and cisplatin (O’Flanagan et al., 2016). In that study, the IGF-1R inhibi- tion induced DNA damage and accumulation of γH2AX. Furthermore, the combination of an IGF-1 inhibitor with VE-821 provided a signifi- cant reduction in the formation of MCF7 cell colonies, relative to the single-agent treatments (O’Flanagan et al., 2016).
Another lethal combination is VE-821 plus (−)-lomaiviticin A, which is an antiproliferative bacterial metabolite that produced SSBs and DSBs in K562 cells (myelogenous leukaemia) (Colis & Herzon, 2016).
High linear energy transfer (LET) carbon ion radiation has also been tested with VE-821 in one healthy cell line (1BR-hTERT) and two cancer cell lines (HeLa and U2OS) (Fujisawa et al., 2015). In that study, G2/M abrogation was more pronounced in the HeLa cells (p53 deficient) than in the U2OS and 1BR-hTERT cells (p53-wt). Interestingly, the cancer cells pre-treated using VE-821 were more sensitive to LET carbon ions than to X-rays (based on a higher number of multiple micronuclei), although VE-821 did not have a significant effect on normal cells (Fujisawa et al., 2015). VE-821 was not only ATRi used in combination with LET. A separate study evaluated a FGFR2-targeted thorium 227 conjugate (FGFR2-TTC) with BAY1895344, which revealed that the combination provided significantly reduced viability in several cancer cell lines (MFM-223 [TNBC], SUM52-PE [breast cancer] and KATO III [gastric cancer]). Moreover, the combination treatment provided
better inhibition of tumour growth in human-derived mouse xenograft models using NCI-H716, SNU-16, and MFM-223 (Wickstroem et al., 2019).
In PANC-1 and MGC-803 cells, VE-821 is able to induce the epithelial-mesenchymal transition through ZEB1 upregulation. Thus, si- multaneous inhibition of ZEB1 and ATR inhibits migration, as well as suppresses proliferative signalling through the accumulation of DNA damage (Song et al., 2018). Thus, synergism leading to induction of DNA damage might be linked to HR, where ZEB1 is also required (Zhang et al., 2014). It has also been demonstrated that ZEB1 inhibition advances CHK1 phosphorylation, thereby slowing down the S-phase (Song et al., 2018).
The strong photosensitizing ability of ATR inhibitors has been con- firmed in cutaneous T-cell lymphoma cells (MyLa200, SeAx, and Mac2a) using psoralen plus ultraviolet A and ultraviolet A alone. In that setting, VX-970 was 10-times more effective than VE-821, although experiments using knockdown of ATR and CHK1 failed to reveal similar findings (especially regarding UVA sensitization). These results suggest that the effects of VX-970 and VE-821 are not related to the ATR-CHK1 pathway, and may instead be related to their chemical class, as another ATRi (AZD6738) had no effect (Biskup, Naym, & Gniadecki, 2016).

⦁ Synthetic lethal interactions and the potentiation of increased replication stress

