molecular cancer therapeutics - the novel atr …...ddr deﬁciencies and synergistic activity in...

MOLECULAR CANCER THERAPEUTICS | SMALL MOLECULE THERAPEUTICS

The Novel ATR Inhibitor BAY 1895344 Is Efficacious asMonotherapyandCombinedwithDNADamage–Inducingor Repair–Compromising Therapies in Preclinical CancerModels A C

Antje M. Wengner, Gerhard Siemeister, Ulrich L€ucking, Julien Lefranc, Lars Wortmann, Philip Lienau,Benjamin Bader, Ulf B€omer, Dieter Moosmayer, Uwe Ebersp€acher, Sven Golfier, Christoph A. Schatz,Simon J. Baumgart, Bernard Haendler, Pascale Lejeune, Andreas Schlicker, Franz von Nussbaum,Michael Brands, Karl Ziegelbauer, and Dominik Mumberg

ABSTRACT◥

The DNA damage response (DDR) secures the integrity of thegenome of eukaryotic cells. DDR deficiencies can promote tumor-igenesis but concurrently may increase dependence on alternativerepair pathways. The ataxia telangiectasia and Rad3-related(ATR) kinase plays a central role in the DDR by activatingessential signaling pathways of DNA damage repair. Here, westudied the effect of the novel selective ATR kinase inhibitor BAY1895344 on tumor cell growth and viability. Potent antiproli-ferative activity was demonstrated in a broad spectrum of humantumor cell lines. BAY 1895344 exhibited strong monotherapyefficacy in cancer xenograft models that carry DNA damagerepair deficiencies. The combination of BAY 1895344 with DNAdamage–inducing chemotherapy or external beam radiotherapy(EBRT) showed synergistic antitumor activity. Combination

treatment with BAY 1895344 and DDR inhibitors achievedstrong synergistic antiproliferative activity in vitro, and combinedinhibition of ATR and PARP signaling using olaparib demon-strated synergistic antitumor activity in vivo. Furthermore, thecombination of BAY 1895344 with the novel, nonsteroidal andro-gen receptor antagonist darolutamide resulted in significantlyimproved antitumor efficacy compared with respective single-agent treatments in hormone-dependent prostate cancer, andaddition of EBRT resulted in even further enhanced antitumorefficacy. Thus, the ATR inhibitor BAY 1895344 may provide newtherapeutic options for the treatment of cancers with certain DDRdeficiencies in monotherapy and in combination with DNAdamage–inducing or DNA repair–compromising cancer thera-pies by improving their efficacy.

IntroductionThe DNA damage response (DDR) consists of multiple and diverse

signaling pathways that secure the integrity of the genome of livingcells (1). Proteins that directly recognize aberrant DNA structuresrecruit and activate kinases of the DDR pathways, which respond to abroad spectrumofDNAdamage and to increased replication stress, forexample, in oncogene-driven cancer cells (2–5). Many cancers harbordefects in DDR pathways, leading to genomic instability that canpromote tumorigenesis and cancer cell growth. Concurrently, thedefects in some signaling pathways may increase the dependence ofcells on alternative repair pathways.

The ataxia telangiectasia and Rad3-related (ATR) kinase belongs tothe phosphoinositide 3 kinase-related kinase family and plays a keyrole in the DNA replication stress response (RSR) pathway of DNAdamage repair to maintain the genomic integrity through DDRactivation (6–9). ATR is indispensable for cell survival by suppressing

replication origin firing, promoting deoxyribonucleotide synthesis,and preventing DNA double-strand break (DSB) formation—ultimately averting mitotic catastrophe via induction of cell-cyclearrest and DNA damage repair (10). Therefore, loss-of-functionmutations in the ATR pathway are very rarely observed, indicatingessentiality for cellular survival (11). ATR is closely related to two otherDDR kinases, ataxia telangiectasia mutated (ATM), and DNA-dependent protein kinase (DNA-PK). Together, these kinases buildthe core of the DDR by responding to different DNA damage insults,which are primarily DSBs for ATM and DNA-PK, and replicationstress (RS) for ATR (12, 13). The ATM gene is frequently mutated intumor cells, suggesting that loss of ATMactivity is beneficial for cancercell survival (11, 14). ATM kinase inactivation leads to increaseddependence on ATR signaling, and combined inactivation of ATMand ATR induces synthetic lethality in cancer cells (15, 16).

The successful use of PARP inhibitors in patients with ovarian andbreast cancer has shown that the blockade of selected DDR compo-nents is an effective strategy to treat cancers with specific types ofDNA-repair deficiencies (17). Previous studies have demonstrated thatATR inhibition is also effective for treating cancers with defectiveDDR, such as homologous recombination (HR) deficiencies (18). Inparticular, cancer cells with high levels of oncogene-driven RS, forexample, due to RAS mutation or MYC amplification, rely on ATRactivity for survival, indicating the central role of ATR in response toRS (19). These findings demonstrate the potential of ATR inhibition tomediate synthetic lethality in cancer cells with increased RS or DDRdefects.

The cancer-killing activity resulting from DNA-damagingagents such as irradiation or chemotherapy or DNA damage

Bayer AG, Pharmaceuticals, Research and Development, Berlin, Germany.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Antje M. Wengner, Bayer AG, Muellerstr. 178, BerlinD-13353, Germany. Phone: 4930-4681-5787; Fax: 4930-4681-8069; E-mail:[email protected]

Mol Cancer Ther 2020;19:26–38

doi: 10.1158/1535-7163.MCT-19-0019

�2019 American Association for Cancer Research.

AACRJournals.org | 26

on June 11, 2020. © 2020 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst October 3, 2019; DOI: 10.1158/1535-7163.MCT-19-0019

http://crossmark.crossref.org/dialog/?doi=10.1158/1535-7163.MCT-19-0019&domain=pdf&date_stamp=2020-1-10

http://crossmark.crossref.org/dialog/?doi=10.1158/1535-7163.MCT-19-0019&domain=pdf&date_stamp=2020-1-10

http://mct.aacrjournals.org/

repair–compromising therapies such as PARP inhibitors (PARPi) orantiandrogen therapy may be increased by the pharmacologic inac-tivation of ATR, as indicated in previous preclinical studies (7, 20–26).Overall, there is strong potential for ATR inhibition as potent anti-cancer therapy in tumors characterized by increased DNA damage,deficiency in DDR or increased RS (7).

Here, we disclose the structure and functional characterization ofthe novel potent and selective ATR inhibitor (ATRi) BAY 1895344,which exhibits strong monotherapy efficacy in cancers with certainDDR deficiencies and synergistic activity in combination with DNAdamage–inducing or DNA repair–compromising therapies, such aschemotherapy, external beam radiotherapy (EBRT), inhibition ofPARP, or antiandrogen treatment.

Materials and MethodsDetailed descriptions of compounds and methods are presented in

the Supplementary Materials and Methods.

CompoundsBAY1895344 (2-[(3R)-3-methylmorpholin-4-yl]-4-(1-methyl-1H-

pyrazol-5-yl)-8-(1H-pyrazol-5-yl)-1,7-naphthyridine) was identifiedand synthesized at Bayer AG (Fig. 1A) as described previously (BAY1895344 is Example 111 in patentWO2016/020320; ref. 27). Ibrutinib,olaparib, rucaparib, niraparib, talazoparib, 5-FU, cisplatin, and car-boplatin were purchased from commercial providers. AZD6738 wassynthesized at WuXi AppTec, and the analytic data of the deliveredmaterial were identical to the reported data for Example 2.02 in patentWO 2011/154737 (28). For chemical structure and additional infor-mation about AZD6738, see ref. 29. M6620 was purchased from abcrGmbH, and the analytic data of the delivered material were identicalwith the reported data for Example 57a (compound IIA-7) in patentWO 2010/071837 (30). For chemical structure and additional infor-mation about M6620, see ref. 31. Darolutamide was synthesized atOrion Corporation.

Cell lines and culture conditionsCancer cell lines were cultivated according to manufacturer's (Sup-

plementary Table S3) protocols at 37�C in a humidified atmospherecontaining 5% CO2. Cell lines were regularly subjected to DNAfingerprinting at DSMZ (Deutsche Sammlung von Mikroorganismenund Zellkulturen) and tested to be free from Mycoplasma contami-nation using MycoAlert (Lonza) directly before use. For in vitroexperiments, cells from passages 3 to 10 were used, and for in vivocell line–derived xenograft (CDX) models, cells from passages 3 to 5were used for inoculation.

