mct-19-0019r2 manuscript revision september 2019 · mct-19-0019r2 manuscript revision september...

MCT-19-0019R2 Manuscript Revision September 2019

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The novel ATR inhibitor BAY 1895344 is efficacious as monotherapy and

combined with DNA damage-inducing or repair-compromising therapies in

preclinical cancer models

Antje M. Wengner1, Gerhard Siemeister1, Ulrich Lücking1, Julien Lefranc1, Lars

Wortmann1, Philip Lienau1, Benjamin Bader1, Ulf Bömer1, Dieter Moosmayer1, Uwe

Eberspächer1, Sven Golfier1, Christoph A. Schatz1, Simon J. Baumgart1, Bernard

Haendler1, Pascale Lejeune1, Andreas Schlicker1, Franz von Nussbaum1, Michael

Brands1, Karl Ziegelbauer1 and Dominik Mumberg1

1Bayer AG, Pharmaceuticals, Research & Development, Berlin, Germany

Correspondence should be addressed to A.M.W ([email protected]), Bayer

AG, Pharmaceuticals Division, Research & Development, Preclinical Research,

Oncology, Muellerstr. 178, 13353 Berlin, Germany

Phone: +49 30 468 15787; Fax: +49 30 468 18069

Conflict of interest statement: All authors are employees and stockholders of

Bayer AG, and inventors on Bayer AG patent applications.

Running title: Anti-tumor effects of ATR inhibition

Key words: ATR; DNA damage response; anti-androgen; radiation; preclinical

cancer models

Financial support: This work was supported by Bayer AG.

Word count: 5081

Number of Figures: 6

Number of references: 61

Supplementary material: supplementary methods, 4 tables, 4 figures

on July 19, 2020. © 2019 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on October 3, 2019; DOI: 10.1158/1535-7163.MCT-19-0019

http://mct.aacrjournals.org/


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Abstract

The DNA damage response (DDR) secures the integrity of the genome of eukaryotic

cells. DDR deficiencies can promote tumorigenesis but concurrently may increase

dependence on alternative repair pathways. The ataxia telangiectasia and Rad3-

related (ATR) kinase plays a central role in the DDR by activating essential signaling

pathways of DNA damage repair. Here, we studied the effect of the novel selective

ATR kinase inhibitor BAY 1895344 on tumor cell growth and viability. Potent anti-

proliferative activity was demonstrated in a broad spectrum of human tumor cell

lines. BAY 1895344 exhibited strong monotherapy efficacy in cancer xenograft

models that carry DNA damage repair deficiencies. The combination of

BAY 1895344 with DNA damage-inducing chemotherapy or external beam radiation

therapy (EBRT) showed synergistic anti-tumor activity. Combination treatment with

BAY 1895344 and DDR inhibitors achieved strong synergistic anti-proliferative

activity in vitro, and combined inhibition of ATR and poly (ADP-ribose) polymerase

(PARP) signaling using olaparib demonstrated synergistic anti-tumor activity in vivo.

Furthermore, the combination of BAY 1895344 with the novel, non-steroidal

androgen receptor antagonist darolutamide resulted in significantly improved anti-

tumor efficacy compared to respective single agent treatments in hormone-

dependent prostate cancer, and addition of EBRT resulted in even further enhanced

anti-tumor efficacy. Thus, the ATR inhibitor BAY 1895344 may provide new

therapeutic options for the treatment of cancers with certain DDR deficiencies in

monotherapy and in combination with DNA damage-inducing or DNA repair-

compromising cancer therapies by improving their efficacy.

232 words





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Introduction

The DNA damage response (DDR) consists of multiple and diverse signaling

pathways that secure the integrity of the genome of living cells (1). Proteins that

directly recognize aberrant DNA structures recruit and activate kinases of the DDR

pathways, which respond to a broad spectrum of DNA damage and to increased

replication stress e.g. in oncogene-driven cancer cells (2-5). Many cancers harbor

defects in DDR pathways leading to genomic instability that can promote

tumorigenesis and cancer cell growth. Concurrently, the defects in some signaling

pathways may increase the dependence of cells on alternative repair pathways.

The ataxia telangiectasia and Rad3-related (ATR) kinase belongs to the

phosphoinositide 3 kinase-related kinase family and plays a key role in the DNA

replication stress response (RSR) pathway of DNA damage repair to maintain the

genomic integrity through DDR activation (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 cycle arrest and DNA damage repair (10).

Therefore, loss-of-function mutations in the ATR pathway are very rarely observed,

indicating essentiality for cellular survival (11). ATR is closely related to two other

DDR kinases, ataxia telangiectasia mutated (ATM) and DNA-dependent protein

kinase (DNA-PK). Together, these kinases build the core of the DDR by responding

to different DNA damage insults, which are primarily DSBs for ATM and DNA-PK,

and replication stress (RS) for ATR (12,13). The ATM gene is frequently mutated in

tumor cells, suggesting that loss of ATM activity is beneficial for cancer cell survival

(11,14). ATM kinase inactivation leads to increased dependence on ATR signaling,

and combined inactivation of ATM and ATR induces synthetic lethality in cancer cells

(15,16).

The successful use of poly (ADP-ribose) polymerase (PARP) inhibitors in ovarian

and breast cancer patients has shown that the blockade of selected DDR

components is an effective strategy to treat cancers with specific types of DNA repair

deficiencies (17). Previous studies have demonstrated that ATR inhibition is also

effective for treating cancers with defective DDR, such as homologous





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recombination (HR) deficiencies (18). In particular, cancer cells with high levels of

oncogene-driven replication stress, e.g. due to RAS mutation or MYC amplification,

rely on ATR activity for survival, indicating the central role of ATR in response to

replication stress (19). These findings demonstrate the potential of ATR inhibition to

mediate synthetic lethality in cancer cells with increased replication stress or DDR

defects.

The cancer-killing activity resulting from DNA-damaging agents such as irradiation or

chemotherapy or DNA damage repair-compromising therapies such as PARP

inhibitors (PARPi) or anti-androgen therapy may be increased by the

pharmacological inactivation 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 replication stress (7).

Here, we disclose the structure and functional characterization of the novel potent

and selective ATR inhibitor (ATRi), BAY 1895344, which exhibits strong

monotherapy efficacy in cancers with certain DDR deficiencies and synergistic

activity in combination with DNA damage-inducing or DNA repair-compromising

therapies, such as chemotherapy, external beam radiation therapy (EBRT), inhibition

of PARP or anti-androgen treatment.





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Materials and Methods

Detailed descriptions of compounds and methods are presented in the

Supplementary Methods.

Compounds

BAY 1895344 (2-[(3R)-3-methylmorpholin-4-yl]-4-(1-methyl-1H-pyrazol-5-yl)-8-(1H-

pyrazol-5-yl)-1,7-naphthyridine) was identified and synthesized at Bayer AG

(Germany; Fig.1A) as described previously (BAY 1895344 is Example 111 in patent

WO 2016/020320 (27)). Ibrutinib, olaparib, rucaparib, niraparib, talazoparib, 5-FU,

cisplatin and carboplatin were purchased from commercial providers. AZD6738 was

synthesized at WuXi AppTec (China), and the analytical data of the delivered

material were identical to the reported data for Example 2.02 in patent WO

2011/154737 (28). For chemical structure and additional information about AZD6738

see (29). M6620 was purchased from abcr GmbH (Germany), and the analytical data

of the delivered material were identical with the reported data for Example 57a

(compound IIA-7) in patent WO 2010/071837 (30). For chemical structure and

additional information about M6620 see (31). Darolutamide was synthesized at Orion

Corporation (Finland).

Cell lines and culture conditions

Cancer cell lines were cultivated according to supplier (Table S3) protocols at 37C

in a humidified atmosphere containing 5% CO2. Cell lines were regularly subjected to

DNA fingerprinting at DSMZ (Deutsche Sammlung von Mikroorganismen und

Zellkulturen) and tested to be free from mycoplasma contamination using MycoAlert

(Lonza) directly before use. For in vitro experiments, cells from passages 3–10 were

used, and for in vivo cell line-derived xenograft (CDX) models cells from passages

3–5 were used for inoculation.





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Affinity and selectivity of BAY 1895344

A time-resolved fluorescence resonance energy transfer (TR-FRET)-based ATR

competition binding assay was used to determine the affinity of BAY 1895344 to

ATR using fluorescent 5-TAMRA-labeled Tracer 1, an ATP-competitive ATRi

(synthesized at Bayer AG) (27). The ratio of the emissions at 570 and 545 nm was

used to evaluate the binding affinity of BAY 1895344 to ATR.

The selectivity of BAY 1895344 was assessed using both an in-house kinase panel

and a KINOMEscan® assay panel (DiscoverX, San Diego, USA) consisting of 468

kinases, as described previously (32).

In vitro experiments

The activity of ATR and ATM kinases was determined by measuring phospho-

Ser139 histone protein H2AX (γH2AX) levels in hydroxyurea-treated HT-29 cells and

neocarzinostatin-treated M059J cells, respectively. PI3K/AKT/mTOR signaling

pathway activity was investigated in MCF7 breast cancer cells by measuring AKT

phosphorylation.

The anti-proliferative activity of BAY 1895344 was evaluated against a panel of 38

cancer cell lines (Table S3). Cell proliferation was measured after 72–96 hours

exposure to BAY 1895344. Cell viability was determined using crystal violet staining

or the CellTiter-Glo® (CTG) cell viability assay (Promega).

