Elimusertib, a Novel ATR Inhibitor, Induces Anti-tumor Effects through Replication Catastrophe in Breast Cancers

Article information

Cancer Res Treat. 2026;58(1):159-174

Publication date (electronic) : 2025 April 7

doi : https://doi.org/10.4143/crt.2024.1105

, Ahrum Min ¹^,², Sohyeon Kim ¹, Seongyeong Kim ¹, Yu-jin Kim ¹, Sujin Ham ¹, Miso Lee ¹, Eunice Yoojin Lee ³, Jinyong Kim ¹^,⁴, Dae-Won Lee ¹^,⁴^,⁵, Kyung-Hun Lee^,¹^,⁴

, Seock-Ah Im ¹^,²^,⁴^,⁵

¹Cancer Research Institute, Seoul National University, Seoul, Korea

²Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea

³Department of Internal Medicine, University of California San Francisco, San Francisco, CA, USA

⁴Department of Internal Medicine, Seoul National University Hospital, Seoul, Korea

⁵Department of Internal Medicine and Translational Medicine, Seoul National University College of Medicine, Seoul, Korea

Correspondence: Kyung-Hun Lee, Department of Internal Medicine, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul 03080, Korea Tel: 82-2-2072-7207 E-mail: kyunghunlee@snu.ac.kr

Received 2024 December 6; Accepted 2025 April 1.

Abstract

Purpose

Sustained cell proliferation and cell cycle acceleration in cancer cells inherently increase DNA damage, which interferes with homeostatic replication and transcription. Ataxia telangiectasia and Rad3-related (ATR) is crucial for initiation of the DNA damage response, and ATR inhibitors, such as elimusertib, induce increased replication stress and DNA damage. We investigated the anti-tumor effects of elimusertib and its mechanism of action in relation to replication stress.

Materials and Methods

Anti-tumor effects were evaluated by MTT assay and colony formation assay in breast cancer cell lines in vitro, in breast cancer cell xenografts in vivo, and in patient-derived xenograft models. Cell cycle was assessed by flow cytometry and BrdU assay was used to measure replicating cells and S-phase progression. Alkaline and neutral comet assay was used to measure single and double-stranded DNA damages, respectively.

Results

Elimusertib delayed S-phase progression in MDA-MB-453 and MDA-MB-231 cells and induced caspase-7–dependent apoptosis. Furthermore, the increase in sub-G1 population in the fluorescence-activated cell sorting analysis and Annexin V assay also confirmed apoptotic cell death. In the BrdU assay, single-stranded DNA (ssDNA) increased in sensitive cells and aberrant ssDNA induced DNA damage in S-phase and eventually caused replication catastrophe. Finally, these anti-tumor effects were proven in in vivo xenograft and patient-derived xenograft models.

Conclusion

Elimusertib had anti-tumor effects and induced replication catastrophe in breast cancer cells with a high replication rate. Moreover, cells under high DNA replication stress were sensitive to elimusertib. Further studies and treatment strategies with elimusertib are warranted for cancers with a high replication rate.

Keywords: ATR inhibitor; DNA damage; Cell cycle progression; Replication stress; Replication catastrophe

Introduction

Sustained and unchecked cellular proliferation is a major hallmark of cancer [1]. Cells employ evolutionarily conserved mechanisms to ensure the faithful replication and transmission of genetic material to their progeny, including the highly complex task of coordinating DNA replication fork progression. Slowing or stalling of replication fork progression—a phenomenon referred to as replication stress—is a primary cause of genomic instability, a second hallmark of cancer that in turn fuels oncogenesis and tumor progression via the accumulation of pathogenic variants in tumor suppressor genes and oncogenes.