Affinity and selectivity of BAY 1895344A time-resolved fluorescence resonance energy transfer (TR-

FRET)-based ATR competition binding assay was used to determinethe affinity of BAY 1895344 to ATR using fluorescent 5-TAMRA-labeled Tracer 1, an ATP-competitive ATRi (synthesized at Bayer AG;ref. 27). The ratio of the emissions at 570 and 545 nm was used toevaluate the binding affinity of BAY 1895344 to ATR.

The selectivity of BAY 1895344 was assessed using both an in-housekinase panel and a KINOMEscan Assay Panel (DiscoverX) consistingof 468 kinases, as described previously (32).

In vitro experimentsThe activity of ATR and ATM kinases was determined by

measuring phospho-Ser139 histone protein H2AX (gH2AX) levels

in hydroxyurea-treated HT-29 cells and neocarzinostatin-treatedM059J cells, respectively. PI3K/AKT/mTOR signaling pathwayactivity was investigated in MCF7 breast cancer cells by measuringAKT phosphorylation.

The antiproliferative activity of BAY 1895344 was evaluated againsta panel of 38 cancer cell lines (Supplementary Table S3). Cell prolif-erationwasmeasured after 72 to 96 hours of exposure to BAY1895344.Cell viability was determined using crystal violet staining or theCellTiter-Glo Cell Viability Assay (Promega).

The antiproliferative activity of BAY 1895344 in combination withdifferent drugs was assessed by determination of combination indexes(CI). The combination of BAY 1895344 (3–300 nmol/L) with cisplatin(100 nmol/L–10 nmol/L) was investigated in HT-29 cells, and thecombination with olaparib (300 nmol/L–30 mmol/L), niraparib (30nmol/L–3mmol/L), rucaparib (300 nmol/L–30mmol/L), or talazoparib(1–100 nmol/L) in MDA-MB-436 cells. Combination studies withBAY 1895344 (10 nmol/L–10 mmol/L) and darolutamide (10 nmol/L–10 mmol/L) were conducted in LAPC-4 cells, in the presence of thesynthetic androgen methyltrienolone R1881 (10 nmol/L). Additionalcombination studies with BAY 1895344 and a selection of compoundswere conducted in a panel of cancer cell lines (SupplementaryTable S4). Cells were treated with a single compound or a combinationof fixed compound ratios for 4 to 6 days, and viability was measuredusing CellTiter-Glo. EC50 values were calculated from triplicate valuesfor each individual combination data point, and the respective iso-bolograms were generated. CIs were calculated according to themedian-effect model (33). A CI of �0.8 was defined as more thanadditive (i.e., synergistic) interaction, and a CI of �1.2 was defined asantagonistic interaction.

The clonogenic combination assay (34) was used to assess theradiosensitization potential of BAY 1895344. LOVO colorectal cancercells were treated with 3 nmol/L BAY 1895344 and different intensitiesof g-radiation, allowed to form colonies for 10 to 14 days and, finally,the colonies were counted to calculate the combination effect.

In vivo studies in CDX modelsAll animal experiments were conducted in accordance with the

German Animal Welfare Act and approved by local authorities. Thein vivo antitumor efficacy and tolerability of BAY 1895344 as mono-therapy/combination therapy were evaluated in CDX subcutaneous ororthotopic xenograft models in mice. Monotherapy experiments wereperformed in GRANTA-519 (in female SCID beige mice), REC-1 (infemale C.B-17 SCID mice), PC-3 (in male NMRI nude mice), LOVO,andA2780 (both in femaleNMRInudemice)models treatedwithBAY1895344 at 50mg/kg [all models; twice daily, 3 days on/4 days off (3on/4off), per os/orally] or at 3, 10, or 30 mg/kg (GRANTA-519; twicedaily, 3on/4off, per os/orally), ibrutinib (REC-1; 20 mg/kg, once daily,per os/orally), AZD6738 (GRANTA-519, REC-1; 50mg/kg, once daily,per os/orally), M6620 (GRANTA-519 and REC-1; 100 mg/kg, oncedaily, per os/orally), or 5-FU (LOVO; 50 mg/kg, once weekly, intra-peritoneally). The combination of BAY 1895344 at 10 or 20 mg/kg[once daily, 2 days on/5 days off (2on/5off), per os/orally.] or 50mg/kg(twice daily, 3on/4off, per os/orally) and carboplatin (50 mg/kg, onceweekly, intraperitoneally) was investigated in IGROV-1 tumor–bearing female nude (nu/nu)mice. The combination of 20 or 50mg/kgBAY1895344 (twice daily, 2on/5off, per os/orally) and EBRT (5Gy, 7.7minutes, once daily on days 12 and 27) was investigated in LOVOtumor–bearing female NMRI nude mice. Combination therapyexperiments with 20 or 50 mg/kg BAY 1895344 (twice daily, 3on/4off, per os/orally) and 20 or 50 mg/kg olaparib (once daily, intra-peritoneally) were performed in MDA-MB-436 and 22Rv1 models in

Antitumor Effects of ATR Inhibition

AACRJournals.org Mol Cancer Ther; 19(1) January 2020 27




B

D10,000

(nmol/L)

1,000

A

C

E10,000

(nmol/L)

1,000

Figure 1.

Dose-dependent antitumor efficacy and unbound plasma levels of BAY 1895344 as monotherapy. A, Chemical structure of BAY 1895344, a highly potent andselective inhibitor of ATR. B,Growth curves of GRANTA-519mantle cell lymphoma tumors in female SCID beigemice (n¼ 10/group) treatedwith vehicle or differentdoses of BAY 1895344 (3, 10, 30, or 50mg/kg, twice daily 3on/4off, per os/orally).C,Growth curves ofGRANTA-519 tumors in female SCID beigemice (n¼ 10/group)treatedwith BAY 1895344 (50mg/kg, twice daily 3on/4off, per os/orally¼MTD), AZD6738 (50mg/kg, oncedaily, per os/orally¼MTD), orM6620 (100mg/kg, oncedaily, per os/orally¼MTD).D, Unbound BAY 1895344 plasma levels in relation to the determined biochemical, cellular mechanistic, and antiproliferative IC50 valuesof BAY 1895344 after repeated dosing with 3, 10, 30, or 50 mg/kg (twice daily, 3on/4off, per os/orally) in GRANTA-519 tumor–bearing female SCID beige mice.Different IC50 values of BAY 1895344: IC50 biochemical (TR-FRET) ¼ 7 nmol/L, IC50 cellular mechanistic (gH2AX in HT-29) ¼ 36 nmol/L, IC50 antiproliferation(GRANTA-519)¼ 30 nmol/L. E, Unbound BAY 1895344 plasma levels in relation to the respective in vitro antiproliferative IC50 values after repeated dosing with theATRis BAY 1895344 (50mg/kg, twice daily 3on/4off, per os/orally), AZD6738 (50mg/kg, oncedaily, per os/orally), andM6620 (100mg/kg, oncedaily, per os/orally)in GRANTA-519 tumor–bearing female SCID beige mice. Symbols refer to the individual measured concentrations after the final single dose on day 29 after tumorinoculation (end of last cycle); the dashed line represents the simulated concentration for 24 hours during twice daily dosing. Antiproliferative IC50 values for BAY1895344, AZD6738, and M6620 in GRANTA-519 cells are 30, 1,000, and 75 nmol/L, respectively. Relative tumor area (T/Crel.area) is defined as the percentage ofthe final tumor area on the day of vehicle group termination versus the initial tumor area (at the time point of starting treatment) of each individual mouse.Survival analysis was performed using the Cox proportional hazards model, and longitudinal data were modeled using second-order polynomial curves withrandom intercepts and slopes for each subject. Finally, dose–response analysis was performed using the three-parameter logistic model. �� , P < 0.01 versus vehicle;�� , P < 0.001 versus vehicle; ##, P < 0.001 versus M6620. p.o., per os, orally; 2 QD, twice daily.

Wengner et al.

Mol Cancer Ther; 19(1) January 2020 MOLECULAR CANCER THERAPEUTICS28




female NOD/SCID and male SCID mice, respectively. Combinationexperiments with 20 mg/kg BAY 1895344 (twice daily, 3on/4off, peros/orally) and 100mg/kg darolutamide (once daily, per os/orally) wereperformed in the hormone-dependent LAPC-4 prostate cancer modelinmale C.B-17 SCIDmice. Castratedmice served here as a control. Fora triple combination treatment, mice received EBRT (5 Gy, every7 days twice) in addition to treatment with BAY 1895344 anddarolutamide.