The anti-proliferative activity of BAY 1895344 in combination with different drugs was

assessed by determination of combination indexes. The combination of

BAY 1895344 (3 – 300 nM) with cisplatin (100 nM – 10 M) was investigated in HT-

29 cells and the combination with olaparib (300 nM – 30 M), niraparib (30 nM –

3 µM), rucaparib (300 nM – 30 µM) or talazoparib (1 – 100 nM) in MDA-MB-436

cells. Combination studies with BAY 1895344 (10 nM – 10 µM) and darolutamide

(10 nM – 10 µM) were conducted in LAPC-4 cells, in the presence of the synthetic

androgen methyltrienolone R1881 (10 nM). Additional combination studies with

BAY 1895344 and a selection of compounds were conducted in a panel of cancer

cell lines (Table S4). Cells were treated with a single compound or a combination of





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fixed compound ratios for 4–6 days, and viability was measured using CellTiter-Glo®.

EC50 values were calculated from triplicate values for each individual combination

data point, and the respective isobolograms were generated. Combination indexes

(CI) were calculated according to the median-effect model (33). A CI of ≤0.8 was

defined as more than additive (i.e. synergistic) interaction, and a CI of ≥1.2 was

defined as antagonistic interaction.

The clonogenic combination assay (34) was used to assess the radio-sensitization

potential of BAY 1895344. LOVO CRC cells were treated with 3 nM BAY 1895344

and different intensities of γ-radiation, allowed to form colonies for 10–14 days and,

finally, the colonies were counted to calculate the combination effect.

In vivo studies in cell line-derived xenograft models

All animal experiments were conducted in accordance with the German Animal

Welfare Act and approved by local authorities. The in vivo antitumor efficacy and

tolerability of BAY 1895344 as mono-/combination therapy were evaluated in cell

line-derived (CDX) subcutaneous or orthotopic xenograft models in mice.

Monotherapy experiments were performed in GRANTA-519 (in female SCID beige

mice), REC-1 (in female C.B-17 SCID mice), PC-3 (in male NMRI nude mice), LOVO

and A2780 (both in female NMRI nude mice) models treated with BAY 1895344 at

50 mg/kg (all models; 2QD, 3 days on/ 4 days off [3on/4off], p.o.) or at 3, 10 or

30 mg/kg (GRANTA-519; 2QD, 3on/4off, p.o.), ibrutinib (REC-1; 20 mg/kg, QD, p.o.),

AZD6738 (GRANTA-519, REC-1; 50 mg/kg, QD, p.o.), M6620 (GRANTA-519 and

REC-1; 100 mg/kg, QD, p.o.) or 5-FU (LOVO; 50 mg/kg, QW, i.p.). The combination

of BAY 1895344 at 10 or 20 mg/kg (QD, 2 days on/ 5 days off [2on/5off], p.o.) or

50 mg/kg (2QD, 3on/4off, p.o.) and carboplatin (50 mg/kg, QW, i.p.) was investigated

in IGROV-1 tumor-bearing female nude (nu/nu) mice. The combination of 20 or

50 mg/kg BAY 1895344 (2QD, 2on/5off, p.o.) and EBRT (5 Gy, 7.7 min, QD on days

12 and 27) was investigated in LOVO tumor-bearing female NMRI nude mice.

Combination therapy experiments with 20 or 50 mg/kg BAY 1895344 (2QD, 3on/4off,

p.o.) and 20 or 50 mg/kg olaparib (QD, i.p.) were performed in MDA-MB-436 and

22Rv1 models in female NOD/SCID and male SCID mice, respectively. Combination

experiments with 20 mg/kg BAY 1895344 (2QD, 3on/4off, p.o.) and 100 mg/kg





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darolutamide (QD, p.o.) were performed in the hormone-dependent LAPC-4 prostate

cancer model in male C.B-17 SCID mice. Castrated mice served here as a control.

For a triple combination treatment, mice received EBRT (5 Gy, Q7Dx2) in addition to

treatment with BAY 1895344 and darolutamide.

To elucidate the in vivo mode-of-action of BAY 1895344, ATR and H2AX

phosphorylation was determined in lysed GRANTA-519 xenograft tumor samples.

For the quantification of circulating ATRis, plasma samples were taken from mice

and measured by LC-MS/MS.

Statistical analyses

Statistical analysis was performed using a linear model estimated with generalized

least squares that included separate variance parameters for each study group

(Fig.2-3). Survival analysis was performed using the Cox proportional-hazards model

and longitudinal data were modelled using second order polynomial curves with

random intercepts and slopes for each subject. Synergy was defined according to

the Bliss Independence Model (35,36) in the analysis of treatment combinations

(Fig.1,4-6). Finally, dose-response analysis was performed using the 3-parameter

logistic model (Fig.1).





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Results

BAY 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 discovered in a high-throughput screening

approach (Fig.1). The binding affinity of BAY 1895344 to its target molecule ATR

was assessed by its ability to displace a fluorescently labeled ATR-binding tracer

from a GST-ATR fusion protein using TR-FRET analysis. The IC50 of BAY 1895344

required to displace the tracer was 7.03.7 nM, indicating a high affinity of

BAY 1895344 to ATR. The selectivity of BAY 1895344, evaluated in an in-house

kinase selectivity panel and KINOMEscan® profiling, demonstrated high selectivity

of BAY 1895344 for ATR (Tables S1-S2). Only for one kinase out of 31 kinases in

the in-house panel, mTOR, the determined IC50 of 3532 nM was of the same order

of magnitude as for ATR. Five other kinases were inhibited with an IC50 value in the

concentration range tested (< 20 µM) and were at least 47-fold less potent compared

to ATR (Table S1). In the KINOMEscan® panel, 1 µM BAY 1895344 exhibited

activity against only six out of 456 kinases (Table S2), and for only three of these

kinases Kd values below 1 µM were determined (mTOR, cyclin G-associated kinase

[GAK] and right open reading frame kinase 2 [RIOK2], Kd = 24, 580 and 660 nM,

respectively). Taken together, these results demonstrated the high selectivity of

BAY 1895344 for ATR.

BAY 1895344 potently and selectively inhibits ATR-mediated DDR

Phospho-Ser139 histone protein H2AX (γH2AX) is a well-known early marker for

DNA damage or DDR activation. Here, the activity of BAY 1895344 against ATR and

ATM was evaluated in a downstream cellular mechanistic assay by determining the

level of γH2AX. In HT-29 cells, BAY 1895344 inhibited hydroxyurea-induced ATR-

dependent H2AX phosphorylation with an IC50 of 36±5 nM. In neocarzinostatin-

treated DNA-PK-deficient M059J cells, BAY 1895344 had no effect on ATM-

dependent H2AX phosphorylation even at 10 μM, demonstrating that it is a potent

and selective inhibitor of ATR kinase activity.





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ATR and mTOR are phosphatidylinositol 3-kinase (PI3K)-related protein kinases.

AKT phosphorylation (pAKT) was used to monitor BAY 1895344-mediated mTOR

inhibition in MCF7 cells. BAY 1895344 inhibited pAKT with an IC50 of 2.20.9 μM,

whereas, two mTOR inhibitors, PI-103 and AZD8055, inhibited pAKT with IC50

values of 7131 nM and 124 nM, respectively. Hence, BAY 1895344 was 30- to

180-fold less active than specific mTOR inhibitors and showed superior selectivity for

ATR compared to the PI3K/AKT/mTOR pathway (>60-fold).

BAY 1895344 inhibits proliferation in a broad spectrum of human cancer cell

lines

In a panel of cancer cell lines of different histological origins harboring various

mutations affecting DDR pathways, BAY 1895344 showed potent anti-proliferative

activity with IC50 values ranging from 9 to 490 nM (Table S3, Fig.S1). A clear

difference between highly sensitive (IC50 <100 nM) and less sensitive cell lines was

observed, and a majority of the sensitive cell lines were found to carry mutations

affecting the ATM pathway independent of their origin (Fig.S1A). Lymphoma

appeared to be the most sensitive cancer type with IC50 values ranging from 9 to

32 nM in mantle cell lymphoma cell lines (Fig.S1B).

BAY 1895344 shows strong anti-tumor efficacy as monotherapy in cell line-

derived xenograft (CDX) models

Anti-tumor efficacy of BAY 1895344 was evaluated in CDX models in mice. Dose-

dependent efficacy was observed in the GRANTA-519 mantle cell lymphoma model

and the maximum tolerated dose (MTD) and optimal dosing schedule of

BAY 1895344 were identified as 50 mg/kg applied twice daily (2QD) for 3 days on

and 4 days off (Fig.1B). In the GRANTA-519 model, treatment with BAY 1895344

applied at MTD demonstrated strong anti-tumor efficacy, whereas the ATR inhibitors

AZD6738 and M6620 applied at MTD achieved only moderate anti-tumor efficacy

(Fig.1C).





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When BAY 1895344 was dosed repeatedly (2QD) at 30 or 50 mg/kg to

GRANTA-519 tumor-bearing mice, the level of unbound BAY 1895344 in plasma

covered the biochemical, cellular mechanistic and anti-proliferative IC50

concentrations determined in vitro, which is in line with the observed strong in vivo

anti-tumor efficacy (Fig.1D). Even at the highest applied dose (MTD), the level of

unbound BAY 1895344 (1.6 µM) was not sufficient to cover the IC50 concentration of

2.2 μM required for the cellular inhibition of the PI3K/AKT/mTOR signaling pathway,

suggesting that the anti-tumor activity of BAY 1895344 was not mediated by mTOR

pathway inhibition. The level of unbound AZD6738 or M6620 in plasma did not

exceed the anti-proliferative IC50 concentrations, indicating that exposure to these

ATRis may not have been sufficient to mediate anti-tumor activity in vivo (Fig.1E).