Several studies have aimed to identify synthetic lethal partners that could be compatible with ATRi sensitization. For example, the overex- pression of MYC has been evaluated in multiple myeloma, as well as its connection to RS and the generation and metabolism of ROS (Cottini et al., 2015). The results revealed that p53-deficient multiple myeloma cell lines (OPM-1, OPM-2, and RPMI/8266) were more sensi- tive to VE-821 treatment than p53-wt multiple myeloma cells (MM.1S and H929). Moreover, MYC-amplification in U266 cells led to the induc- tion of RS and increased the sensitivity to VE-821, while MYC silencing in MM.1S and H929 cells blocked the effect of VE 821. Piperlongumine induces the production of ROS and is a potential anti-cancer agent (Karki, Hedrick, Kasiappan, Jin, & Safe, 2017; Thongsom, Suginta, Lee, Choe, & Talabnin, 2017), which exhibits lethal co-activity with ATRi via ROS generation. This combination also provided increased sensitiv- ity in MYC-overexpressing multiple myeloma cell lines (H929, OPM-2, MM.1S, and RPMI/8266), while U266 cells with normal MYC expression exhibited no significant difference (Cottini et al., 2015). A stronger apo- ptotic response to VE-821 was also confirmed in multiple myeloma- derived cells with high levels of endogenous DNA damage (OPM-2, MM.1S, and RPMI/8266), relative to a normal lymphoblastoid B cell line (LINF903) and a myeloma cell line with a low levels of DNA damage (U266) (Herrero & Gutiérrez, 2017).
Another study revealed that FGFR2 stimulation in K-Ras-driven
mouse Y1 malignant cells (high levels of Ras activity) disrupted proteostasis and enhanced tonic RS, which offers another possible syn- ergistic partner for DDR inhibitors. Interestingly, FGFR2 is overstim- ulated in many cancer types, including gastric, colorectal, and breast cancers (Babina & Turner, 2017; Turner & Grose, 2010). Furthermore, VE-821 treatment with FGFR2 stimulation reduced the cell viability the Ewing’s sarcoma family of tumours (A673, RD-ES, SK-N-MC, and TC-32), which are Ras-driven cancers. The potentiation of the ATRi’s ef- fect was enhanced by MAPK-ERK1/2 overstimulation through FGFR2 ac- tivation (Dias et al., 2019).
It has been hypothesized that VE-821 is intimately involved in the reduction of HR in JJN3-HR and U266-HR cells. These results indicate that the ATR and ATM kinases could compensate for the lack of the other, and that multiple myeloma cells rely on an intact HR pathway after DNA end resection (i.e., the repair could no longer be completed via non-homologous end joining [NHEJ]) (Herrero & Gutiérrez, 2017). Thus, VE-821 was evaluated as monotherapy for cancer based on HR de- ficiencies (Middleton et al., 2015). In that model, VE-821 conferred

L. Gorecki et al. / Pharmacology & Therapeutics xxx (2020) xxx 9

sensitivity via HR deficiencies (i.e., related to ATM, BRCA2, or X-ray re- pair cross-complemented protein 3 [XRCC3] deficiency) and base exci- sion repair deficiencies (BER, XRCC1). Surprisingly, high expression of DNA-PKcs also resulted in hypersensitivity to VE-821, which is hypo- thetically related to genomic instability caused by competition between HR and NHEJ to reliance on ATR for tumour growth (Middleton et al., 2015). Similarly, another synthetic lethal interaction was observed be- tween ATRis and inactivated DNA polymerase theta (POLQ), which plays an important role in microhomology-mediated end joining (MMEJ) for repair of