To elucidate the in vivo mode of action of BAY 1895344, ATR andH2AX phosphorylation was determined in lysed GRANTA-519 xeno-graft tumor samples. For the quantification of circulating ATRis,plasma samples were taken from mice and measured by LC-MS/MS.

Statistical analysisStatistical analysis was performed using a linear model estimated

with generalized least squares that included separate variance para-meters for each study group (Figs. 2 and 3). Survival analysis wasperformed using the Cox proportional hazards model, and longitu-dinal data were modeled using second-order polynomial curves withrandom intercepts and slopes for each subject. Synergy was definedaccording to the Bliss Independence Model (35, 36) in the analysis oftreatment combinations (Figs. 1, 4–6). Finally, dose–response analysiswas performed using the three-parameter logistic model (Fig. 1).

ResultsBAY 1895344 is a highly selective and potent inhibitor of ATR

BAY 1895344 (2-[(3R)-3-methylmorpholin-4-yl]-4-(1-methyl-1H-pyrazol-5-yl)-8-(1H-pyrazol-5-yl)-1,7-naphthyridine) was dis-covered in a high-throughput screening approach (Fig. 1). Thebinding affinity of BAY 1895344 to its target molecule ATR wasassessed by its ability to displace a fluorescently labeled ATR-binding tracer from a GST–ATR fusion protein using TR-FRETanalysis. The IC50 of BAY 1895344 required to displace the tracerwas 7.0 � 3.7 nmol/L, indicating a high affinity of BAY 1895344to ATR. The selectivity of BAY 1895344, evaluated in an in-housekinase selectivity panel and KINOMEscan profiling, demonstratedhigh selectivity of BAY 1895344 for ATR (Supplementary Tables S1and S2). Only for one kinase out of 31 kinases in the in-housepanel, mTOR, the determined IC50 of 35 � 32 nmol/L was of thesame order of magnitude as for ATR. Five other kinases wereinhibited with an IC50 value in the concentration range tested (<20mmol/L) and were at least 47-fold less potent compared with ATR(Supplementary Table S1). In the KINOMEscan panel, 1 mmol/LBAY 1895344 exhibited activity against only six of 456 kinases(Supplementary Table S2), and for only three of these kinases Kd

values below 1 mmol/L were determined [mTOR, cyclin G-associated kinase (GAK) and right open reading frame kinase 2(RIOK2), Kd ¼ 24, 580, and 660 nmol/L, respectively]. Takentogether, these results demonstrated the high selectivity of BAY1895344 for ATR.

BAY 1895344 potently and selectively inhibits ATR-mediatedDDR

Phospho-Ser139 histone protein H2AX (gH2AX) is a well-knownearly marker for DNA damage or DDR activation. Here, the activity ofBAY 1895344 against ATR and ATM was evaluated in a downstreamcellular mechanistic assay by determining the level of gH2AX. In HT-29 cells, BAY 1895344 inhibited hydroxyurea-induced ATR-dependent H2AX phosphorylation with an IC50 of 36 � 5 nmol/L.In neocarzinostatin-treated DNA-PK–deficient M059J cells, BAY

1895344 had no effect on ATM-dependent H2AX phosphorylationeven at 10 mmol/L, demonstrating that it is a potent and selectiveinhibitor of ATR kinase activity.

ATR and mTOR are PI3K-related protein kinases. AKT phosphor-ylation (pAKT) was used to monitor BAY 1895344–mediated mTORinhibition inMCF7 cells. BAY1895344 inhibited pAKTwith an IC50 of2.2 � 0.9 mmol/L, whereas two mTOR inhibitors, PI-103 andAZD8055, inhibited pAKT with IC50 values of 71 � 31 nmol/L and12� 4 nmol/L, respectively. Hence, BAY 1895344 was 30- to 180-foldless active than specific mTOR inhibitors and showed superior selec-tivity for ATR compared with the PI3K/AKT/mTOR pathway (>60-fold).

BAY 1895344 inhibits proliferation in a broad spectrum ofhuman cancer cell lines

In a panel of cancer cell lines of different histologic origins har-boring various mutations affecting DDR pathways, BAY 1895344showed potent antiproliferative activity with IC50 values ranging from9 to 490 nmol/L (Supplementary Fig. S1; Supplementary Table S3). Aclear difference between highly sensitive (IC50 < 100 nmol/L) and lesssensitive cell lines was observed, and a majority of the sensitive celllines were found to carry mutations affecting the ATM pathwayindependent of their origin (Supplementary Fig. S1A). Lymphomaappeared to be the most sensitive cancer type with IC50 values rangingfrom 9 to 32 nmol/L in mantle cell lymphoma cell lines (Supplemen-tary Fig. S1B).

BAY 1895344 shows strong antitumor efficacy as monotherapyin CDX models

Antitumor efficacy of BAY 1895344 was evaluated in CDX modelsin mice. Dose-dependent efficacy was observed in the GRANTA-519mantle cell lymphoma model, and the MTD and optimal dosingschedule of BAY 1895344 were identified as 50 mg/kg applied twicedaily for 3on/4off (Fig. 1B). In the GRANTA-519 model, treatmentwith BAY 1895344 applied at MTD demonstrated strong antitumorefficacy, whereas the ATR inhibitors AZD6738 and M6620 applied atMTD achieved only moderate antitumor efficacy (Fig. 1C).

When BAY 1895344 was dosed repeatedly (twice daily) at 30 or50 mg/kg to GRANTA-519 tumor–bearing mice, the level of unboundBAY 1895344 in plasma covered the biochemical, cellularmechanistic,and antiproliferative IC50 concentrations determined in vitro, which isin line with the observed strong in vivo antitumor efficacy (Fig. 1D).Even at the highest applied dose (MTD), the level of unbound BAY1895344 (1.6 mmol/L) was not sufficient to cover the IC50 concentra-tion of 2.2 mmol/L required for the cellular inhibition of the PI3K/AKT/mTOR signaling pathway, suggesting that the antitumor activityof BAY 1895344 was not mediated by mTOR pathway inhibition. Thelevel of unbound AZD6738 or M6620 in plasma did not exceed theantiproliferative IC50 concentrations, indicating that exposure to theseATRis may not have been sufficient to mediate antitumor activityin vivo (Fig. 1E).

Antitumor efficacy of BAY 1895344 as a single agent was furtherevaluated in CDX models of different indications characterized byDDR defects or oncogenes potentially mediating RS (SupplementaryTable S3). BAY 1895344 applied at MTD resulted in strong antitumorefficacy as demonstrated in the A2780 ovarian cancer, PC-3 prostatecancer, LOVO colorectal cancer, and REC-1 mantle cell lymphomamodels (Fig. 2). The efficacy of BAY 1895344 clearly exceeded theefficacy of other treatments, as 5-FU (5-fluorouracil) failed to inhibitLOVO tumor growth, and AZD6738, M6620, and the BTK inhibitoribrutinib showed significantly weaker efficacy (P < 0.001) in the REC-1






Figure 2.

Antitumor efficacy of BAY 1895344 asmonotherapy. The optimal dose andtreatment schedule for BAY 1895344was50 mg/kg (twice daily 3on/4off, per os/orally), based on efficacy and tolerabilityin the GRANTA-519 human mantle celllymphoma xenograft model in femaleSCID beige mice. 5-FU, ibrutinib,AZD6738, and M6620 were used at theirknown MTDs on the basis of publisheddata (39, 44). All treatments were welltolerated without toxicities (no death,acceptable body weight loss). A, Growthcurves ofA2780 human ovarian tumors infemale NMRI nude mice (n ¼ 10/group)treated with vehicle or BAY 1895344. B,Relative tumor areas (T/Crel.area) of theA2780 tumors shown inA at study end.C,Growth curves of PC-3 human prostatetumors in male NMRI nude mice (n ¼ 10/group) treated with vehicle or BAY1895344. D, Relative tumor areas of thePC-3 tumors shown in C at study end. E,Growth curves of LOVOhuman colorectaltumors in female NMRI nude mice (n ¼10/group) treated with vehicle, BAY1895344, or 5-FU (50 mg/kg, once week-ly, intraperitoneally). F, Relative tumorareas of the LOVO tumors shown in E atstudy end. G, Growth curves of REC-1human mantle cell lymphoma tumors infemale C.B-17 SCID mice (n ¼ 10/group)treated with vehicle, BAY 1895344, ibru-tinib (20 mg/kg, once daily, per os/oral-ly), AZD6738 (50 mg/kg, once daily, peros/orally), or M6620 (100 mg/kg, oncedaily, per os/orally). H, Relative tumorareas of the REC-1 tumors shown in G onday 28 after tumor inoculation. Relativetumor area is defined as the percentageof the final tumor area on day of controltermination versus the initial tumor area(at the time point of starting treatment)of each individual mouse. Statistical anal-ysis was performed using a linear modelestimated with generalized least squaresthat included separate variance para-meters for each study group. �� , P <0.001 versus vehicle; 5-FU, 5-fluorouracil;i.p., intraperitoneally; PD, progressive dis-ease; p.o., per os/orally; PR, partialresponse; QD, once daily; 2 QD, twicedaily; QW, once weekly; SD, stabledisease.