Anti-tumor efficacy of BAY 1895344 as a single agent was further evaluated in CDX

models of different indications characterized by DDR defects or oncogenes

potentially mediating replication stress (Table S3). BAY 1895344 applied at MTD

resulted in strong anti-tumor efficacy as demonstrated in the A2780 ovarian cancer,

PC-3 prostate cancer, LOVO colorectal cancer and REC-1 mantle cell lymphoma

models (Fig.2). The efficacy of BAY 1895344 clearly exceeded the efficacy of other

treatments, as 5-FU (5-fluorouracil) failed to inhibit LOVO tumor growth, and

AZD6738, M6620 and the BTK inhibitor ibrutinib showed significantly weaker efficacy

(p < 0.001) in the REC-1 model. All treatments with BAY 1895344 were well-

tolerated over a treatment time of 11–59 days without toxicities and acceptable body

weight loss (10–13%) (Fig.S2).

In order to analyze the association between in vivo anti-tumor efficacy and the

inhibition of ATR, ATR phosphorylation (pATR) as a direct measure of ATR kinase

activity (37) and H2AX phosphorylation as an indirect measure of ATR-mediated

DNA repair were determined in GRANTA-519 tumor xenografts after treatment with

BAY 1895344 at different doses and dosing schedules. pATR levels decreased

remarkably 24–48 hours after a single or repeated treatment with 50 mg/kg

BAY 1895344, demonstrating direct ATR kinase inhibition (Fig.3A). γH2AX levels

clearly increased 3–48 hours after a single or repeated treatment with 50 mg/kg

BAY 1895344, indicating that ATR kinase inhibition may induce DNA damage by

abrogation of DDR signaling (Fig.3B).





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Further studies demonstrated that the BAY 1895344-evoked increase in H2AX

phosphorylation was time-, dose- and dosing schedule-dependent (Fig.3C-D). A

single dose (QDx1) of 30 mg/kg BAY 1895344 increased the level of γH2AX peaking

at 8–24 h (Fig.3C), whereas a single dose of 10 mg/kg or repeated dosing with

3 mg/kg (2QDx2 + QDx1) was not sufficient to block ATR and cause DNA damage.

In contrast, repeated doses of 10 or 30 mg/kg BAY 1895344 increased and

prolonged H2AX phosphorylation compared to a single dose of 30 mg/kg. Increasing

treatment cycles with BAY 1895344 led to decreased levels of H2AX

phosphorylation (Fig.3D), highlighting the importance of the time point chosen for the

determination of a pharmacodynamic signal (γH2AX) to demonstrate target

engagement.

In summary, BAY 1895344 displayed strong activity as monotherapy in different

CDX models. Dose-dependent anti-tumor efficacy in vivo correlated with compound

exposure in plasma, the reduction of ATR phosphorylation and the induction of DNA

damage, demonstrating the anticipated mechanism of action of ATR inhibition with

BAY 1895344.

BAY 1895344 shows synergistic activity in combination with chemotherapy or

external beam radiation therapy (EBRT)

To assess the potential synergistic activity of ATR inhibition with different DNA

damage-inducing chemotherapeutic agents or the tubulin stabilizer docetaxel, anti-

proliferation-based in vitro combination studies were performed in cancer cell lines of

different genetic backgrounds and tumor types. In HT-29 colorectal cancer (CRC)

cells, the interaction of BAY 1895344 with cisplatin was strongly synergistic with a

combination index (CI) of 0.14 (Fig.4A). Further combinations with cisplatin,

bleomycin, or SN-38 were also strongly synergistic (Table S4). In contrast, the

combination of docetaxel and BAY 1895344 achieved a CI of 1.26, indicating an

antagonistic interaction. The CIs for all in vitro combination treatments are

summarized in Table S4.





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The in vivo anti-tumor efficacy of BAY 1895344 in combination with the

chemotherapy drug carboplatin was investigated in the IGROV-1 ovarian cancer

model. Very low doses of BAY 1895344 (10 or 20 mg/kg, QD, 2 on/ 5 off = 7 or 14%

of MTD) had to be used in combination treatment with carboplatin at MTD to achieve

tolerability. BAY 1895344 monotherapy at MTD showed good anti-tumor efficacy,

while sub-MTD doses of BAY 1895344 or carboplatin alone showed only weak

efficacy. Combination treatment with BAY 1895344 and carboplatin clearly enhanced

anti-tumor efficacy pointing towards a synergistic effect (80% confidence, Fig.4B-C)

at acceptable tolerability (Fig.S3A). Although the combination treatments were more

effective than respective monotherapies, the efficacy of the tolerated combination

treatment did not exceed tumor growth inhibition achieved with BAY 1895344

monotherapy at MTD.

The in vitro radio-sensitization potential of BAY 1895344 was assessed in LOVO

CRC cells using the clonogenic assay. Treatment with a non-effective dose of

BAY 1895344 in combination with different intensities of γ-radiation clearly reduced

the colony-forming ability of LOVO cells compared to radiation alone, confirming the

radio-sensitization effect of BAY 1895344 (Fig.4D).

The in vivo anti-tumor efficacy of BAY 1895344 in combination with EBRT was

investigated in LOVO CRC xenografts. Sub-MTD treatment with BAY 1895344 (20 or

50 mg/kg, 2QD, 2on/5off = 30 or 60% of MTD) or EBRT alone resulted in weak to

moderate anti-tumor efficacy. Combination treatment with each tested BAY 1895344

dose and EBRT showed strong anti-tumor efficacy and a synergistic effect (95%

confidence, Fig.4D). Moreover, combination treatment with 50 mg/kg BAY 1895344

and EBRT was more efficacious than radiation alone, as determined on the day of

combination group termination. All treatments in the LOVO model were well-tolerated

with a maximum body weight loss of <10% (Fig.S3B).

The rationale for the combination of BAY 1895344 with DNA damage-inducing

cancer therapies is supported by the observed synergistic interactions. The observed

radio-sensitizing effect of ATR inhibition, that however does not have an impact on

the safety of EBRT, supports further clinical assessment. However, the tolerability of

simultaneous systemic chemotherapy-induced DNA damage and ATR inhibition

needs careful evaluation to minimize safety risks.





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BAY 1895344 shows additive to synergistic activity in combination with

different DDR inhibitors in vitro and synergy with the PARP1 inhibitor olaparib

in vivo

The anti-proliferative activity of BAY 1895344 was further assessed in combination

with different DDR pathway inhibitors (DDRis) in cancer cell lines of different genetic

backgrounds and tumor types. To assess the potential synergistic activity of ATR

inhibition together with PARP inhibition, BRCA1-deficient MDA-MB-436 breast

cancer cells were treated with BAY 1895344 in combination with the PARPis

olaparib, niraparib, rucaparib and talazoparib. The in vitro interaction of

BAY 1895344 with all tested PARPis was strongly synergistic (Fig.5A-D). Further

combinations with DDRis targeting ATM, CHEK1, DNA-PK and WEE1 demonstrated

additive to highly synergistic interactions in ATM- and MRE11A-proficient HT29 and

PC-3 cells (CIs summarized in Table S4).

The in vivo anti-tumor efficacy of BAY 1895344 in combination with the PARPi

olaparib was investigated using two different CDX models. In the PARPi-sensitive

MDA-MB-436 breast cancer model, monotherapy with BAY 1895344 at MTD or sub-

MTD (40%) (50 or 20 mg/kg, 2QD, 3on/4 off) or olaparib at MTD (50 mg/kg, QD)

effectively inhibited tumor growth. Combination treatment with BAY 1895344 at sub-

MTD and olaparib at MTD showed potent anti-tumor efficacy, pointing towards a

synergistic combination effect (90.5% confidence, Fig.5E-F). All treatments in the

MDA-MB-436 model were well-tolerated with a maximum body weight loss of <10%

(Fig.S3C). In the PARPi-resistant 22Rv1 prostate cancer model, monotherapy with

BAY 1895344 at MTD or sub-MTD (40%) resulted in moderate or weak anti-tumor

efficacy. Monotherapy with olaparib at MTD or sub-MTD (40%) failed to inhibit tumor

growth, confirming the PARPi resistance of this model. Combination treatment with

BAY 1895344 and olaparib at sub-MTDs (40%) delayed tumor growth compared to

vehicle and olaparib monotherapy, indicating a synergistic combination effect (95%

confidence level, Fig.5 G-H). When looking at final tumor weight, the combination

treatment showed improved efficacy also compared to the respective BAY 1895344

monotherapy at sub-MTD (Fig.5H). However, the efficacy achieved by BAY 1895344

monotherapy at MTD was comparable to the efficacy achieved by combination





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therapy in the PARPi resistant tumor model 22Rv1. All treatments were well-

tolerated with a maximum body weight loss of <10% (Fig.S3D).

These results support the rationale for the combination of BAY 1895344 treatment

with DNA repair-compromising cancer therapies. However, the tolerability of

simultaneous systemic inhibition of parallel DDR pathways needs careful evaluation

of dose and treatment schedule to ensure safety.

BAY 1895344 shows improved anti-tumor activity in combination with the AR

antagonist darolutamide in vitro and in vivo

In LAPC-4 prostate cancer cells, the in vitro interaction of BAY 1895344 with the AR

antagonist darolutamide achieved a CI of 0.82, indicating an additive interaction

(Fig.6A). Furthermore, combination treatment with BAY 1895344 and darolutamide

led to more pronounced downregulation of several genes involved in DNA repair,

including BRCA1, EXO1 and MCM10, in comparison to respective monotherapies

(Fig.S4).