The ataxia telangiectasia and Rad3-related (ATR) protein is a member of the phosphoinositide 3 kinase-related kinase family that, together with its closely related kinases ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase. Both kinases respond to DNA double-stranded breaks (DSBs). However, ATR plays a critical role in the cellular response to replication stress and DNA damage [2]. ATR acts as a replication stress checkpoint as a surveillant by monitoring and responding to problems that arise during DNA replication. When DNA replication is stalled or slowed due to stress or damage, ATR is activated and coordinates a series of cellular responses to stabilize the replication fork, promote DNA repair, and control cell cycle progression. This helps maintain genomic integrity and prevent further damage [3]. The generation of single-stranded DNA (ssDNA) as a result of replication fork stalling or DNA DSBs leads to ssDNA binding by the replication protein A (RPA) complex, which recruits ATR to promote downstream DNA repair, replication fork stabilization, and transient cell cycle arrest [4,5].

Given the critical function of ATR in promoting cellular survival during replication stress, ATR has emerged as a potential novel therapeutic target in anti-tumor drug development. Studies have demonstrated that suppression of ATR function in cancer cells inhibited tumor growth and increased cell death as a result of increased sensitivity to replicative stress, suggesting that low levels of selective ATR inhibition may suppress cancer cell proliferation while sparing normal cells [6,7]. It has also been demonstrated that loss of ATR function increases sensitization to other therapeutic modalities including radiation and chemotherapy, supporting the potential for ATR inhibition in combinatorial anti-tumor therapy [6,8]. Accordingly, several selective small molecule ATR inhibitors (ATRis) have been developed and are under clinical investigation for both lymphomas and solid tumors [8-10].

Targeting ATR has shown promising anti-tumor effects in preclinical models, and several ATRis are currently undergoing phase I-III clinical trials as potential cancer treatments [11]. Berzosertib (M6620, VE-822, VX-970), which was derived from the previous version VE-821, underwent phase I-II trial as a monotherapy or in combination with various chemotherapeutic agents in triple-negative breast cancer, urothelial carcinoma, castration-resistant prostate cancer, small cell lung cancer, as well as high-grade serous ovarian cancer [12-15]. Ceralasertib (AZD6738), derived from AZ20, underwent phase I-II trials in tumors with various genetic traits such as BRCA1/2-mutation, ATM-mutation, and with poly(ADP-ribose) polymerase (PARP)-inhibitor–resistance and is now being evaluated in a phase III trial in non–small cell lung cancer (NCT05450692) [16-19].

Additionally, several other orally available ATRis have been discovered, including gartisertib (M4344, VX-803) and tuvusertib (M1774), elimusertib (BAY1895344), camonsertib (RP-3500) [20-23]. These inhibitors are currently in preclinical development and have advanced to phase I/II clinical trials, being evaluated both as monotherapies and in combination with DNA-damaging agents or molecular targeted treatments. ATRis have demonstrated promising anti-tumor activity in clinical trials, particularly when combined with chemotherapy, PARP inhibitors, or immunotherapy. While ATM loss appears to be a useful biomarker for predicting responses, further research is needed to identify additional predictive biomarkers for the successful implementation of ATRi-based therapies [22,24-27].

Elimusertib, a novel selective inhibitor of ATR, has shown high efficacy as both monotherapy and in combination with chemotherapy in cancer cells with defective responses to DNA damage, such as ATM loss [21,28,29]. Despite its potency, detailed mechanisms underlying its anti-tumor effects remain to be investigated. Moreover, it is difficult to define loss-of-function of ATM both preclinically and clinically [30]. A deeper understanding of the mechanisms of action, as well as molecularly and clinically targetable tumors, is crucial. In the present study, we sought to investigate the mechanisms of the anti-tumor activities of elimusertib, particularly in breast cancer, characterized by high rates of replicative stress and genomic instability [8,9,28].

Materials and Methods

1. Cell lines and reagents

Cell line mutation data were obtained from the online public dataset DepMap (depmap.org/portal).

Human breast cancer cell lines (MDA-MB-453, HCC1143, HCC1954, HCC38, HCC1395, BT-20, MDA-MB-157, and MDA-MB-231) were purchased from the American Type Culture Collection (ATCC) and verified by short tandem repeat analysis.