DSBs (Chiarle et al., 2011). That study evaluated 11 breast cancer cell lines with POLQ overexpression (including 7 lines with ≥5-fold expression), which revealed lethal effects after simul- taneous inhibition of POLQ and ATR. Moreover, ATR inhibition in POLQ- knockdown BT-474 cells (p53-wt) resulted in potentiation of VX-970 toxicity (Z. Wang et al., 2019). The search for synthetic lethal interac- tions broadened to incorporate translesion polymerase Pol ζ. A combination of VE-821 plus cisplatin revealed sensitization of cisplatin-resistant cells (MDA-MB-468), while the response to VE-821 plus cisplatin was improved further in cells with loss of Pol ζ or p53. Le- thal synergism was also observed with a PARPi, similar to the results for VE-821 plus cisplatin, although the authors mentioned that this synergy could probably also harm normal cells (Mohni et al., 2015). A recent study has compared cisplatin responses in MCF7-based two- and three-dimensional cell culture models, which revealed a distinctive in- crease in cisplatin resistance using the three-dimensional culture model. The cisplatin resistance was p53-independent and was associ- ated with increased translesion polymerase capacity. However, VE-821 treatment decreased the cellular viability in the three- dimensional via attenuated translesion polymerase activity. Moreover, VE-821 was able to reverse cisplatin-based induction of REV3L, which was related to treatment resistance (Gomes et al., 2019).
The relationship between ATR inhibition and cells with alternative lengthening of telomeres (ALT) has also been discussed. For example, ALT-positive cells could be hypersensitive to ATR inhibition (Flynn et al., 2015), as ALT-positive cells exhibited greater sensitivity to VE- 821 than cells with active telomerase (TEL). It has also been suggested that ATR inhibition can cause fragility of ALT telomeres, as ALT- positive osteosarcoma cells (U2OS, SAOS2, CAL72, NOS1, and HUO9) and glioblastoma cells (MGG119) were hypersensitive to VE-821, while TEL-positive cells (MG63 and SJSA1) exhibited only moderate sensitivity (IC50 values: ~0.8 μM for ALT-positive cells versus ~9 μM for TEL-positive cells) (Flynn et al., 2015). However, a similar study sug- gested that VE-821 hypersensitivity was unrelated to ALT activity (Deeg, Chung, Bauer, & Rippe, 2016), based on comparisons of VE-821 activity in TEL-positive cell lines (HeLa, HCT116, and MG63; IC50 values of 0.9–3.3 μM) and ALT-positive cells (U2OS, CAL72, and SAOS2; IC50 values of 0.7–7.0 μM). Moreover, subsequent experiments using U2OSALT and U2OSTEL cells revealed no significant difference in VE-821 sensitivity. Finally, it has been proposed that additional cellular factors must be involved in ATR sensitivity, as ALT activity alone is insufficient to provide synthetic lethality synergism (Deeg et al., 2016).
Mutations in ARID1A are an interesting synthetic lethal partner for ATR inhibition, as they exert one of the most common molecular alterations in human cancer. In vitro and in vivo models revealed that VX-970 had no effect against mice with normal HCT116 xenografts (ARID1A+/+), although growth was substantially inhibited in ARID1A−/− tumours (Williamson et al., 2016). Another study also re- vealed that ATRi treatment (VE-821 or VX-970) was significantly more effective against gastric cancer organoids with ARID1A mutations (Yan et al., 2018), although VX-970 provided superior efficacy.