In the hormone-dependent LAPC-4 CDX model, BAY 1895344 monotherapy at sub-

MTD (40%) showed a weak to moderate anti-tumor effect, while darolutamide

monotherapy exhibited good anti-tumor efficacy. Combination treatment with

BAY 1895344 and darolutamide resulted in enhanced anti-tumor efficacy, showing

significant improvement compared to darolutamide monotherapy on the day of

combination group termination (Fig.6B). All treatments were well-tolerated with a

maximum body weight loss of <10% (Fig.S3E). The triple combination of

BAY 1895344, darolutamide and EBRT resulted in further enhanced anti-tumor

efficacy compared to respective monotherapies and all dual combination treatments

and was even more effective than castration (Fig.6C).

These results support the rationale for the combination treatment of BAY 1895344

with antiandrogen therapy and the addition of EBRT may even further enhance the

efficacy of this combination.





16

Discussion

Cancer cells rely on DDR pathways to survive genomic instability in particular by

activating ATR kinase (38). In this study, we have shown that BAY 1895344 is a

selective low-nanomolar inhibitor of ATR kinase activity, which potently inhibits the

proliferation of a broad spectrum of human tumor cell lines of different origins

harboring DDR defects, including ATM aberrations. We have demonstrated strong in

vivo anti-tumor efficacy of BAY 1895344 in a variety of xenograft models of different

tumor indications carrying defects in DNA repair, thus validating the concept of

synthetic lethality of tumor-intrinsic DNA repair deficiencies and ATR blockade.

However, further preclinical and clinical investigations are required to understand

which DDR defects have the strongest impact on the sensitivity to ATR inhibition.

BAY 1895344 demonstrated superiority compared to the ATRis AZD6738 and

M6620 (6,29,39). Its high potency and selectivity resulted in stronger anti-tumor

activity as monotherapy in a variety of tumor types with DDR defects. We could

demonstrate that the plasma exposure of BAY 1895344 clearly covered the anti-

proliferative concentration levels (IC50) of tumor cells. In contrast, the plasma

exposure of AZD6738 and M6620 did not exceed the concentrations needed for anti-

proliferative activity in tumor cells, which is in line with their weak in vivo anti-tumor

efficacy in monotherapy (Fig.1C,E).

In addition to its potential as monotherapy, BAY 1895344 is likely to be particularly

efficacious in combination treatment with DNA damage-inducing or DNA repair-

compromising therapies, as indicated in earlier studies (40-44). Here, we have

demonstrated in preclinical tumor models that BAY 1895344 exhibits synergistic anti-

tumor efficacy with EBRT in colorectal cancer, with the PARPi olaparib in PARPi-

sensitive, HR-defective (BRCA1 mutant) breast cancer as well as in PARPi-resistant

prostate cancer and enhanced anti-tumor activity in combination with the AR

antagonist darolutamide in hormone-dependent prostate cancer. The combination of

BAY 1895344 with the chemotherapeutic drug carboplatin was associated with dose-

dependent toxicity. This restricted overall anti-tumor efficacy and limited the potential

therapeutic value of this combination, in particular since BAY 1895344 monotherapy

applied at the MTD was more efficacious than the combination therapy with

carboplatin.





17

The efficacy of EBRT in cancer therapy is mainly due to the ability of ionizing

radiation to produce DSBs leading to cell death in tumor tissue. However, the use of

EBRT is limited by the sensitivity of normal healthy tissue to radiation which can

cause acute and chronic toxicities. As ionizing radiation also causes an extensive

amount of less cytotoxic DNA lesions, such as DNA single-strand breaks (SSBs)

(45), recent research has focused on the radio-sensitization of tumors by combining

radiation with DDR-targeted agents, and these studies have demonstrated promising

preclinical efficacy (25,46-49). In this study, BAY 1895344 and EBRT combination

treatment of LOVO CRC xenografts, which have several defects in DDR and

mismatch repair (MMR) (Table S3), increased the efficacy of EBRT without affecting

tolerability or increasing toxicity. This mechanism-based potential of combining

EBRT-induced DNA damage and blockade of ATR-mediated DNA repair with

BAY 1895344 suggests a powerful new treatment option for patients resistant to

radiation, and a possibility to widen the therapeutic window of radiation therapy.

PARPis prevent the repair of SSBs by trapping inactivated PARP onto SS-DNA,

resulting in the generation of DSBs during DNA replication, thus activating DNA

repair processes mediated by homologous recombination. In tumors with a

homologous recombination deficiency (HRD), e.g. a BRCA1/2 mutation, PARPi-

induced accumulation of replication errors and DSBs leads to increased genetic

instability, and ultimately, cell death (50). The first approval of a PARPi, olaparib,

was granted by the FDA in 2014 for use in ovarian cancer patients with

deleterious/suspected deleterious germline BRCA1/2 mutations. However, the

efficacy of PARPis is limited by drug resistance (51-53). Not all BRCA1/2 mutation

carriers respond to PARP inhibition, and even responders often relapse. Different

mechanisms potentially causing PARPi resistance have been described (51,54,55).

Previous studies have demonstrated that ATR inhibition may be effective in treating

cancers with HRD (18). However, ATR is also involved in HR-independent repair

pathways indicating the potential of ATRis to improve the anti-tumor activity of

PARPi. PARP and ATR bind to DNA SSBs and initiate distinct DNA repair

mechanisms to inhibit cytotoxic DNA DSB formation. PARP inhibition blocks the

DNA repair complex formation via base-excision repair (BER) and induces PARP

trapping, which leads to cytotoxic PARP-DNA complexes resulting in DNA breaks.

ATR inhibition affects the activation of different DNA repair pathways, including HR,





18

as part of the replication stress response. Combined inhibition of ATR and PARP is

expected to be highly synergistic by blocking central, but independent, DNA repair

pathways. Therefore, combined inhibition of ATR and PARP could help patients who

have relapsed on PARPi therapy by overcoming PARPi resistance but could also

enhance anti-tumor activity in PARPi-sensitive patients. Overall, combined inhibition

of ATR and PARP represents a valuable strategy for the improvement of PARPi

therapy (56). Here, combined inhibition of ATR with BAY 1895344 and PARP with

olaparib resulted in significant reduction of tumor size in PARPi-sensitive, HR-

deficient MDA-MB-436 breast cancer xenografts, and in PARPi-resistant 22Rv1

prostate cancer xenografts. Parallel inhibition of different DDR pathways by

combining a PARPi, such as olaparib, with the ATR kinase inhibitor BAY 1895344,

thus presents novel treatment opportunities in cancer patients with HRDs sensitive

or resistant to PARPi therapy. In fact, a combination of an ATRi with a PARPi is

currently under clinical investigation (NCT03462342). Other DDRi combinations may

also have clinical impact, as indicated in our in vitro combination studies where the

interaction of BAY 1895344 with DNA-PK, CHK1/2 and WEE1 inhibitors was strongly

synergistic. However, well-tolerated and efficacious combination treatment regimens

need to be established first (57,58).

AR signaling is a key regulator of the DDR in prostate cancer (21,24). In recent

studies, androgen depletion has been shown to result in the down-regulation of DDR

genes, impairing DNA repair and increasing sensitivity to radiation (24). AR activity

has also been reported to be induced by DNA damage, promoting the resolution of

DSBs (59). Thus, AR inhibition can contribute to an HRD-phenotype, resulting in

sensitivity to DDR inhibition. For example, the AR antagonist enzalutamide in

combination with the PARPi olaparib downregulated the expression of DDR genes,

resulting in reduced HR efficiency in AR-positive prostate cancer cells (22).

Furthermore, combination treatment of enzalutamide with the Chk1/2 inhibitor

AZD7762 showed synergistic activity in the inhibition of AR-CDC6-ATR-Chk1

signaling, induction of ATM phosphorylation, and apoptosis in hormone-dependent

prostate cancer cells (21). Here, we investigated for the first time the combination of

ATR inhibition and anti-androgen therapy. Combined treatment with BAY 1895344

and darolutamide, a novel non-steroidal AR antagonist (60) which has successfully

completed a clinical phase III trial in the indication of non-metastatic prostate cancer





19

(61), demonstrated significantly reduced expression of DDR genes in prostate

cancer cells in vitro and significantly improved anti-tumor efficacy in the hormone-

dependent LAPC-4 prostate cancer CDX model in vivo. Adding EBRT to the

combination treatment even further enhanced the therapeutic efficacy. Therefore,

induction of DNA damage by radiation and reduced DNA repair capability by the AR

antagonist darolutamide combined with the ATR inhibitor BAY 1895344 could

present a new treatment option for patients with hormone-dependent prostate

cancer.

In summary, BAY 1895344 is a novel ATR kinase inhibitor which exhibits strong

monotherapy efficacy in cancers with DDR deficiencies of different tumor histologies,

as well as synergistic activity in combination with DNA damage-inducing or DNA

repair-compromising therapies. This may provide new treatment options and improve

the therapeutic efficacy of standard-of-care therapies. BAY 1895344 is currently

under clinical investigation in patients with advanced solid tumors and lymphomas

(NCT03188965).





20

Acknowledgements

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

the preparation of this manuscript, funded by Bayer AG. Darolutamide is jointly

developed by Bayer AG (Germany) and Orion Corporation (Finland). Special thanks

go to Lisa Ehremann, Stephanie Osterberg, Sarah Böttcher, Angela Bieseke and

Nils Guthof for excellent technical support.