Other human breast cancer cells (MCF7, T-47D, MDA-MB-468, HCC1937, HCC70, BT-474, SK-BR-3, HCC1419, Hs 578T, and BT-549) were purchased from Korean Cell Line Bank and confirmed using DNA fingerprint analysis. All cell lines were banked and passaged for less than 6 months before use and maintained in a moistened atmosphere of 5% CO₂ and 37℃ in RPMI-1640 (Welgene Inc.) complemented with 10% fetal bovine serum (GIBCO, Thermo Fisher Scientific Inc.) and 10 μg/mL gentamicin (Cellgro).

Elimusertib was provided by Bayer AG in powder form and which was then dissolved in dimethyl sulfoxide (DMSO) to form 10 mmol/L stock solution.

2. Cell growth inhibition assay

Cells were seeded in 96-well plates at a density ranging from 1 to 7×10³ cells per well and incubated overnight at 37°C. The cells were then treated with varying concentrations of elimusertib (0-1 μmol/L) for 5 days. Following treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole (MTT) solution was added up each well. Cells were incubated at 37°C for 4 hours prior to measuring cell viability (VersaMax microplate reader at 540 nm; Molecular Devices). Obtained results were used to calculate IC₅₀ values via PRISM software (GraphPad Inc.).

3. Colony formation assay

Cells were seeded into 6-well plates and incubated for 48 hours at 37°C in 5% CO₂ prior to treatment with varying concentrations of elimusertib (0, 1, 10, 25, and 50 nmol/L). Cells were incubated for up to 14 days at which the presence of colonies was analyzed by staining with a 0.1% Coomassie blue solution (Sigma-Aldrich) and counted using a GelCount automatic plate scanner (Oxford Optronics GelCount). The cell survival rate and IC₅₀ of elimusertib were calculated using PRISM software (GraphPad Inc.).

4. Western blot analysis

Whole cell protein lysates (20 μg) were loaded and subjected to electrophoresis on a 7%-15% sodium dodecyl sulfate– polyacrylamide gel. Separated proteins were transferred onto nitrocellulose membranes followed by overnight incubation with primary antibodies at 4℃. Antibody binding was detected using a chemiluminescence system labeled probes as horseradish peroxidase–linked secondary antibody according to the manufacturer’s protocols (Amersham Biosciences).

The following is a list of primary antibodies used in this study: anti-RPA32 (mouse, sc-56770, Santa Cruz Biotechnology), anti-caspase 7 (rabbit, #9492, Cell Signaling Technology), anti-pH2AX (mouse, #05-636, Millipore), anti-PARP (mouse, 556494, BD Biosciences), anti-pRPA32(Ser4/Ser8) (mouse, #A300-245A, Bethyl Laboratories), anti-pCDK2(Tyr15) (rabbit, ab76146, Abcam), anti-CDK2 (rabbit, #2546, Cell Signaling Technology), and anti-actin (mouse, A3853, Millipore).

5. Cell cycle analysis

Cells were treated with elimusertib or vehicle control DMSO for indicated time, harvested, and fixed in 70% ethanol prior to storage in –20°C for at least 2 days. To quantify DNA content, cells were treated with 10 μg/mL RNase A (Sigma-Aldrich) for 20 minutes, followed by staining with 20 μg/mL propidium iodide (PI; Sigma-Aldrich) and subsequent flow cytometry analysis on a BD FACSCalibur cytometer (BD Biosciences). A total of 10,000 cells were counted per experimental group. Apoptosis was quantified via Annexin V–binding assay performed in accordance with the manufacturer’s instructions (BD Pharmingen).

6. BrdU assay

To quantify cell proliferation, cells were treated with 20 mmol/L BrdU for 2 hours before harvesting and incubation with a FITC-conjugated anti-BrdU antibody (host, BD Biosciences) [31]. Cells were stained with 20 μg/mL PI (Sigma-Aldrich) prior to flow cytometry analysis on a BD FACSCalibur flow cytometer (BD Biosciences). To measure S-phase progression, cells were incubated with 20 mmol/L of BrdU for 2 hours prior to treatment with elimusertib, followed by anti-BrdU and PI staining as above. Cells were analyzed via flow cytometry on a FACSCalibur cytometer (BD Biosciences).