⦁ Transition to clinical trials

The use of VX-970 or other ATRis is based on cancer cells’ higher levels of RS or DNA damage, which could enhance sensitivity to conven- tional treatment (Cottini et al., 2015; Herrero & Gutiérrez, 2017). Similar

logic has been applied to oncogenes that induce RS (e.g., mutations af- fecting MYC, RAS, or Cyclin E) (Cottini et al., 2015; Muralidharan et al., 2016; Schmitt et al., 2017; Luis I. Toledo, Murga, Zur, et al., 2011). Defi- ciencies in the ATR-CHK1 downstream pathway, or overexpression of ATR or CHK1, are also potential triggers for the rational use of VX-970. For example, upregulation of CDC25A, as well as high levels of ATR or CHK1, induce sensitivity to ATRis (Abdel-Fatah et al., 2015; Alsubhi et al., 2016; Pitts, Davis, Eckhardt, & Bradshaw-Pierce, 2014; Ruiz et al., 2016), which is related to ATM-p53 pathway alterations, as all preclinical studies have indicated that ATM or p53-deficient cell lines are more vulnerable to ATRis. This is important because ATM-p53 muta- tions are often present in various cancers (Choi, Kipps, & Kurzrock, 2016; Schmitt et al., 2017). Nevertheless, ATM expression alone should not be a limiting factor, as some exceptions have been reported, espe- cially regarding the lack of reliance on TP53 status (Kurmasheva et al., 2018; Pires et al., 2012). Thus, in addition to ATM mutations, it is pru- dent to consider other genes that can promote synthetic lethality with ATRis, which include inactivating mutations in PTEN, XRCC1, ERCC4, BRCA1, BRCA2, and ARID1A (Al-Subhi et al., 2018; Middleton et al., 2015; Mohni et al., 2015; Mohni, Kavanaugh, & Cortez, 2014; Yan et al., 2018). Moreover, depletion of dNTP provides useful synergism with VX-970, which may also support the clinical use of ATRis, although it is important to note that dNTP production can be restored in cancer cells, and should not be the only target (Elliott et al., 2019). Defects in HR in general provide distinctive synergism, as VX-970 has markedly higher toxicity in HR-deficient cell lines (Huntoon et al., 2013; Krajewska et al., 2015; Prevo et al., 2012; Tu et al., 2018).
Several kinome library screens using CRISPR-Cas9 (Nagel et al., 2019; Z. Wang et al., 2019) or shRNA (Combès et al., 2019; Elliott et al., 2019; Jossé et al., 2014) have clearly favoured ATR inhibition to address chemotherapy or radiotherapy resistance, or to potentiate the efficacy of anticancer treatments. However, sensitization varies based on tumour-specific alterations, which makes it important to specifically and rationally target the use of ATRi treatment. Hypothetically, all in- ducers of RS or DNA damage could be combined with VX-970, and pre- clinical studies have indicated that VX-970 provided highly enhanced radiosensitization, despite the fact that radiotherapy mainly induces DSBs, which should primarily involve a response from ATM or DNA- PKcs (Fokas et al., 2012; Leszczynska et al., 2016; Pires et al., 2012; Rifﬂe et al., 2017; Tu et al., 2018). This radiosensitization might be ex- plained through a compensatory ATR-activated HR pathway in ATM-null cells (Krajewska et al., 2015; Prevo et al., 2012). Moreover, VX-970 was particularly efficient in radioresistant hypoxic tumours, as hypoxic conditions produce elevated RS that increases vulnerability to ATR inhibition (Bindra et al., 2004; Chan et al., 2008; Olcina, Lecane, & Hammond, 2010; Scanlon & Glazer, 2015). Hypofractionated radiother- apy exhibits distinctive synergism with VE-821/VX-970 treatment (E.
M. Chen et al., 2018; Fokas et al., 2012; Middleton et al., 2018), and
these strategies are being explored by combining VX-970 and radio- therapy in two phase I trials (NCT02589522 and NCT02567422).
Combining ATR inhibition with RS-inducing drugs is promising strategy, with clinical trials evaluating combinations of VX-970 with DNA crosslinking agents (cisplatin or carboplatin), Top I and Top II in- hibitors (topotecan, irinotecan, or etoposide), a nucleoside analogues (gemcitabine), a PARP inhibitor (veliparib), and a taxane analogue (do- cetaxel) (Table 1). The preclinical results for VE-821/VX-970 highlight the potential effectiveness of this approach, as their combination with DNA crosslinking agents was particularly effective in various cancer cell lines, especially in p53-deficient cells (Huntoon et al., 2013; Kurmasheva et al., 2018; Mohni et al., 2015; O’Flanagan et al., 2016; Reaper et al., 2011). Sensitization was also observed in lung cancer xe- nografts (Hall et al., 2014; Nagel et al., 2019); in xenografts involving rhabdoid tumours, sarcomas, neuroblastoma, and brain tumours (Kurmasheva et al., 2018); in oesophageal tumours (Leszczynska et al., 2016; Shi et al., 2018); and in colorectal cancer (Combès et al., 2019). Similarly, Top inhibitors have been extensively used for

10 L. Gorecki et al. / Pharmacology & Therapeutics xxx (2020) xxx

Table 1
Summary of VX-970 clinical trials collected from ClinicalTrials.gov (as of January, 2020).