Author Contributions

Conceptualization, A.M.W., G.S., U.L., J.L., L.W., P.L., B.B., U.B., D.Mo. U.E., S.G.,

C.A.S., B.H., P.Le., A.S., F.v.N., M.B., K.Z., D.M.; Methodology, A.M.W., G.S., U.L.,

J.L., L.W., P.L., B.B., U.B., D.Mo. U.E., S.G., C.A.S., S.J.B., B.H., P.Le;

Investigation, A.M.W., G.S., U.L., J.L., L.W., S.J.B., P.L., B.B., U.B., S.G., C.A.S.,

B.H., P.Le.; Writing, A.M.W., G.S., B.B., U.B., B.H., P.L.; Writing-Review & Editing,

A.M.W., G.S., U.L., J.L., L.W., P.L., B.B., U.B., D.Mo. U.E., S.G., C.A.S., B.H., P.Le.,

A.S., F.v.N., M.B., K.Z., D.M.; Funding Acquisition, K.Z., D.M.; Resources, A.M.W.,

G.S., U.L., J.L., L.W., P.L., B.B., U.B., D.Mo., U.E., S.G., C.A.S., S.J.B., B.H., P.Le.,

A.S., F.v.N., M.B., K.Z., D.M.; Supervision, A.M.W., U.L., F.v.N., M.B., K.Z., D.M.





21

References

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

Mol Cell 2010;40(2):179-204 doi 10.1016/j.molcel.2010.09.019.

2. Karnitz LM, Zou L. Molecular Pathways: Targeting ATR in Cancer Therapy. Clin

Cancer Res 2015;21(21):4780-5 doi 10.1158/1078-0432.CCR-15-0479.

3. Marechal A, Zou L. DNA damage sensing by the ATM and ATR kinases. Cold

Spring Harb Perspect Biol 2013;5(9) doi 10.1101/cshperspect.a012716.

4. Taylor EM, Lindsay HD. DNA replication stress and cancer: cause or cure? Future

Oncol 2016;12(2):221-37 doi 10.2217/fon.15.292.

5. Yazinski SA, Zou L. Functions, Regulation, and Therapeutic Implications of the ATR

Checkpoint Pathway. Annu Rev Genet 2016;50:155-73 doi 10.1146/annurev-genet-

121415-121658.

6. Foote KM, Lau A, Nissink JWM. Drugging ATR: progress in the development of

specific inhibitors for the treatment of cancer. Future Med Chem 2015;7(7):873-91

doi 10.4155/fmc.15.33.

7. Lecona E, Fernandez-Capetillo O. Targeting ATR in cancer. Nat Rev Cancer 2018

doi 10.1038/s41568-018-0034-3.

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(1):43 doi 10.1186/s13045-

019-0733-6.

9. Pollard J, Curtin N, editors. Targeting the DNA Damage Response for Anti-Cancer

Therapy. 1 ed: Humana Press; 2018. IX, 401 p.

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(2):225-31.

11. Gaillard H, Garcia-Muse T, Aguilera A. Replication stress and cancer. Nat Rev

Cancer 2015;15(5):276-89 doi 10.1038/nrc3916.

12. Cimprich KA, Shin TB, Keith CT, Schreiber SL. cDNA cloning and gene mapping of

a candidate human cell cycle checkpoint protein. Proceedings of the National

Academy of Sciences of the United States of America 1996;93(7):2850-5.

13. Bentley NJ, Holtzman DA, Flaggs G, Keegan KS, DeMaggio A, Ford JC, et al. The

Schizosaccharomyces pombe rad3 checkpoint gene. EMBO J 1996;15(23):6641-51.

14. Choi M, Kipps T, Kurzrock R. ATM Mutations in Cancer: Therapeutic Implications.

Mol Cancer Ther 2016;15(8):1781-91 doi 10.1158/1535-7163.MCT-15-0945.

15. Menezes DL, Holt J, Tang Y, Feng J, Barsanti P, Pan Y, et al. A synthetic lethal

screen reveals enhanced sensitivity to ATR inhibitor treatment in mantle cell

lymphoma with ATM loss-of-function. Mol Cancer Res 2015;13(1):120-9 doi

10.1158/1541-7786.MCR-14-0240.

16. Min A, Im SA, Jang H, Kim S, Lee M, Kim DK, et al. AZD6738, A Novel Oral

Inhibitor of ATR, Induces Synthetic Lethality with ATM Deficiency in Gastric

Cancer Cells. Mol Cancer Ther 2017;16(4):566-77 doi 10.1158/1535-7163.MCT-16-

0378.

17. Bouwman P, Jonkers J. The effects of deregulated DNA damage signalling on cancer

chemotherapy response and resistance. Nat Rev Cancer 2012;12(9):587-98 doi

10.1038/nrc3342.

18. Krajewska M, Fehrmann RS, Schoonen PM, Labib S, de Vries EG, Franke L, et al.

ATR inhibition preferentially targets homologous recombination-deficient tumor

cells. Oncogene 2015;34(26):3474-81 doi 10.1038/onc.2014.276.





22

19. Gilad O, Nabet BY, Ragland RL, Schoppy DW, Smith KD, Durham AC, et al.

Combining ATR suppression with oncogenic Ras synergistically increases genomic

instability, causing synthetic lethality or tumorigenesis in a dosage-dependent manner.

Cancer Res 2010;70(23):9693-702 doi 10.1158/0008-5472.CAN-10-2286.

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 and Rad3

related (ATR) protein kinase as potential anticancer agents. J Med Chem

2011;54(7):2320-30 doi 10.1021/jm101488z.

21. Karanika S, Karantanos T, Li L, Wang J, Park S, Yang G, et al. Targeting DNA

Damage Response in Prostate Cancer by Inhibiting Androgen Receptor-CDC6-ATR-

Chk1 Signaling. Cell Rep 2017;18(8):1970-81 doi 10.1016/j.celrep.2017.01.072.

22. Li L, Chang W, Yang G, Ren C, Park S, Karantanos T, et al. Targeting poly(ADP-

ribose) polymerase and the c-Myb-regulated DNA damage response pathway in

castration-resistant prostate cancer. Sci Signal 2014;7(326):ra47 doi

10.1126/scisignal.2005070.

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 J

Cancer 2012;107(2):291-9 doi 10.1038/bjc.2012.265.

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(11):1245-53 doi 10.1158/2159-8290.CD-13-0172.

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 of ATR.

Nat Chem Biol 2011;7(7):428-30 doi 10.1038/nchembio.573.

26. Li L, Karanika S, Yang G, Wang J, Park S, Broom BM, et al. Androgen receptor

inhibitor-induced "BRCAness" and PARP inhibition are synthetically lethal for

castration-resistant prostate cancer. Sci Signal 2017;10(480) doi

10.1126/scisignal.aam7479.

27. Wortmann L, Lücking U, Lefranc J, Briem H, Koppitz M, Eis K, et al.; 2-(Morpholin-

4-yl)-1,7-naphthyridines patent WO2016020320. 2016.

28. Foote KM, Nissink JW, Turner P; Morpholino pyrimidines and their use in therapy

patent WO 2011/154737. 2011.

29. Foote KM, Nissink JWM, McGuire T, Turner P, Guichard S, Yates JWT, et al.

Discovery and Characterization of AZD6738, a Potent Inhibitor of Ataxia

Telangiectasia Mutated and Rad3 Related (ATR) Kinase with Application as an

Anticancer Agent. J Med Chem 2018;61(22):9889-907 doi

10.1021/acs.jmedchem.8b01187.

30. Charrier JD, Durrant S, Kay D, Knegtel R, MacCormick S, Mortimore M, et al.;

Pyrazine derivatives useful as inhibitors of atr kinase patent WO 2010/071837. 2010.

31. Knegtel R, Charrier JD, Durrant S, Davis C, O'Donnell M, Storck P, et al. Rational

Design of 5-(4-(Isopropylsulfonyl)phenyl)-3-(3-(4-

((methylamino)methyl)phenyl)isoxazol-5-yl )pyrazin-2-amine (VX-970, M6620):

Optimization of Intra- and Intermolecular Polar Interactions of a New Ataxia

Telangiectasia Mutated and Rad3-Related (ATR) Kinase Inhibitor. J Med Chem

2019;62(11):5547-61 doi 10.1021/acs.jmedchem.9b00426.

32. Politz O, Siegel F, Barfacker L, Bomer U, Hagebarth A, Scott WJ, et al. BAY

1125976, a selective allosteric AKT1/2 inhibitor, exhibits high efficacy on AKT

signaling-dependent tumor growth in mouse models. Int J Cancer 2017;140(2):449-59

doi 10.1002/ijc.30457.





23

33. Chou TC. Theoretical basis, experimental design, and computerized simulation of

synergism and antagonism in drug combination studies. Pharmacol Rev

2006;58(3):621-81 doi 10.1124/pr.58.3.10.

34. Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of

cells in vitro. Nat Protoc 2006;1(5):2315-9 doi 10.1038/nprot.2006.339.

35. Bliss CI. The toxicity of poisons applied jointly. Ann Appl Biol 1939;26:585-615 doi

https://doi.org/10.1111/j.1744-7348.1939.tb06990.x.

36. Foucquier J, Guedj M. Analysis of drug combinations: current methodological

landscape. Pharmacol Res Perspect 2015;3(3):e00149 doi 10.1002/prp2.149.