Wengner et al.





model. All treatments with BAY 1895344 were well tolerated over atreatment time of 11 to 59 days without toxicities and acceptable bodyweight loss (10%–13%; Supplementary Fig. S2).

To analyze the association between in vivo antitumor efficacy andthe inhibition of ATR, ATR phosphorylation (pATR) as a directmeasure of ATR kinase activity (37) and H2AX phosphorylation as

an indirect measure of ATR-mediated DNA repair were determined inGRANTA-519 tumor xenografts after treatment with BAY 1895344 atdifferent doses and dosing schedules. pATR levels decreased remark-ably 24 to 48 hours after a single or repeated treatment with 50 mg/kgBAY 1895344, demonstrating direct ATR kinase inhibition (Fig. 3A).gH2AX levels clearly increased 3 to 48 hours after a single or repeated

A B

C

D

Figure 3.

Induction of ATR phosphorylation (pATR) and DNA damage in GRANTA-519 xenograft tumors after treatment with BAY 1895344. Intratumor pATR (A) and gH2AXprotein levels (B–D) were determined in GRANTA-519 tumors in female SCID beigemice at different time points (n¼ 3–4) after last treatmentwith BAY 1895344 (peros/orally) at 50mg/kg, oncedaily or twice daily for 2 days followedby oncedaily for 1 day (A), 50mg/kg, once daily or twice daily for 2 days followedbyonce daily for1 day (B), 10 or 30 mg/kg, once daily or 3, 10, or 30 mg/kg, twice daily for 2 days followed by once daily for 1 day (dose-dependent gH2AX phosphorylation; C), or30mg/kg for 1 to 3 treatment cycles: twice daily for 2 days followed by once daily for 1 day, twice daily for 3 days on and 4 days off followed by twice daily for 2 daysandoncedaily for one day, twice daily for 3 days on and4 daysoff followedby another twice daily for 3 dayson and4days off and twice daily for 2 days andonce dailyfor 1 day (schedule-dependent gH2AX phosphorylation; D). p.o., per os/orally; QD, once daily; 2 QD, twice daily.






Figure 4.

Antitumor efficacy of BAY 1895344 in combination with chemotherapy or EBRT. A, Isobologram for the in vitro combination effect of BAY 1895344 and cisplatin onthe proliferation of human HT-29 colorectal cancer cells (n ¼ 3). B, Growth curves of IGROV-1 human ovarian tumors in female nude (nu/nu) mice (n ¼ 10/group)treated with BAY 1895344 (10 or 20mg/kg, once daily, 2 days on/ 5 days off, per os/orally; 50mg/kg, twice daily, 3 days on/4 days off, per os/orally) in combinationwith carboplatin (50 mg/kg, once weekly, intraperitoneally). C,Weights of the IGROV-1 tumors described in B at study end. D, Survival curves of LOVO colorectalcancer cells after treatment with 3 nmol/L BAY 1895344 and different doses of X-ray radiation determined using the clonogenic assay. E, Growth curves of LOVOhuman colorectal tumors in female nude mice (n ¼ 10/group) treated with BAY 1895344 (20 or 50 mg/kg, twice daily 2on/5off, per os/orally) in combinationwith g-radiation (EBRT). EBRT (5 Gy) was applied directly on the subcutaneously growing tumors on days 12 and 27 after tumor inoculation. Relative tumor area(T/Crel.area) is defined as the percentage of the final tumor area on day of control termination versus the initial tumor area (at the time point of starting treatment) ofeach individual mouse. Asterisks indicate statistical significance, evaluated by survival analysis using the Cox proportional hazardsmodel and longitudinal dataweremodeled using second-order polynomial curves with random intercepts and slopes for each subject. � , P < 0.05; �� , P < 0.01; �� , P < 0.001 versus vehicle unlessindicated otherwise. EC50, half-maximal effective concentration; Gy, Gray; i.p. intraperitoneally; p.o., per os/orally; QD, oncedaily; 2QD, twice daily; QW, onceweekly.

Wengner et al.





treatment with 50 mg/kg BAY 1895344, indicating that ATR kinaseinhibition may induce DNA damage by abrogation of DDR signaling(Fig. 3B).

Further studies demonstrated that the BAY 1895344-evokedincrease in H2AX phosphorylation was time-, dose-, and dosingschedule–dependent (Fig. 3C and D). A single dose (once daily �1) of 30mg/kg BAY1895344 increased the level of gH2AXpeaking at 8to 24 hours (Fig. 3C), whereas a single dose of 10 mg/kg or repeateddosing with 3 mg/kg (twice daily � 2 þ once daily � 1) was notsufficient to block ATR and cause DNA damage. In contrast, repeateddoses of 10 or 30mg/kg BAY 1895344 increased and prolonged H2AXphosphorylation compared with a single dose of 30 mg/kg. Increasingtreatment cycles with BAY 1895344 led to decreased levels of H2AXphosphorylation (Fig. 3D), highlighting the importance of the timepoint chosen for the determination of a pharmacodynamic signal(gH2AX) to demonstrate target engagement.

In summary, BAY 1895344 displayed strong activity as monother-apy in different CDX models. Dose-dependent antitumor efficacyin vivo correlated with compound exposure in plasma, the reductionof ATR phosphorylation and the induction of DNA damage, dem-onstrating the anticipatedmechanism of action ofATR inhibitionwithBAY 1895344.

BAY 1895344 shows synergistic activity in combination withchemotherapy or EBRT

To assess the potential synergistic activity of ATR inhibition withdifferent DNA damage–inducing chemotherapeutic agents or thetubulin stabilizer docetaxel, antiproliferation-based in vitro combina-tion studies were performed in cancer cell lines of different geneticbackgrounds and tumor types. In HT-29 colorectal cancer cells, theinteraction of BAY 1895344 with cisplatin was strongly synergisticwith a CI of 0.14 (Fig. 4A). Further combinations with cisplatin,bleomycin, or SN-38 were also strongly synergistic (SupplementaryTable S4). In contrast, the combination of docetaxel and BAY 1895344achieved a CI of 1.26, indicating an antagonistic interaction. The CIsfor all in vitro combination treatments are summarized in Supple-mentary Table S4.

The in vivo antitumor efficacy of BAY 1895344 in combination withthe chemotherapy drug carboplatin was investigated in the IGROV-1ovarian cancermodel.Very lowdoses of BAY1895344 (10 or 20mg/kg,once daily, 2on/5off ¼ 7% or 14% of MTD) had to be used incombination treatment with carboplatin at MTD to achieve tolera-bility. BAY 1895344 monotherapy at MTD showed good antitumorefficacy, whereas sub-MTDdoses of BAY1895344 or carboplatin aloneshowed onlyweak efficacy. Combination treatment with BAY1895344and carboplatin clearly enhanced antitumor efficacy pointing toward asynergistic effect (80% confidence; Fig. 4B and C) at acceptabletolerability (Supplementary Fig. S3A). Although the combinationtreatments were more effective than respective monotherapies, theefficacy of the tolerated combination treatment did not exceed tumorgrowth inhibition achieved with BAY 1895344 monotherapy at MTD.

The in vitro radiosensitization potential of BAY 1895344 wasassessed in LOVO colorectal cancer cells using the clonogenic assay.Treatment with a noneffective dose of BAY 1895344 in combinationwith different intensities of g-radiation clearly reduced the colony-forming ability of LOVO cells compared with radiation alone, con-firming the radiosensitization effect of BAY 1895344 (Fig. 4D).