7. Comet assay

The extent of DNA damage was measured by an alkaline comet assay using the Trevigen Comet Assay Kit (Trevigen) according to the manufacturer’s protocols. Tail lengths were measured using the Comet assay IV program (Andor Technology).

8. Immunofluorescence assay

Cells were seeded on a coverslip coated with poly-L-lysine and incubated with 40 mmol/L EdU (Invitrogen, Thermo Fisher Scientific Inc.) for 2 hours before treatment with elimusertib. After 48 hours, the cells were fixed in 3.7% paraformaldehyde, permeabilized with 0.5% Triton X-100 in phosphate buffered saline (Welgene Inc.) and incubated with primary antibodies 4°C overnight. Cells were mounted onto glass sides with aqueous mounting media (Vector Laboratories) and visualized with a STELLARIS 5 laser scanning microscope (Leica Biosystems).

The following is a list of primary antibodies used in this study: anti-pH2AX (mouse, #05-636, Millipore), anti-pRPA32 (Ser4/Ser8) (mouse, #A300-245A, Bethyl Laboratories).

9. In vivo xenograft model

All animal experiments were performed at Seoul National University’s Institute for Experimental Animals under institutional guidelines and approval from the Institutional Animal Care and Use Committee (IACUC) (Protocol #19-0176-S1A1). MDA-MB-231 tissue was implanted into 5-week-old female BALB/c athymic nude mice (Orient Bio Inc.) via subcutaneous injection. Tumor height and width were measured twice a week with a caliper and volume was estimated using the formula (width²×height)/2. Body weight was also measured (daily/weekly/etc.). When tumors reached a size of 200 mm³, mice were randomly divided into the following experimental groups (n=5 mice per group): 30 mg/kg elimusertib, 50 mg/kg elimusertib, or vehicle control (40% polyethylene glycol and 60% distilled water). Elimusertib was administered twice daily for 4 weeks according to a 3 days on/4 days off schedule via oral gavage. At the end of the observation period, mice were euthanized via CO₂ asphyxiation and the tumors were harvested for further analysis.

10. Patient-derived xenograft model

After implanting the tumor tissue from the patient into NSG mice (P0) and when the tumor volume reached 1,000 mm³, it was divided in small pieces to store as P1 [14]. Twenty-six female BALB/c athymic nude 6-week-old mice were implanted with tissue of P1. Tumors (IGX-216, gastric cancer with F2813fs ATM mutation; X151, breast cancer with Lys2777* BRCA2 mutation) were minced and implanted into 6-week-old BALB/c athymic nude mice (Orient Bio Inc.) via subcutaneous injection. Tumor size/volume and body weight were measured weekly as above. When the tumor volume reached 200 mm³, mice were randomly assigned to receive either 40 mg/kg elimusertib or vehicle control (IGX-216: eight per group, X151: six per group). elimusertib (40 mg/kg) was administered twice daily for 4 weeks according to a 3 days on/4 days off schedule via oral gavage. Tumor volume was calculated as (width²×height)/2. At the end of the observation period, mice were euthanized via CO₂ asphyxiation and the tumors were harvested for further analysis.

11. Immunohistochemistry

Paraffin-embedded xenograft tumor tissues were deparaffinized with xylene and rehydrated with ethanol. Ki-67 levels were measured by staining 4 μm tissue sections with rabbit anti–Ki-67 (1:200, Invitrogen). To measure apoptosis, we performed a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) labeling assay using the ApopTag In Situ Apoptosis Detection Kit (Chemicon International) following the manufacturer’s protocol. Stained slides were scanned at ×400. Positive cells for each three slides was counted per 100 cells. Counting is based on differentiation between brown (positive) and blue counter staining, was used for analysis of Ki-67 and TUNEL signals.