37. Liu S, Shiotani B, Lahiri M, Marechal A, Tse A, Leung CC, et al. ATR

autophosphorylation as a molecular switch for checkpoint activation. Mol Cell

2011;43(2):192-202 doi 10.1016/j.molcel.2011.06.019.

38. Halazonetis TD, Gorgoulis VG, Bartek J. An oncogene-induced DNA damage model

for cancer development. Science 2008;319(5868):1352-5 doi

10.1126/science.1140735.

39. Hall AB, Newsome D, Wang Y, Boucher DM, Eustace B, Gu Y, et al. Potentiation of

tumor responses to DNA damaging therapy by the selective ATR inhibitor VX-970.

Oncotarget 2014;5(14):5674-85 doi 10.18632/oncotarget.2158.

40. Zeng L, Beggs RR, Cooper TS, Weaver AN, Yang ES. Combining Chk1/2 Inhibition

with Cetuximab and Radiation Enhances In Vitro and In Vivo Cytotoxicity in Head

and Neck Squamous Cell Carcinoma. Mol Cancer Ther 2017;16(4):591-600 doi

10.1158/1535-7163.MCT-16-0352.

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 selective

sensitization of pancreatic tumors to radiation. Cell Death Dis 2012;3:e441 doi

10.1038/cddis.2012.181.

42. Vendetti FP, Karukonda P, Clump DA, Teo T, Lalonde R, Nugent K, et al. ATR

kinase inhibitor AZD6738 potentiates CD8+ T cell-dependent antitumor activity

following radiation. J Clin Invest 2018;128(9):3926-40 doi 10.1172/JCI96519.

43. Wallez Y, Dunlop CR, Johnson TI, Koh SB, Fornari C, Yates JWT, et al. The ATR

Inhibitor AZD6738 Synergizes with Gemcitabine In Vitro and In Vivo to Induce

Pancreatic Ductal Adenocarcinoma Regression. Mol Cancer Ther 2018;17(8):1670-82

doi 10.1158/1535-7163.MCT-18-0010.

44. Vendetti FP, Lau A, Schamus S, Conrads TP, O'Connor MJ, Bakkenist CJ. The orally

active and bioavailable ATR kinase inhibitor AZD6738 potentiates the anti-tumor

effects of cisplatin to resolve ATM-deficient non-small cell lung cancer in vivo.

Oncotarget 2015;6(42):44289-305 doi 10.18632/oncotarget.6247.

45. Rothkamm K, Lobrich M. Evidence for a lack of DNA double-strand break repair in

human cells exposed to very low x-ray doses. Proceedings of the National Academy

of Sciences of the United States of America 2003;100(9):5057-62 doi

10.1073/pnas.0830918100.

46. Barazzuol L, Jena R, Burnet NG, Meira LB, Jeynes JC, Kirkby KJ, et al. Evaluation

of poly (ADP-ribose) polymerase inhibitor ABT-888 combined with radiotherapy and

temozolomide in glioblastoma. Radiat Oncol 2013;8:65 doi 10.1186/1748-717X-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(12):2405-14 doi 10.1158/1535-

7163.MCT-11-0469.

48. Senra JM, Telfer BA, Cherry KE, McCrudden CM, Hirst DG, O'Connor MJ, et al.

Inhibition of PARP-1 by olaparib (AZD2281) increases the radiosensitivity of a lung



https://doi.org/10.1111/j.1744-7348.1939.tb06990.x



24

tumor xenograft. Mol Cancer Ther 2011;10(10):1949-58 doi 10.1158/1535-

7163.MCT-11-0278.

49. Dillon MT, Barker HE, Pedersen M, Hafsi H, Bhide SA, Newbold KL, et al.

Radiosensitization by the ATR Inhibitor AZD6738 through Generation of Acentric

Micronuclei. Mol Cancer Ther 2017;16(1):25-34 doi 10.1158/1535-7163.MCT-16-

0239.

50. O'Connor MJ. Targeting the DNA Damage Response in Cancer. Mol Cell

2015;60(4):547-60 doi 10.1016/j.molcel.2015.10.040.

51. Fojo T, Bates S. Mechanisms of resistance to PARP inhibitors--three and counting.

Cancer Discov 2013;3(1):20-3 doi 10.1158/2159-8290.CD-12-0514.

52. Lord CJ, Ashworth A. Mechanisms of resistance to therapies targeting BRCA-mutant

cancers. Nat Med 2013;19(11):1381-8 doi 10.1038/nm.3369.

53. Sonnenblick A, de Azambuja E, Azim HA, Jr., Piccart M. An update on PARP

inhibitors--moving to the adjuvant setting. Nat Rev Clin Oncol 2015;12(1):27-41 doi

10.1038/nrclinonc.2014.163.

54. D'Andrea AD. Mechanisms of PARP inhibitor sensitivity and resistance. DNA Repair

(Amst) 2018;71:172-6 doi 10.1016/j.dnarep.2018.08.021.

55. Gogola E, Rottenberg S, Jonkers J. Resistance to PARP Inhibitors: Lessons from

Preclinical Models of BRCA-Associated Cancer. Annu Rev Cancer Biol 2019;3:235-

54 doi https://doi.org/10.1146/annurev-cancerbio-030617-050232.

56. Yazinski SA, Comaills V, Buisson R, Genois M-M, Nguyen HD, Ho CK, et al. ATR

inhibition disrupts rewired homologous recombination and fork protection pathways

in PARP inhibitor-resistant BRCA-deficient cancer cells. Genes Dev 2017;31(3):318-

32 doi 10.1101/gad.290957.116.

57. Fang Y, McGrail DJ, Sun C, Labrie M, Chen X, Zhang D, et al. Sequential Therapy

with PARP and WEE1 Inhibitors Minimizes Toxicity while Maintaining Efficacy.

Cancer Cell 2019;35(6):851-67 e7 doi 10.1016/j.ccell.2019.05.001.

58. Sanjiv K, Hagenkort A, Calderon-Montano JM, Koolmeister T, Reaper PM,

Mortusewicz O, et al. Cancer-Specific Synthetic Lethality between ATR and CHK1

Kinase Activities. Cell Rep 2016;14(2):298-309 doi 10.1016/j.celrep.2015.12.032.

59. Goodwin JF, Schiewer MJ, Dean JL, Schrecengost RS, de Leeuw R, Han S, et al. A

hormone-DNA repair circuit governs the response to genotoxic insult. Cancer Discov

2013;3(11):1254-71 doi 10.1158/2159-8290.CD-13-0108.

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 targeting

resistance mechanisms to androgen signaling-directed prostate cancer therapies. Sci

Rep 2015;5:12007 doi 10.1038/srep12007.

61. Fizazi K, Shore N, Tammela TL, Ulys A, Vjaters E, Polyakov S, et al. Darolutamide

in Nonmetastatic, Castration-Resistant Prostate Cancer. N Engl J Med

2019;380(13):1235-46 doi 10.1056/NEJMoa1815671.



https://doi.org/10.1146/annurev-cancerbio-030617-050232



25

Figure legends

Figure 1. Dose-dependent anti-tumor efficacy and unbound plasma levels of

BAY 1895344 as monotherapy.

(A) Chemical structure of BAY 1895344, a highly potent and selective inhibitor of ATR.

(B) Growth curves of GRANTA-519 mantle cell lymphoma tumors in female SCID beige

mice (n = 10/group) treated with vehicle or different doses of BAY 1895344 (3, 10, 30 or

50 mg/kg, 2QD 3 days on/ 4 days off, p.o.).

(C) Growth curves of GRANTA-519 tumors in female SCID beige mice (n = 10/group)

treated with BAY 1895344 (50 mg/kg, 2QD 3 days on/ 4 days off, p.o. = MTD), AZD6738

(50 mg/kg, QD, p.o. = MTD) or M6620 (100 mg/kg, QD, p.o. = MTD).

(D) Unbound BAY 1895344 plasma levels in relation to the determined biochemical, cellular

mechanistic and anti-proliferative IC50values of BAY 1895344 after repeated dosing with 3,

10, 30 or 50 mg/kg (2QD, 3 days on/ 4 days off, p.o.) in GRANTA-519 tumor-bearing female

SCID beige mice. Different IC50 values of BAY 1895344: IC50 biochemical (TR-FRET) =

7 nM, IC50 cellular mechanistic (H2AX in HT-29) = 36 nM, IC50 anti-proliferation (GRANTA-

519) = 30 nM.

(E) Unbound BAY 1895344 plasma levels in relation to the respective in vitro anti-

proliferative IC50 values after repeated dosing with the ATRis BAY 1895344 (50 mg/kg, 2QD

3 days on/ 4 days off, p.o.), AZD6738 (50 mg/kg, QD, p.o.) and M6620 (100 mg/kg, QD,

p.o.) 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 tumor

inoculation (end of last cycle); the dashed line represents the simulated concentration for

24 hours during twice daily dosing. Anti-proliferative IC50 values for BAY 1895344, AZD6738

and M6620 in GRANTA-519 cells are 30 nM, 1000 nM and 75 nM, respectively.

Relative tumor area (T/Crel.area) is defined as the percentage of the 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 modelled using second order

polynomial curves with random intercepts and slopes for each subject. Finally, dose-

response analysis was performed using the 3-parameter logistic model.





26

**, p < 0.01 vs vehicle; ***, p < 0.001 vs vehicle; ##, p < 0.001 vs M6620; 2QD, twice daily;

MTD, maximum tolerated dose; p.o., per os, orally.