The in vivo antitumor efficacy of BAY 1895344 in combination withEBRT was investigated in LOVO colorectal cancer xenografts. Sub-MTD treatment with BAY 1895344 (20 or 50 mg/kg, twice daily, 2on/5off ¼ 30% or 60% of MTD) or EBRT alone resulted in weak to

moderate antitumor efficacy. Combination treatment with each testedBAY 1895344 dose and EBRT showed strong antitumor efficacy and asynergistic effect (95% confidence, Fig. 4D). Moreover, combinationtreatment with 50 mg/kg BAY 1895344 and EBRT was more effica-cious than radiation alone, as determined on the day of combinationgroup termination. All treatments in the LOVO model were welltolerated with a maximum body weight loss of <10% (SupplementaryFig. S3B).

The rationale for the combination of BAY 1895344 with DNAdamage–inducing cancer therapies is supported by the observedsynergistic interactions. The observed radiosensitizing effect of ATRinhibition, that however does not have an impact on the safety ofEBRT, supports further clinical assessment. However, the tolerabilityof simultaneous systemic chemotherapy-induced DNA damage andATR inhibition needs careful evaluation to minimize safety risks.

BAY 1895344 shows additive to synergistic activity incombination with different DDR inhibitors in vitro and synergywith the PARP1 inhibitor olaparib in vivo

The antiproliferative activity of BAY 1895344 was furtherassessed in combination with different DDR pathway inhibitors(DDRi) in cancer cell lines of different genetic backgrounds andtumor types. To assess the potential synergistic activity of ATRinhibition together with PARP inhibition, BRCA1-deficient MDA-MB-436 breast cancer cells were treated with BAY 1895344 incombination with the PARPis olaparib, niraparib, rucaparib, andtalazoparib. The in vitro interaction of BAY 1895344 with all testedPARPis was strongly synergistic (Fig. 5A–D). Further combinationswith DDRis targeting ATM, CHEK1, DNA-PK, and WEE1 dem-onstrated additive to highly synergistic interactions in ATM- andMRE11A-proficient HT29 and PC-3 cells (CIs summarized inSupplementary Table S4).

The in vivo antitumor efficacy of BAY 1895344 in combinationwiththe PARPi olaparib was investigated using two different CDXmodels.In the PARPi-sensitive MDA-MB-436 breast cancer model, mono-therapywithBAY1895344 atMTDor sub-MTD(40%; 50 or 20mg/kg,twice daily, 3on/4off) or olaparib at MTD (50 mg/kg, once daily)effectively inhibited tumor growth. Combination treatment with BAY1895344 at sub-MTD and olaparib at MTD showed potent antitumorefficacy, pointing toward a synergistic combination effect (90.5%confidence; Fig. 5E and F). All treatments in theMDA-MB-436modelwere well tolerated with a maximum body weight loss of <10%(Supplementary Fig. S3C). In the PARPi-resistant 22Rv1 prostatecancer model, monotherapy with BAY 1895344 at MTD or sub-MTD (40%) resulted in moderate or weak antitumor efficacy. Mono-therapy with olaparib at MTD or sub-MTD (40%) failed to inhibittumor growth, confirming the PARPi resistance of this model. Com-bination treatment with BAY 1895344 and olaparib at sub-MTDs(40%) delayed tumor growth compared with vehicle and olaparibmonotherapy, indicating a synergistic combination effect (95% con-fidence level; Fig. 5G andH). When looking at final tumor weight, thecombination treatment showed improved efficacy also compared withthe respective BAY 1895344 monotherapy at sub-MTD (Fig. 5H).However, the efficacy achieved by BAY1895344monotherapy atMTDwas comparable with the efficacy achieved by combination therapy inthe PARPi-resistant tumor model 22Rv1. All treatments were welltolerated with a maximum body weight loss of <10% (SupplementaryFig. S3D).

These results support the rationale for the combination of BAY1895344 treatment with DNA repair–compromising cancer therapies.However, the tolerability of simultaneous systemic inhibition of






Figure 5.

Antitumor efficacy of BAY 1895344 incombination with PARPis. Isobologramsfor the in vitro combination effect of BAY1895344 and the PARPis olaparib (A),niraparib (B), rucaparib (C), and talazo-parib (D) on the proliferation of humanMDA-MB-436 breast cancer cells (n ¼ 3).E, Growth curves of MDA-MB-436 humanbreast cancer tumors in femaleNOD/SCIDmice (n ¼ 10/group) treated with BAY1895344 (20 or 50 mg/kg, twice daily,3 days on/4 days off, per os/orally) incombination with olaparib (50 mg/kg,intraperitoneally, once daily). F, Weightsof theMDA-MB-436 tumors described inEat study end. G, Growth curves of 22Rv1human prostate tumors inmale SCIDmice(n¼ 10/group) treated with BAY 1895344(20 or 50 mg/kg, twice daily, 3 days on/4 days off, per os/orally) in combinationwith olaparib (20 or 50mg/kg, once daily,intraperitoneally).H,Weights of the 22Rv1tumors described in G at study end. Rel-ative tumor area (T/Crel.area) is defined asthe percentage of the final tumor area onday of control termination versus the ini-tial tumor area (at the timepoint of start-ing treatment) of each individual mouse.Asterisks indicate statistical significance,evaluated by survival analysis using theCox proportional hazards model and lon-gitudinal data were modeled using sec-ond-order polynomial curves with ran-dom intercepts and slopes for each sub-ject. � , P < 0.05; �� , P < 0.01; �� , P < 0.001versus vehicle unless indicated otherwise.EC50, half-maximal effective concentra-tion; i.p. intraperitoneally; p.o., per os/orally; QD, once daily; 2 QD, twice daily.

Wengner et al.





parallel DDR pathways needs careful evaluation of dose and treatmentschedule to ensure safety.

BAY 1895344 shows improved antitumor activity incombination with the AR antagonist darolutamide in vitro andin vivo

In LAPC-4 prostate cancer cells, the in vitro interaction of BAY1895344 with the AR antagonist darolutamide achieved a CI of 0.82,indicating an additive interaction (Fig. 6A). Furthermore, combina-tion treatment with BAY 1895344 and darolutamide led to morepronounced downregulation of several genes involved in DNA repair,including BRCA1, EXO1, andMCM10, in comparison with respectivemonotherapies (Supplementary Fig. S4).

In the hormone-dependent LAPC-4 CDX model, BAY 1895344monotherapy at sub-MTD (40%) showed a weak to moderate anti-tumor effect, whereas darolutamide monotherapy exhibited goodantitumor efficacy. Combination treatment with BAY 1895344 anddarolutamide resulted in enhanced antitumor efficacy, showing sig-nificant improvement compared with darolutamide monotherapy onthe day of combination group termination (Fig. 6B). All treatmentswere well tolerated with a maximum body weight loss of <10%

(Supplementary Fig. S3E). The triple combination of BAY 1895344,darolutamide, and EBRT resulted in further enhanced antitumorefficacy compared with respective monotherapies and all dual com-bination treatments and was even more effective than castration(Fig. 6C).

These results support the rationale for the combination treatment ofBAY 1895344 with antiandrogen therapy and the addition of EBRTmay even further enhance the efficacy of this combination.

DiscussionCancer cells rely onDDR pathways to survive genomic instability in

particular by activating ATR kinase (38). In this study, we have shownthat BAY 1895344 is a selective low-nanomolar inhibitor of ATRkinase activity, which potently inhibits the proliferation of a broadspectrum of human tumor cell lines of different origins harboringDDR defects, including ATM aberrations. We have demonstratedstrong in vivo antitumor efficacy of BAY 1895344 in a variety ofxenograft models of different tumor indications carrying defects inDNA repair, thus validating the concept of synthetic lethality oftumor-intrinsic DNA-repair deficiencies and ATR blockade.

A

C

BFigure 6.

Antitumor efficacy of BAY 1895344 incombination with antiandrogen ther-apy. A, Isobologram for the in vitrocombination effect of BAY 1895344and darolutamide on the proliferationof human LAPC-4 prostate cancercells (n ¼ 3). B, Growth curves ofhormone-dependent LAPC-4 humanprostate tumors in male SCID mice(n ¼ 11/group) treated with BAY1895344 (20 mg/kg, twice daily 3on/4off, per os/orally) in combinationwith darolutamide (100 mg/kg, oncedaily, per os/orally). C, Growth curvesof LAPC-4 tumors in male SCID mice(n ¼ 10/group) treated with BAY1895344 (20 mg/kg, twice daily 3on/4off, per os/orally) in combinationwith darolutamide (100 mg/kg, oncedaily, per os/orally) and EBRT (5 Gy,every 7 days twice). Relative tumorarea (T/Crel.area) is defined as the per-centage of the final tumor area on dayof control termination versus the initialtumor area (at the time point of start-ing treatment) of each individualmouse. Asterisks indicate statisticalsignificance, evaluated by survivalanalysis using the Cox proportionalhazards model and longitudinal datawere modeled using second-orderpolynomial curves with random inter-cepts and slopes for each subject.� , P < 0.05; �� , P < 0.01; �� , P <0.001 versus vehicle or as indicated.EC50, half-maximal effective concen-tration; Gy, Gray; p.o., per os/orally;QD, once daily; 2 QD, twice daily;Q7D�2, every 7 days twice.