Clinical trial identifiere

Phase Co-treatment Patient segment

NCT02157792 I standard chemotherapyd advanced solid tumors
NCT02487095a I/II topotecan small cell lung cancer and extrapulmonary small cell cancers
NCT02567409b II cisplatin + gemcitabine metastatic urothelial cancer
NCT02567422 I cisplatin + radiation locally advanced head and neck squamous cell carcinoma
NCT02589522 I whole brain radiation brain metastases from non-small cell or small cell lung cancer, or neuroendocrine tumors
NCT02595892 II gemcitabine recurrent ovarian, primary peritoneal, or fallopian tube cancer
NCT02595931 I irinotecan solid tumors that are metastatic or cannot be removed by surgery
NCT02627443 I/II carboplatin + gemcitabine Recurrent and metastatic ovarian, primary peritoneal, or fallopian tube cancer
NCT02723864 I veliparib + cisplatin refractory solid tumors
NCT03309150 I monotherapy; carboplatin + paclitaxel advanced solid tumors
NCT03517969 II carboplatin; carboplatin + docetaxel metastatic castration-resistant prostate cancer
NCT03641313 II irinotecan progressive, metastatic, or unresectable TP53 mutant gastric or gastroesophageal junction cancer
NCT03641547 I radiotherapy + cisplatin + capecitabine oesophageal cancer

NCT03704467c I/II avelumab, carboplatin, paclitaxel, gemcitabine, doxorubicin, bevacizumab
PARPi-resistant, recurrent, ovarian cancer

NCT03718091 II monotherapy selected solid tumors
NCT03896503 II topotecan small cell lung cancer and extrapulmonary small cell cancers
NCT04052555 I radiation breast cancer
NCT04216316 I/II carboplatin + gemcitabine + avelumab non-small cell lung cancer
a Published results (Thomas et al., 2018).
b Terminated study.
c Completed study with only 3 patients – no results available.
d Cisplatin; carboplatin; cisplatin + gemcitabine; cisplatin + etoposide; irinotecan; gemcitabine.
e Aligned according to NCT numbers from the lowest to highest (from the oldest to newest).

advanced chemosensitization (Abu-Sanad et al., 2015; Hall et al., 2014; Huntoon et al., 2013; Jossé et al., 2014; Schmitt et al., 2017), with Top I inhibitors having the greatest potential because they mainly generate DNA SSBs. This treatment was effective in lung and colon cancer xeno- grafts (Jossé et al., 2014; Nagel et al., 2019), as well as in a phase I study involving advanced solid tumours (Thomas et al., 2018). Gemcitabine (a nucleoside analogue) also provided a compelling in- crease in the sensitivity of cancer cell lines (Fokas et al., 2012; Fordham et al., 2018; Hall et al., 2014; Huntoon et al., 2013; Middleton et al., 2015; Prevo et al., 2012) and in pancreatic cancer xeno- grafts (Fokas et al., 2012). Finally, PARP inhibitors have provided favourable combined effects in cancer cells (Abu-Sanad et al., 2015; Huntoon et al., 2013; Schmitt et al., 2017) and lung allografts (Schmitt et al., 2017).
One potential major complication of ATRi-based strategies is a substantial increase in the toxicity of chemotherapy or radiotherapy. A relevant example is the combination of crosslinking agents and O6- benzylguanine (an O6-alkylguanine-DNA alkyltransferase inhibitor), which reached phase III clinical trials but displayed unacceptable toxic- ity in those trials (Blumenthal et al., 2015). The most promising and advanced ATRi options (AZD6738 and VX-970) have been well toler- ated as monotherapy (Bradbury, Hall, Curtin, & Drew, 2019; Foote et al., 2015; Mei et al., 2019), and the existing animal studies have not identified any substantial side-effects related to VX-970 alone or in combination with chemotherapy or radiotherapy. Decreased ATR levels might even reduce the risk of cancer recurrence based on data from mouse models (Murga et al., 2009; Schoppy et al., 2012). Thus, VX- 970 appears to be a safe drug without any marked limitations, although clinical trial data are needed to address this point, especially given the potentially long-term consequences of combining inhibition of the DNA damage response with DNA-damaging agents.