Figure 2. Anti-tumor efficacy of BAY 1895344 as monotherapy. The optimal dose and

treatment schedule for BAY 1895344 was 50 mg/kg (2QD 3 days on/ 4 days off, p.o.), based

on efficacy and tolerability in the GRANTA-519 human mantle cell lymphoma xenograft

model in female SCID beige mice. 5-FU, ibrutinib, AZD6738 and M6620 were used at their

known maximal tolerated doses based on published data (39,44). All treatments were well-

tolerated without toxicities (no death, acceptable body weight loss).

(A) Growth curves of A2780 human ovarian tumors in female NMRI nude mice

(n = 10/group) treated with vehicle or BAY 1895344.

(B) Relative tumor areas of the A2780 tumors shown in (A) at study end.

(C) Growth curves of PC-3 human prostate tumors in male NMRI nude mice (n = 10/group)

treated with vehicle or BAY 1895344.

(D) Relative tumor areas of the PC-3 tumors shown in (C) at study end.

(E) Growth curves of LOVO human colorectal tumors in female NMRI nude mice

(n = 10/group) treated with vehicle, BAY 1895344 or 5-FU (50 mg/kg, QW, i.p.).

(F) Relative tumor areas of the LOVO tumors shown in (E) at study end.

(G) Growth curves of REC-1 human mantle cell lymphoma tumors in female C.B-17 SCID

mice (n = 10/group) treated with vehicle, BAY 1895344, ibrutinib (20 mg/kg, QD, p.o.),

AZD6738 (50 mg/kg, QD, p.o.) or M6620 (100 mg/kg, QD, p.o.).

(H) Relative tumor areas of the REC-1 tumors shown in (G) on day 28 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) of

each individual mouse. Statistical analysis was performed using a linear model estimated

with generalized least squares that included separate variance parameters for each study

group.





27

***, p < 0.001 vs vehicle; 5-FU, 5-fluorouracil; i.p., intraperitoneally; PD, progressive disease;

p.o., per os/ orally; PR, partial response; QD, once daily; 2QD, twice daily; QW, once

weekly; SD, stable disease.

Figure 3. Induction of ATR phosphorylation (pATR) and DNA damage in GRANTA-519

xenograft tumors after treatment with BAY 1895344. Intra-tumor (A) pATR and (B-D)

γH2AX protein levels were determined in GRANTA-519 tumors in female SCID beige mice

at different time points (n = 3-4) after last treatment with BAY 1895344 (p.o.) at

(A) 50 mg/kg, QD or 2QD for 2 days followed by QD for one day,

(B) 50 mg/kg, QD or 2QD for 2 days followed by QD for one day,

(C) 10 or 30 mg/kg, QD or 3, 10 or 30 mg/kg, 2QD for 2 days followed by QD for one day

(dose-dependent γH2AX phosphorylation), or

(D) 30 mg/kg for 1-3 treatment cycles: 2QD for 2 days followed by QD for one day, 2QD for

3 days on and 4 days off followed by 2QD for 2 days and QD for one day, 2QD for 3 days on

and 4 days off followed by another 2QD for 3 days on and 4 days off and 2QD for 2 days

and QD for one day) (schedule-dependent γH2AX phosphorylation).

p.o., per os/ orally; QD, once daily; 2QD, twice daily.

Figure 4. Anti-tumor efficacy of BAY 1895344 in combination with chemotherapy or

external beam radiation therapy (EBRT).

(A) Isobologram for the in vitro combination effect of BAY 1895344 and cisplatin on the

proliferation of human HT-29 colorectal cancer cells (n = 3).

(B) Growth curves of IGROV-1 human ovarian tumors in female nude (nu/nu) mice

(n = 10/group) treated with BAY 1895344 (10 or 20 mg/kg, QD, 2 days on/ 5 days off, p.o.;

50 mg/kg, 2QD, 3 days on/ 4 days off, p.o.) in combination with carboplatin (50 mg/kg, QW,

i.p.).

(C) Weights of the IGROV-1 tumors described in (B) at study end.





28

(D) Survival curves of LOVO CRC cells after treatment with 3 nM BAY 1895344 and different

doses of X-radiation determined using the clonogenic assay.

(E) Growth curves of LOVO human colorectal tumors in female nude mice (n = 10/group)

treated with BAY 1895344 (20 or 50 mg/kg, 2QD 2 days on/ 5 days off, p.o.) in combination

with γ-radiation (EBRT). EBRT (5 Gy) was applied directly on the subcutaneously growing

tumors on days 12 and 27 after tumor inoculation.



each individual mouse. Asterisks indicate statistical significance, evaluated by survival

analysis using the Cox proportional-hazards model and longitudinal data were modelled

using second order polynomial curves with random intercepts and slopes for each subject. *,

p < 0.05; **, p < 0.01; ***, p < 0.001 vs vehicle unless indicated otherwise.

CI, combination index; EC50, half-maximal effective concentration; Gy, Gray; i.p.

intraperitoneally; p.o., per os/ orally; QD, once daily; 2QD, twice daily; QW, once weekly.

Figure 5. Anti-tumor efficacy of BAY 1895344 in combination with PARPis.

Isobolograms for the in vitro combination effect of BAY 1895344 and the PARPis (A)

olaparib, (B) niraparib, (C) rucaparib and (D) talazoparib on the proliferation of human

MDA-MB-436 breast cancer cells (n = 3).

(E) Growth curves of MDA-MB-436 human breast cancer tumors in female NOD/SCID mice

(n = 10/group) treated with BAY 1895344 (20 or 50 mg/kg, 2QD, 3 days on/ 4 days off, p.o.)

in combination with olaparib (50 mg/kg, i.p., QD).

(F) Weights of the MDA-MB-436 tumors described in (E) at study end.

(G) Growth curves of 22Rv1 human prostate tumors in male SCID mice (n = 10/group)

treated with BAY 1895344 (20 or 50 mg/kg, 2QD, 3 days on/ 4 days off, p.o.) in combination

with olaparib (20 or 50 mg/kg, QD, i.p.).

(H) Weights of the 22Rv1 tumors described in (G) at study end.







29



using second order polynomial curves with random intercepts and slopes for each subject.

*, p < 0.05; **, p < 0.01; ***, p < 0.001 vs vehicle unless indicated otherwise.

CI, combination index; EC50, half-maximal effective concentration; i.p. intraperitoneally; p.o.,

per os/ orally; QD, once daily; 2QD, twice daily.

Figure 6. Anti-tumor efficacy of BAY 1895344 in combination with anti-androgen

therapy.

(A) Isobologram for the in vitro combination effect of BAY 1895344 and darolutamide on the

proliferation of human LAPC-4 prostate cancer cells (n = 3).

(B) Growth curves of hormone-dependent LAPC-4 human prostate tumors in male SCID

mice (n = 11/group) treated with BAY 1895344 (20 mg/kg, 2QD 3 days on/ 4 days off, p.o.)

in combination with darolutamide (100 mg/kg, QD, p.o.).

(C) Growth curves of LAPC-4 tumors in male SCID mice (n = 10/group) treated with

BAY 1895344 (20 mg/kg, 2QD 3 days on/ 4 days off, p.o.) in combination with darolutamide

(100 mg/kg, QD, p.o.) and EBRT (5 Gy, Q7Dx2).





using second order polynomial curves with random intercepts and slopes for each subject.

*, p < 0.05; **, p < 0.01; ***, p < 0.001 vs vehicle or as indicated.

CI, combination index; EC50, half-maximal effective concentration; Gy, Gray; p.o., per os/

orally; QD, once daily; 2QD, twice daily; Q7Dx2, every 7 days twice.




6 8 10 12 14 16 18 20 22 24 26 28 300

50

100

150

200

250

Days after tumor inoculation

Tum

orar

ea(m

m2 ,

mea

n±SD

)

BAY 1895344 dose-response in GRANTA-519

Vehicle

10 mg/kg (T/Crel.area = 0.66)30 mg/kg (T/Crel.area = 0.19)50 mg/kg (T/Crel.area = 0.03)

3 mg/kg (T/Crel.area = 0.79)

***

******

**

0 5 10 15 20 251

10

100

1000

10000

Time (h)

Unbo

und

plas

ma

conc

entra

tion

(nM

) Exposure of BAY 1895344 in plasma

2 x 50 mg/kg2 x 3 mg/kg

IC50 H2AX phosphorylation (HT-29)

2 x 10 mg/kg2 x 30 mg/kg

IC50 anti-proliferation (GRANTA-519)

IC50 biochemical (TR-FRET)

0 5 10 15 20 251

10

100

1000

10000

Time (h)

Unbo

und

plas

ma

conc

entra

tion

(nM

) Exposure of ATRis in plasma (GRANTA-519)

IC50 anti-proliferation AZD6738

BAY 1895344 2x50 mg/kg - simulated

AZD6738 (50 mg/kg)

M6620 (100 mg/kg)

IC50 anti-proliferation M6620

BAY 1895344 50 mg/kg - last single dose

IC50 anti-proliferation BAY 1895344

18 20 22 24 26 28 300

50

100

150

200


Tum

orar

ea(m

m2 ,

mea

n±SD

)

ATRis in GRANTA-519

Vehicle

BAY 1895344 (T/Crel.area = -0.02)AZD6738 (T/Crel.area = 0.42)M6620 (T/Crel.area = 0.70)

***

**

##

A

B C

D E

Figure 1




4 6 8 10 12 14 160

50

100

150

200

250

Time after tumor inoculation [days]

Tum

or

are

a (

mm

2, m

ean±S

D)

A2780

Vehicle BAY 1895344 (T/Crel.area = 0.13)

***

7 14 21 280

50

100

150

200

250


Tum

or

are

a (

mm

2, m

ean±S

D)