However, further preclinical and clinical investigations are required tounderstand which DDR defects have the strongest impact on thesensitivity to ATR inhibition.

BAY 1895344 demonstrated superiority compared with the ATRisAZD6738 and M6620 (6, 29, 39). Its high potency and selectivityresulted in stronger antitumor activity as monotherapy in a variety oftumor types with DDR defects. We could demonstrate that the plasmaexposure of BAY 1895344 clearly covered the antiproliferative con-centration levels (IC50) of tumor cells. In contrast, the plasma exposureof AZD6738 andM6620 did not exceed the concentrations needed forantiproliferative activity in tumor cells, which is in line with their weakin vivo antitumor efficacy in monotherapy (Fig. 1C and E).

In addition to its potential asmonotherapy, BAY1895344 is likely tobe particularly efficacious in combination treatment with DNA dam-age–inducing or DNA repair–compromising therapies, as indicated inearlier studies (40–44). Here, we have demonstrated in preclinicaltumor models that BAY 1895344 exhibits synergistic antitumorefficacy with EBRT in colorectal cancer, with the PARPi olaparib inPARPi-sensitive, HR-defective (BRCA1-mutant) breast cancer as wellas in PARPi-resistant prostate cancer and enhanced antitumor activityin combination with the AR antagonist darolutamide in hormone-dependent prostate cancer. The combination of BAY 1895344 with thechemotherapeutic drug carboplatin was associated with dose-dependent toxicity. This restricted overall antitumor efficacy andlimited the potential therapeutic value of this combination, in partic-ular because BAY 1895344 monotherapy applied at the MTD wasmore efficacious than the combination therapy with carboplatin.

The efficacy of EBRT in cancer therapy is mainly due to theability of ionizing radiation to produce DSBs leading to cell death intumor tissue. However, the use of EBRT is limited by the sensitivityof normal healthy tissue to radiation, which can cause acute andchronic toxicities. As ionizing radiation also causes an extensiveamount of less cytotoxic DNA lesions, such as DNA single-strandbreaks (SSB; ref. 45), recent research has focused on the radio-sensitization of tumors by combining radiation with DDR-targetedagents, and these studies have demonstrated promising preclinicalefficacy (25, 46–49). In this study, BAY 1895344 and EBRT com-bination treatment of LOVO colorectal cancer xenografts, whichhave several defects in DDR and mismatch repair (SupplementaryTable S3), increased the efficacy of EBRT without affecting toler-ability or increasing toxicity. This mechanism-based potential ofcombining EBRT-induced DNA damage and blockade of ATR-mediated DNA repair with BAY1895344 suggests a powerful newtreatment option for patients resistant to radiation, and a possibilityto widen the therapeutic window of radiotherapy.

PARPis prevent the repair of SSBs by trapping inactivated PARPonto SS-DNA, resulting in the generation of DSBs during DNAreplication, thus activating DNA-repair processes mediated by HR.In tumors with a homologous recombination deficiency (HRD), forexample, a BRCA1/2 mutation, PARPi-induced accumulation ofreplication errors and DSBs leads to increased genetic instability, andultimately, cell death (50). The first approval of a PARPi, olaparib, wasgranted by the FDA in 2014 for use in patients with ovarian cancerwithdeleterious/suspected deleterious germline BRCA1/2mutations.How-ever, the efficacy of PARPis is limited by drug resistance (51–53). Notall BRCA1/2 mutation carriers respond to PARP inhibition, and evenresponders often relapse. Different mechanisms potentially causingPARPi resistance have been described (51, 54, 55). Previous studieshave demonstrated that ATR inhibition may be effective in treatingcancers with HRD (18). However, ATR is also involved in HR-independent repair pathways indicating the potential of ATRis to

improve the antitumor activity of PARPi. PARP and ATR bind toDNA SSBs and initiate distinct DNA-repair mechanisms to inhibitcytotoxic DNA DSB formation. PARP inhibition blocks the DNA-repair complex formation via base-excision repair and induces PARPtrapping, which leads to cytotoxic PARP–DNA complexes resulting inDNA breaks. ATR inhibition affects the activation of different DNA-repair pathways, including HR, as part of the RSR. Combined inhi-bition of ATR and PARP is expected to be highly synergistic byblocking central, but independent, DNA-repair pathways. Therefore,combined inhibition of ATR and PARP could help patients who haverelapsed on PARPi therapy by overcoming PARPi resistance but couldalso enhance antitumor activity in PARPi-sensitive patients. Overall,combined inhibition of ATR and PARP represents a valuable strategyfor the improvement of PARPi therapy (56). Here, combined inhibi-tion of ATR with BAY 1895344 and PARP with olaparib resulted insignificant reduction of tumor size in PARPi-sensitive, HR-deficientMDA-MB-436 breast cancer xenografts, and in PARPi-resistant22Rv1 prostate cancer xenografts. Parallel inhibition of different DDRpathways by combining a PARPi, such as olaparib, with the ATRkinase inhibitor BAY 1895344, thus presents novel treatment oppor-tunities in patients with cancer with HRDs sensitive or resistant toPARPi therapy. In fact, a combination of an ATRi with a PARPi iscurrently under clinical investigation (NCT03462342). Other DDRicombinationsmay also have clinical impact, as indicated in our in vitrocombination studies where the interaction of BAY 1895344 withDNA-PK, CHK1/2, and WEE1 inhibitors was strongly synergistic.However, well-tolerated and efficacious combination treatment regi-mens need to be established first (57, 58).

AR signaling is a key regulator of theDDR inprostate cancer (21, 24).In recent studies, androgen depletion has been shown to result in thedownregulation of DDR genes, impairing DNA repair and increasingsensitivity to radiation (24). AR activity has also been reported to beinduced by DNA damage, promoting the resolution of DSBs (59).Thus, AR inhibition can contribute to anHRD phenotype, resulting insensitivity to DDR inhibition. For example, the AR antagonist enza-lutamide in combination with the PARPi olaparib downregulated theexpression of DDR genes, resulting in reduced HR efficiency in AR-positive prostate cancer cells (22). Furthermore, combination treat-ment of enzalutamide with the Chk1/2 inhibitor AZD7762 showedsynergistic activity in the inhibition of AR–CDC6–ATR–Chk1 sig-naling, induction of ATM phosphorylation, and apoptosis in hor-mone-dependent prostate cancer cells (21). Here, we investigated forthe first time the combination of ATR inhibition and antiandrogentherapy. Combined treatment with BAY 1895344 and darolutamide, anovel nonsteroidal AR antagonist (60) that has successfully completeda clinical phase III trial in the indication of nonmetastatic prostatecancer (61), demonstrated significantly reduced expression of DDRgenes in prostate cancer cells in vitro and significantly improvedantitumor efficacy in the hormone-dependent LAPC-4 prostate cancerCDXmodel in vivo. Adding EBRT to the combination treatment evenfurther enhanced the therapeutic efficacy. Therefore, induction ofDNA damage by radiation and reduced DNA-repair capability by theAR antagonist darolutamide combined with the ATR inhibitor BAY1895344 could present a new treatment option for patients withhormone-dependent prostate cancer.

In summary, BAY 1895344 is a novel ATR kinase inhibitor thatexhibits strongmonotherapy efficacy in cancers withDDRdeficienciesof different tumor histologies, as well as synergistic activity in com-bination with DNA damage–inducing or DNA repair–compromisingtherapies. This may provide new treatment options and improve thetherapeutic efficacy of standard-of-care therapies. BAY 1895344 is

Wengner et al.





currently under clinical investigation in patients with advanced solidtumors and lymphomas (NCT03188965).

Disclosure of Potential Conflicts of InterestAll authors are employed at and have ownership interest (including patents) in

Bayer AG.