⦁ VX-970 in clinical trials

Preclinical data have indicated that VX-970 was effective as mono- therapy and in combination therapy, although 17 of 18 clinical trials have focused on combination therapy (Table 1), presumably in an effort to provide robust anticancer effects. The only trial to evaluate VX-970
monotherapy (NCT03718091) was a phase II trial involving patients with various solid tumours who were enrolled based on specific bio- markers/mutations. The cases were grouped according to whether they involved ATM mutations, BRCA1 mutations, BRCA2 mutations, other known HR gene mutations, MYC or Cyclin E1 amplification, and ARID1A mutation. The other 17 clinical trials are evaluating VX-970 combined with chemotherapy or radiotherapy.
The only published results are for a single trial (NCT02487095), which evaluated 21 patients with advanced solid tumours and defective ATM-p53 signalling and/or with MYC and Cyclin E1 activation. This strategy was based on several recent studies (Al-Ahmadie et al., 2014; Hall et al., 2014; Jossé et al., 2014), and the phase I clinical data revealed promising outcomes after treatment using VX-970 plus topotecan (Thomas et al., 2018). There were no limiting toxicities at the highest applied doses of topotecan (1.25 mg/m2 intravenously on days 1–5) or VX-970 (210 mg/m2 intravenously on days 2 and 5) throughout the 21-day cycles. This combination seems to have been well tolerated, without substantial signs of additional toxicity, and myelosuppression was the most common adverse effect, similar to topotecan monother- apy. Seven of 8 patients with stable disease either experienced improve- ment or maintained their stable disease, while 3 of 5 patients with refractory SCLC exhibited a partial response or stable disease (Thomas et al., 2018). Nevertheless, additional clinical data are necessary, espe- cially from a broader group of patients, as the existing trials have predominantly targeted patients with advanced-stage, metastatic, re- current, or treatment-resistant cancers.
Another study (NCT02157792) evaluated patients with advanced solid tumours with inclusion criteria: ATM or TP53 loss of function, and BRCA1/BRCA2 mutations. The patients received dual therapy using VX-970 plus irinotecan, gemcitabine, or cisplatin, or triple therapy using VX-970 plus gemcitabine/cisplatin, cisplatin/etoposide, or cis- platin/carboplatin. A separate trial (NCT03641313) evaluated patients with gastric or gastroesophageal junction cancers and TP53 mutations, who were treated using VX-970 plus irinotecan. That study aimed to monitor other concomitant DDR defects, including HR-related mutations (e.g., involving ATM, BRCA1, and BRCA2) and other factors. Similarly, two other trials (NCT03517969 and NCT04052555) aimed to evaluate the relationship with HR deficiency. A recent trial

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(NCT04216316) included patients with ATM-proficient or ATM- deficient squamous non-SCLC, and evaluated dual combination therapy using VX-970 plus avelumab/carboplatin/gemcitabine. One terminated study (NCT02567409) aimed to identify predictors of treatment re- sponse, which included mutations affecting p53, p21, and ERCC2.