PC-3

Vehicle BAY 1895344 (T/Crel.area = -0.02)

***

7 14 21 280

50

100

150

200


Tum

or

are

a (

mm

2, m

ean±S

D)

LOVO

Vehicle

BAY 1895344 (T/Crel.area = 0.13)

5-FU (T/Crel.area = 0.90)

***

14 21 28 35 420

50

100

150

200

250


Tum

or

are

a (

mm

2, m

ean±S

D)

REC-1

Vehicle

BAY 1895344 (***, day 28 vs all other groups;

T/Crel.area = -0.14)

Ibrutinib (***, day 28; T/Crel.area = 0.22)

AZD6738 (***, day 28; T/Crel.area = 0.52)

M6620 (***, day 28; T/Crel.area = 0.53)

Vehicle BAY 189534410

100

1000

Rela

tive tum

or

are

a [%

]

A2780

PD

SD

PR

Vehicle BAY 189534410

100

1000

Rela

tive tum

or

are

a [%

]

PC-3

PD

SD

PR

Vehicle BAY 1895344 5-FU10

100

1000

Rela

tive tum

or

are

a [%

]

LOVO

PD

SD

PR

Vehicle BAY 1895344 Ibrutinib AZD6738 M662010

100

1000

Rela

tive tum

or

are

a [%

]

REC-1

PD

SD

PR

A B

C D

E F

G H

Figure 2




3 h 7 h 24 h 48 h0102030γH2AX protein level (% of GAPDH, mean±SD) Vehicle BAY1895344 (QDx1) BAY1895344 (2QDx2 + QDx1)BAY 1895344020406080100 Time (h)γH2AX protein level (% of GAPDH, mean+SD) 0.5 1 3 8 24 48 72Vehicle 10 mg/kg QDx1 30 mg/kg QDx130 mg/kg 2QDx2 + QDx110 mg/kg 2QDx2 + QDx13 mg/kg 2QDx2 + QDx1020,00040,00060,00080,000 Time (h)γH2AX protein level (% of GAPDH, mean+SD) BAY 18953443 8 24 2QD 3 days on/ 4 days off, 3 cycles2QD 3 days on/ 4 days off, 2 cycles2QD 3 days on, 1 cycleVehicle3 h 7 h 24 h 48 h5.0×1061.0×1071.5×1072.0×107Phosphorylated ATR (chemiluminescence intensity, mean±SD) Vehicle BAY1895344 (QDx1) BAY1895344 (2QDx2 + QDx1)A BCD Figure 3




0

2×

10

-7

4×

10

-7

6×

10

-7

0

2×10-6

4×10-6

6×10-6

8×10-6

EC50 (BAY 1895344, M)

EC

50 (

Cis

pla

tin, M

)CImin = 0.14

40 nM BAY 1895344 + 560 nM cisplatin

8 10 12 14 16 18 20 22 24 26 2820

40

60

80

100


Tum

or

are

a (

mm

2, m

ean±S

D)

IGROV-1

Vehicle!

BAY 1895344 (10 mg/kg; T/Crel.area = 0.79)


BAY 1895344 (10 mg/kg) + carboplatin (***, day 28 vs vehicle;

*, vs BAY 1895344; *, vs carboplatin; T/Crel.area = 0.49)

BAY 1895344 (20 mg/kg) + carboplatin (***, day 28 vs vehicle;

***, vs BAY 1895344; ***, vs carboplatin; T/Crel.area = 0.37)

BAY 1895344 (50 mg/kg) (***, day 28; T/Crel.area = 0.28)

Carboplatin (T/Crel.area = 0.79)

10 20 30 40 50 60 70 800

50

100

150

200

250


Tum

or

are

a (

mm

2, m

ean ±

SD

)

LOVO

Vehicle


BAY 1895344 (50 mg/kg) (**, day 39; T/Crel.area = 0.55)

Radiation (***, day 39; T/Crel.area = 0.44)

BAY 1895344 (50 mg/kg) + radiation

(***, day 39 vs vehicle; ***, vs BAY 1895344 (50 mg/kg);

***, vs radiation; T/Crel.area = -0.05)

BAY 1895344 (20 mg/kg) + radiation

(***, day 39 vs vehicle; ***, vs BAY 1895344 (20 mg/kg);

*, vs radiation; T/Crel.area = 0.22)

5 Gy5 Gy

IGROV-1

0

100

200

300

400

Tum

or

weig

ht (m

g, m

ean±S

D)

Vehicle

BAY 1895344

(mg/kg)

Carbo-

platinBAY 1895344

(mg/kg)

+ carboplatin

20 5010 2010

0 1 2 3 40.00001

0.0001

0.001

0.01

0.1

1

Radiation dose (Gy)

Surv

ivin

g fra

ction

LOVO

Vehicle BAY 1895344 (3 nM)

A

B C

D E

Figure 4




0 2×10-8 4×10-8 6×10-8 8×10-8 1×10-702×10-64×10-66×10-68×10-6 EC50 (BAY 1895344, M)EC 50 (Olaparib, M) CImin = 0.3614 nM BAY 1895344 + 1.4 µM olaparib 0.0 4.0×10-8 8.0×10-8 1.2×10-7 1.6×10-701×10-52×10-53×10-5 EC50 (BAY 1895344, M)EC 50 (Rucaparib, M) CImin = 0.3538 nM BAY 1895344 + 3.8 µM rucaparib 40 50 60 70 80050100150200 Days after tumor inoculationTumor area (mm2 , mean±SD) MDA-MB-436 Vehicle!BAY 1895344 (20 mg/kg) ( ***, day 83; T/Crel.area = 0.57)BAY 1895344 (50 mg/kg) (***, day 83; T/Crel.area = 0.38)Olaparib (***, day 83; T/Crel.area = 0.29)BAY 1895344 (20 mg/kg) + olaparib (***, day 83 vs vehicle; ***, vs BAY 1895344; **, vs olaparib; T/Crel.area = -0.03)10 12 14 16 18 20 22 24050100150200250 Days after tumor inoculationTumor area (mm2 , mean±SD) 22Rv1Vehicle BAY 1895344 (20 mg/kg) ( *, day 24; T/Crel.area = 0.73)Olaparib (20 mg/kg)Olaparib (50 mg/kg)BAY 1895344 (50 mg/kg) (***, day 24; T/Crel.area = 0.42)BAY 1895344 (20 mg/kg) + olaparib (20 mg/kg)(***, day 24 vs vehicle; ***, vs olaparib; T/Crel.area = 0.54)0.0 5.0×10-8 1.0×10-7 1.5×10-701×10-62×10-63×10-6 EC50 (BAY 1895344, M)EC 50 (Niraparib, M) CImin = 0.3529 nM BAY 1895344 + 320 nM niraparib 0.0 4.0×10-8 8.0×10-8 1.2×10-7 1.6×10-702×10-84×10-86×10-88×10-8 EC50 (BAY 1895344, M)EC 50 (Talazoparib, M) CImin = 0.3726 nM BAY 1895344 + 13 nM talazoparib 0500100015002000Tumor weight (mg, mean±SD) MDA-MB-436Vehicle BAY 1895344(mg/kg) Olaparib BAY 1895344(20 mg/kg)+ olaparib20 5022Rv10500100015002000Tumor weight (mg, mean±SD) Vehicle BAY 1895344(mg/kg) Olaparib(mg/kg) BAY 1895344(20 mg/kg)+ olaparib(20 mg/kg)20 5020 50

A BC DE FG H Figure 5




0 2×10-9 4×10-9 6×10-9 8×10-901×10-72×10-73×10-7 EC50 (BAY 1895344, M)EC 50 (Darolutamide, M) CImin = 0.827.6 nM BAY 1895344 + 280 nM darolutamide 25 50 75 10050100150200250 Days after tumor inoculationTumor area [mm2 , mean+SD] VehicleCastration (***, day 53; T/Crel.area = 0.16)BAY 1895344 (***, day 53; T/Crel.area = 0.69)Darolutamide (***, day 53; T/Crel.area = 0.36)Radiation (***, day 53; T/Crel.area = 0.35)BAY 1895344 + radiation (***, day 53 vs. vehicle; T/Crel.area = 0.19)Darolutamide + radiation (***, day 53 vs vehicle; T/Crel.area = 0.02; ***, day 81 vs radiation; ***, day 81 vs darolutamide)BAY 1895344 + darolutamide (***, day 53 vs vehicle; T/Crel.area = 0.17; **, day 81 vs darolutamide)BAY 1895344 + darolutamide + radiation (***, day 53 vs vehicle; T/Crel.area = 0.01; ***, day 81 vs radiation; ***, day 81 vs darolutamide; ***, day 102 vs BAY 1895344 + radiation)LAPC-4 30 40 50 60 70 80 90 100050100150200250 Days after tumor inoculationTumor area (mm2 , mean±SD) LAPC-4 Vehicle BAY 1895344 (*, day 51; T/Crel.area = 0.66)Darolutamide (***, day 51; T/Crel.area = 0.25)BAY 1895344 + darolutamide (***, day 51 vs vehicle; T/Crel.area = 0.15; ***, day 103 vs BAY 1895344; *, day 103 vs darolutamide)Castration (***, day 51)A BC Figure 6




Published OnlineFirst October 3, 2019.Mol Cancer Ther Antje M. Wengner, Gerhard Siemeister, Ulrich Lücking, et al. repair-compromising therapies in preclinical cancer modelsmonotherapy and combined with DNA damage-inducing or The novel ATR inhibitor BAY 1895344 is efficacious as

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