Authors’ ContributionsConception and design: A.M. Wengner, G. Siemeister, U. L€ucking, J. Lefranc,L. Wortmann, P. Lienau, U. Ebersp€acher, P. Lejeune, F. von Nussbaum, D. MumbergDevelopment of methodology: A.M.Wengner, G. Siemeister, U. L€ucking, J. Lefranc,L. Wortmann, D. Moosmayer, U. Ebersp€acher, B. Haendler, P. LejeuneAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): A.M. Wengner, G. Siemeister, J. Lefranc, U. B€omer, U. Ebersp€acher,S. Golfier, C.A. Schatz, S.J. Baumgart, B. Haendler, P. LejeuneAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.M. Wengner, G. Siemeister, J. Lefranc, L. Wortmann,P. Lienau, B. Bader, U. B€omer, U. Ebersp€acher, C.A. Schatz, S.J. Baumgart,B. Haendler, P. Lejeune, A. Schlicker, F. von NussbaumWriting, review, and/or revision of the manuscript: A.M. Wengner,G. Siemeister, U. L€ucking, J. Lefranc, L. Wortmann, P. Lienau, B. Bader,

U. B€omer, U. Ebersp€acher, B. Haendler, P. Lejeune, A. Schlicker, F. vonNussbaum, K. Ziegelbauer, D. MumbergAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): A.M. Wengner, U. L€ucking, J. Lefranc, U. Ebersp€acherStudy supervision: A.M. Wengner, J. Lefranc, P. Lienau, P. Lejeune, M. Brands,K. ZiegelbauerOther (synthesis of BAY 1895344): U. L€ucking

AcknowledgmentsAurexel Life Sciences Ltd. (www.aurexel.com) is thanked for editorial assistance in

the preparation of this article, funded by Bayer AG. Darolutamide is jointly developedby Bayer AG and Orion Corporation. Special thanks go to Lisa Ehremann, StephanieOsterberg, Sarah Boettcher, Angela Bieseke, Nils Guthof, Tatsuo Sugawara, BerndHartmann, Lisa Bartnitzky and Kathleen Stadelmann for excellent technical support.This work was supported by Bayer AG.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 14, 2019; revised July 5, 2019; accepted September 27, 2019;published first October 3, 2019.

References1. Ciccia A, Elledge SJ. The DNA damage response: making it safe to play with

knives. Mol Cell 2010;40:179–204.2. Karnitz LM, Zou L. Molecular pathways: targeting ATR in cancer therapy.

Clin Cancer Res 2015;21:4780–5.3. Marechal A, Zou L. DNA damage sensing by the ATM and ATR kinases.

Cold Spring Harb Perspect Biol 2013;5. doi: 10.1101/cshperspect.a012716.4. Taylor EM, Lindsay HD. DNA replication stress and cancer: cause or cure?

Future Oncol 2016;12:221–37.5. Yazinski SA, Zou L. Functions, regulation, and therapeutic implications of the

ATR checkpoint pathway. Annu Rev Genet 2016;50:155–73.6. Foote KM, Lau A, Nissink JWM. Drugging ATR: progress in the development of

specific inhibitors for the treatment of cancer. FutureMed Chem 2015;7:873–91.7. Lecona E, Fernandez-Capetillo O. Targeting ATR in cancer. Nat Rev Cancer

2018;18:586–95.8. Mei L, Zhang J, He K, Zhang J. Ataxia telangiectasia and Rad3-related inhibitors

and cancer therapy: where we stand. J Hematol Oncol 2019;12:43.9. Pollard J, Curtin N, editors. Targeting the DNAdamage response for anti-cancer

therapy. 1st ed. Totowa, NJ: Humana Press; 2018.10. Durocher D, Jackson SP. DNA-PK, ATM and ATR as sensors of DNA damage:

variations on a theme? Curr Opin Cell Biol 2001;13:225–31.11. Gaillard H, Garcia-Muse T, Aguilera A. Replication stress and cancer. Nat Rev

Cancer 2015;15:276–89.12. Cimprich KA, Shin TB, Keith CT, Schreiber SL. cDNA cloning and gene

mapping of a candidate human cell cycle checkpoint protein. Proc Natl AcadSci U S A 1996;93:2850–5.

13. Bentley NJ, HoltzmanDA, Flaggs G, Keegan KS, DeMaggio A, Ford JC, et al. TheSchizosaccharomyces pombe rad3 checkpoint gene. EMBO J 1996;15:6641–51.

14. Choi M, Kipps T, Kurzrock R. ATM mutations in cancer: therapeutic implica-tions. Mol Cancer Ther 2016;15:1781–91.

15. Menezes DL, Holt J, Tang Y, Feng J, Barsanti P, Pan Y, et al. A synthetic lethalscreen reveals enhanced sensitivity to ATR inhibitor treatment in mantle celllymphoma with ATM loss-of-function. Mol Cancer Res 2015;13:120–9.

16. Min A, Im SA, Jang H, Kim S, Lee M, Kim DK, et al. AZD6738, a novel oralinhibitor of ATR, induces synthetic lethality with ATM deficiency in gastriccancer cells. Mol Cancer Ther 2017;16:566–77.

17. Bouwman P, Jonkers J. The effects of deregulated DNA damage signalling oncancer chemotherapy response and resistance. Nat Rev Cancer 2012;12:587–98.

18. KrajewskaM, Fehrmann RS, Schoonen PM, Labib S, de Vries EG, Franke L, et al.ATR inhibition preferentially targets homologous recombination-deficienttumor cells. Oncogene 2015;34:3474–81.

19. Gilad O, Nabet BY, Ragland RL, Schoppy DW, Smith KD, Durham AC, et al.Combining ATR suppression with oncogenic Ras synergistically increasesgenomic instability, causing synthetic lethality or tumorigenesis in a dosage-dependent manner. Cancer Res 2010;70:9693–702.

20. Charrier JD, Durrant SJ, Golec JM, Kay DP, Knegtel RM, MacCormick S, et al.Discovery of potent and selective inhibitors of ataxia telangiectasia mutated andRad3 related (ATR) protein kinase as potential anticancer agents. J Med Chem2011;54:2320–30.

21. Karanika S, Karantanos T, Li L, Wang J, Park S, Yang G, et al. Targeting DNAdamage response in prostate cancer by inhibiting androgen receptor-CDC6-ATR-Chk1 signaling. Cell Rep 2017;18:1970–81.

22. Li L, ChangW, Yang G, Ren C, Park S, Karantanos T, et al. Targeting poly(ADP-ribose) polymerase and the c-Myb-regulated DNA damage response pathway incastration-resistant prostate cancer. Sci Signal 2014;7:ra47.

23. Pires IM, Olcina MM, Anbalagan S, Pollard JR, Reaper PM, Charlton PA, et al.Targeting radiation-resistant hypoxic tumour cells through ATR inhibition. Br JCancer 2012;107:291–9.

24. Polkinghorn WR, Parker JS, Lee MX, Kass EM, Spratt DE, Iaquinta PJ, et al.Androgen receptor signaling regulates DNA repair in prostate cancers.Cancer Discov 2013;3:1245–53.

25. Reaper PM, Griffiths MR, Long JM, Charrier JD, Maccormick S, Charlton PA,et al. Selective killing of ATM- or p53-deficient cancer cells through inhibition ofATR. Nat Chem Biol 2011;7:428–30.

26. Li L, Karanika S, Yang G, Wang J, Park S, Broom BM, et al. Androgen receptorinhibitor-induced "BRCAness" and PARP inhibition are synthetically lethal forcastration-resistant prostate cancer. Sci Signal 2017;10. doi: 10.1126/scisignal.aam7479.

27. Wortmann L, L€ucking U, Lefranc J, Briem H, Koppitz M, Eis K, et al.inventors; Bayer Pharma Aktiengesellschaft, assignee. 2-(Morpholin-4-yl)-1,7-naphthyridines. Patent WO/2016/020320. 2016 Feb 11.

28. Foote KM, Nissink JW, Turner P, inventors; AstraZeneca AB, assignee.Morpholino pyrimidines and their use in therapy. Patent WO 2011/154737.2011 Dec 15.

29. Foote KM, Nissink JWM, McGuire T, Turner P, Guichard S, Yates JWT, et al.Discovery and characterization of AZD6738, a potent inhibitor of ataxiatelangiectasia mutated and Rad3 related (ATR) kinase with application as ananticancer agent. J Med Chem 2018;61:9889–907.