⦁ Interconnectivity with AZD6738, BAY1895433, and VX-803

The preferred method for administering VX-970 is intravenous infu- sion, while other ATRis can be administered orally. Similar to VX-970, VX-803 (M4344, developed by Vertex and bought by Merck) displayed good antiproliferative activity in 92 cancer cell lines (ED50 of approxi- mately 110 nM), and provided good in vivo results as monotherapy or in combination with talazoparib (a PARPi) (Zenke et al., 2019). These data supported the use of VX-803 in two phase I clinical trials as mono- therapy or in combination with carboplatin for advanced solid tumours (NCT02278250), and in combination with niraparib (a PARPi) against ovarian cancer (NCT04149145).
Another interesting ATRi is BAY1895344 (developed by Bayer), which has been tested in two phase I trials. The in vitro and in vivo data revealed strong antiproliferative activity, with good synergism when it was combined with a PARPi (olaparib), a LET-based strategy (FGFR2 TTC), and darolutamide (a nonsteroidal androgen receptor an- tagonist) (Luecking et al., 2017; Wengner et al., 2020; Wickstroem et al., 2019). The first trial (NCT03188965) evaluated BAY1895344 as monotherapy for advanced solid tumours and lymphomas, which revealed good efficacy in patients with DDR defects that were related to ATM loss and/or ATM deleterious mutations. The other trial (NCT04095273) evaluated the tolerability of BAY1895344 in combina- tion with pembrolizumab (a humanized antibody) for advanced solid tumours.
AZD6738 (or ceralasertib) has been developed by AstraZeneca as optimized orally available drug from their initial lead the AZ20 molecule (Foote et al., 2013, 2018). Numerous preclinical studies evaluated AZD6738, which was found to have antitumor activity in p53-deficient and ATM-deficient models, as both monotherapy and in combination with RS-inducing treatments (Bradbury et al., 2019; Karnitz & Zou, 2015; Mei et al., 2019). There are currently 25 trials evaluating AZD6738, including 16 phase II studies, two phase I/II studies, and seven phase I studies (1 has been completed). Similar to VX-970, AZD6738 has been used in trials that evaluated advanced, recurrent, treatment-resistant, and metastatic cancers. Several trials have evalu- ated HR-related defects (ATM, BRCA1/2, TP53, and/or ARID1A mutations) or hypoxia markers, although these cases were less common than in the trials evaluating VX-970. Moreover, AZD6738 has predominantly been evaluated in combination with olaparib (16 trials), with the synergism between the ATR and PARP inhibition being related to ATM/p53 defi- ciency. In contrast, VX-970 has only been evaluated with a PARPi in one clinical trial. Combinations of AZD6738 with standard chemo- therapy or radiotherapy have been less frequent, with studies tending to focus on using monoclonal antibodies for immunotherapy. Co- administration of AZD6738 with recently approved acalabrutinib (Bruton’s tyrosine kinase inhibitor) has also been incorporated into tri- als as treatment for non-Hodgkin’s lymphoma.

⦁ Conclusion

Conventional cancer treatment still focuses on cytotoxic chemother- apy and radiotherapy, although there is currently a shift towards more targeted therapy (Gavande et al., 2016). Thus, there is a desire to develop strategies that selectively target one or more pathways in can- cer cells while sparing healthy cells. In this context, small-molecule in- hibitors and monoclonal antibodies have better targeting of cancer cells and are generally safer than DNA-damaging agents. However, this selectivity typically means that each drug will only benefit a small subset of patients or tumours. Thus, there are two possible approaches

to advancing targeted treatment using small-molecule drugs. The first approach involves targeting defective pathways in cancer cells and exploiting synthetic lethal interactions that selectively kill the cancer cells. The second approach relies on identifying synergistic drug combi- nations that provide enhanced antitumor activity. Strategies that target ATR inhibition would seem to fit well within both approaches.
Preclinical data regarding VX-970, AZD6738, and BAY1895344 have
indicated that they provide selective and substantial inhibition of ATR. It is also possible that additional drugs might be developed to provide po- tent and selective inhibition of ATR with balanced physicochemical and pharmacokinetic properties. For example, VX-803/M4344 is an orally administered and potent ATRi, which was recently reported by Zenke et al. at the 2019 American Association for Cancer Research meeting, and these properties might balance any shortcomings related to the in- travenous administration of VX-970. However, it remains unclear whether intravenous administration is an issue, as most potential drug partners are also administered intravenously, and dose titration is likely simpler using intravenous methods. Clinical trials have already indi- cated that strategies targeting loss of ATM or HR deficiency are effective, which suggests that VX-970 could be especially useful for difficult can- cer cases. Examples of these cases include resistance to chemotherapy or radiotherapy, relapsed tumours, haematological cancers, and cases that require adjuvant therapy after surgical intervention. Positive find- ings from the trials evaluating ATRis may help provide oncologists with a new tool for improving patient survival in these challenging cases and give them new hope for survival.

Funding

This study was supported by the InoMed project (Reg. No. CZ.02.1.01/0.0/0.0/18_069/0010046) co-funded by the European Union and by Grant from the Czech Science Foundation (19-07674S).

Declaration of Competing Interest

The authors declare that there are no conﬂicts of interest. The au- thors also declare that the manuscript has not been published and is not under consideration for publication elsewhere.

Acknowledgment

The authors are grateful to Ian McColl MD, PhD for assistance with the manuscript.

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