30 Charrier JD, Durrant S, Kay D, Knegtel R, MacCormick S, Mortimore M, et al.inventors; Vertex Pharmaceuticals Incorporated, assignee. Pyrazine derivativesuseful as inhibitors of ATR kinase. Patent WO/2010/071837. 2010 Jun 24.

31. Knegtel R, Charrier JD,Durrant S, Davis C, O'DonnellM, Storck P, et al. Rationaldesign of 5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-((methylamino)methyl)phe-nyl)isoxazol-5-yl)pyrazin-2-amine (VX-970,M6620): optimization of intra- andintermolecular polar interactions of a new ataxia telangiectasia mutated andRad3-related (ATR) kinase inhibitor. J Med Chem 2019;62:5547–61.

32. Politz O, Siegel F, Barfacker L, Bomer U, Hagebarth A, Scott WJ, et al. BAY1125976, a selective allosteric AKT1/2 inhibitor, exhibits high efficacy onAKT signaling-dependent tumor growth in mouse models. Int J Cancer 2017;140:449–59.






33. Chou TC. Theoretical basis, experimental design, and computerized simulationof synergism and antagonism in drug combination studies. Pharmacol Rev 2006;58:621–81.

34. Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assayof cells in vitro. Nat Protoc 2006;1:2315–9.

35. Bliss CI. The toxicity of poisons applied jointly. Ann Appl Biol 1939;26:585–615.36. Foucquier J, Guedj M. Analysis of drug combinations: current methodological

landscape. Pharmacol Res Perspect 2015;3:e00149.37. Liu S, Shiotani B, Lahiri M, Marechal A, Tse A, Leung CC, et al. ATR autopho-

sphorylation as a molecular switch for checkpoint activation. Mol Cell 2011;43:192–202.

38. Halazonetis TD, Gorgoulis VG, Bartek J. An oncogene-induced DNA damagemodel for cancer development. Science 2008;319:1352–5.

39. Hall AB, NewsomeD,Wang Y, Boucher DM, Eustace B, Gu Y, et al. Potentiationof tumor responses to DNA damaging therapy by the selective ATR inhibitorVX-970. Oncotarget 2014;5:5674–85.

40. Zeng L, Beggs RR,Cooper TS,Weaver AN, YangES. CombiningChk1/2 inhibitionwith cetuximab and radiation enhances in vitro and in vivo cytotoxicity in headand neck squamous cell carcinoma. Mol Cancer Ther 2017;16:591–600.

41. Fokas E, Prevo R, Pollard JR, Reaper PM, Charlton PA, Cornelissen B, et al.Targeting ATR in vivo using the novel inhibitor VE-822 results in selectivesensitization of pancreatic tumors to radiation. Cell Death Dis 2012;3:e441.

42. Vendetti FP, Karukonda P, Clump DA, Teo T, Lalonde R, Nugent K, et al. ATRkinase inhibitor AZD6738 potentiates CD8þ T cell-dependent antitumoractivity following radiation. J Clin Invest 2018;128:3926–40.

43. Wallez Y, Dunlop CR, Johnson TI, Koh SB, Fornari C, Yates JWT, et al. TheATR inhibitor AZD6738 synergizes with gemcitabine in vitro and in vivo toinduce pancreatic ductal adenocarcinoma regression. Mol Cancer Ther 2018;17:1670–82.

44. Vendetti FP, Lau A, Schamus S, Conrads TP, O'Connor MJ, Bakkenist CJ. Theorally active and bioavailable ATR kinase inhibitor AZD6738 potentiates theanti-tumor effects of cisplatin to resolve ATM-deficient non-small cell lungcancer in vivo. Oncotarget 2015;6:44289–305.

45. RothkammK, LobrichM. Evidence for a lack ofDNAdouble-strand break repairin human cells exposed to very low x-ray doses. Proc Natl Acad Sci U S A 2003;100:5057–62.

46. Barazzuol L, Jena R, Burnet NG,Meira LB, Jeynes JC, Kirkby KJ, et al. Evaluationof poly (ADP-ribose) polymerase inhibitor ABT-888 combined with radiother-apy and temozolomide in glioblastoma. Radiat Oncol 2013;8:65.

47. Sarcar B, Kahali S, Prabhu AH, Shumway SD, Xu Y, Demuth T, et al.Targeting radiation-induced G(2) checkpoint activation with the Wee-1

inhibitor MK-1775 in glioblastoma cell lines. Mol Cancer Ther 2011;10:2405–14.

48. Senra JM, Telfer BA, Cherry KE,McCrudden CM,Hirst DG, O'ConnorMJ, et al.Inhibition of PARP-1 by olaparib (AZD2281) increases the radiosensitivity of alung tumor xenograft. Mol Cancer Ther 2011;10:1949–58.

49. Dillon MT, Barker HE, Pedersen M, Hafsi H, Bhide SA, Newbold KL, et al.Radiosensitization by the ATR inhibitor AZD6738 through generation ofacentric micronuclei. Mol Cancer Ther 2017;16:25–34.

50. O'ConnorMJ. Targeting the DNA damage response in cancer. Mol Cell 2015;60:547–60.

51. Fojo T, Bates S. Mechanisms of resistance to PARP inhibitors–three andcounting. Cancer Discov 2013;3:20–3.

52. Lord CJ, Ashworth A. Mechanisms of resistance to therapies targeting BRCA-mutant cancers. Nat Med 2013;19:1381–8.

53. Sonnenblick A, de Azambuja E, Azim HA Jr, Piccart M. An update on PARPinhibitors–moving to the adjuvant setting. Nat Rev Clin Oncol 2015;12:27–41.

54. D'Andrea AD. Mechanisms of PARP inhibitor sensitivity and resistance.DNA Repair 2018;71:172–6.

55. Gogola E, Rottenberg S, Jonkers J. Resistance to PARP inhibitors: lessons frompreclinical models of BRCA-associated cancer. Annu Rev Cancer Biol 2019;3:235–54.

56. Yazinski SA, Comaills V, Buisson R, Genois M-M, Nguyen HD, Ho CK, et al.ATR inhibition disrupts rewired homologous recombination and fork protectionpathways in PARP inhibitor-resistant BRCA-deficient cancer cells. Genes Dev2017;31:318–32.

57. Fang Y,McGrail DJ, Sun C, Labrie M, Chen X, ZhangD, et al. Sequential therapywith PARP and WEE1 inhibitors minimizes toxicity while maintaining efficacy.Cancer Cell 2019;35:851–67.

58. Sanjiv K, Hagenkort A, Calderon-Montano JM, Koolmeister T, Reaper PM,MortusewiczO, et al. Cancer-specific synthetic lethality betweenATR andCHK1kinase activities. Cell Rep 2016;14:298–309.

59. Goodwin JF, SchiewerMJ, Dean JL, Schrecengost RS, de Leeuw R, Han S, et al. Ahormone-DNA repair circuit governs the response to genotoxic insult. CancerDiscov 2013;3:1254–71.

60. Moilanen AM, Riikonen R, Oksala R, Ravanti L, Aho E, Wohlfahrt G, et al.Discovery of ODM-201, a new-generation androgen receptor inhibitor targetingresistancemechanisms to androgen signaling-directed prostate cancer therapies.Sci Rep 2015;5:12007.

61. Fizazi K, Shore N, Tammela TL, Ulys A, Vjaters E, Polyakov S, et al. Darolu-tamide in nonmetastatic, castration-resistant prostate cancer. N Engl J Med2019;380:1235–46.


Wengner et al.




2020;19:26-38. Published OnlineFirst October 3, 2019.Mol Cancer Ther Antje M. Wengner, Gerhard Siemeister, Ulrich Lücking, et al. Compromising Therapies in Preclinical Cancer Models−

Inducing or Repair−Monotherapy and Combined with DNA Damage The Novel ATR Inhibitor BAY 1895344 Is Efficacious as

Updated version

10.1158/1535-7163.MCT-19-0019doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2019/10/03/1535-7163.MCT-19-0019.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/19/1/26.full#ref-list-1

This article cites 55 articles, 19 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/19/1/26.full#related-urls

This article has been cited by 1 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/19/1/26To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-19-0019

http://mct.aacrjournals.org/content/suppl/2019/10/03/1535-7163.MCT-19-0019.DC1

http://mct.aacrjournals.org/content/19/1/26.full#ref-list-1

http://mct.aacrjournals.org/content/19/1/26.full#related-urls

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/19/1/26


molecular cancer therapeutics - the novel atr …...ddr deﬁciencies and synergistic activity in...

Documents