12. Statistical analysis

SigmaPlot ver. 9.0 (Systat Software Inc.) was used for statistical analysis. Results were expressed as the mean±standard error (SE). A two-sided Student’s t test was used when appropriate. p-values < 0.05 were considered statistically significant.

Results

1. Elimusertib exerts anti-tumor effects on breast cancer cell lines by delaying cell cycle progression and inducing apoptosis

In response to replication stress in proliferating cells, ATR orchestrates a robust DNA damage response and subsequent cell cycle arrest, and inhibition of ATR could result in anti-proliferative effects. To test the anti-proliferative effects of elimusertib in breast cancer, we treated breast cancer cell lines with varying concentrations of elimusertib and performed an MTT assay. Of the 18 cell lines, MDA-MB-231, a triple-negative breast cancer cell line (IC₅₀=100 nM), and MDA-MB-453, a human epidermal growth factor receptor 2 (HER2)-amplified cell line (IC₅₀=46 nM), displayed high sensitivity to elimusertib with IC₅₀ equal or lower than 100 nM, which is the achievable plasma concentration in humans [29]; meanwhile, T-47D, a hormone receptor–positive cell line, displayed a low sensitivity to elimusertib, with IC₅₀ of 650 nM (Fig. 1A and B). We also confirmed the anti-proliferative effect of elimusertib using a colony forming assay, which showed that elimusertib suppressed the proliferation of MDA-MB-453 and MDA-MB-231 but not that of T-47D cells (S1 Table, S2A and S2B Fig.).

Fig. 1.

Elimusertib inhibited the proliferation of breast cancer cells by inducing S-phase arrest and apoptosis. (A) Cytotoxicity of elimusertib as a monotherapy in breast cancer cell lines. Following incubation with the indicated concentrations of elimusertib for 5 days, cell viability was accessed by MTT assay. (B) IC₅₀ values for 18 cell lines. (C) Table of mutations affecting genes associated with ataxia telangiectasia and Rad3-related (ATR) inhibitor sensitivity in MDA-MB-453, MDA-MB-231 and T-47D cells. (D) The DNA content was assessed by flow cytometry. Breast cancer cells were treated for 5 days with elimusertib. (E) Apoptotic cells were assessed by flow cytometry. Breast cancer cells were treated with elimusertib for 5 days. ^*p < 0.05. (F) Protein expression was measured by Western blotting. Breast cancer cells were treated with elimusertib (0.05 μmol/L) for 5 days. PARP, poly(ADP-ribose) polymerase.

Prior to assessing ATR inhibition, protein expression levels of relevant DNA damage response factors were confirmed across the selective cell lines (Fig. 1C).

To specifically assess whether ATR inhibition via elimusertib leads to cell cycle arrest, we performed a cell cycle analysis by measuring DNA content with flow cytometry-detected PI. In MDA-MB-453 and MDA-MB-231, which are highly sensitive to elimusertib, we observed an accumulation of cells in the S-phase relative to the less sensitive T-47D cell line. We also observed an increase in the sub-G1 population in MDA-MB-453 and MDA-MB-231 relative to T-47D (Fig. 1D). To examine whether this increase in the sub-G1 population was due to increased apoptosis, we performed staining for Annexin V, which showed a dose-dependent increase in apoptosis in MDA-MB-453 and MDA-MB-231 (Fig. 1E). Consistent with this result, we also observed a dose-dependent increase in the expression of the cleaved forms of PARP and caspase-7, which are upregulated during apoptosis, in MDA-MB-453 and MDA-MB-231 (Fig. 1F).

2. Elimusertib delays the S-phase progression

To investigate the mechanism by which ATR inhibition leads to the accumulation of S-phase cells, we performed BrdU staining followed by flow cytometry analysis. In contrast to the increased S-phase population in MDA-MB-453 as assessed by PI staining, we unexpectedly observed a decrease in BrdU incorporation in MDA-MB-453, but not in T-47D, with the treatment of elimusertib (Fig. 2A). We postulated that this discrepancy could be attributed to a delay in S-phase progression induced by elimusertib, leading to decreased BrdU uptake if pulsed either with or after elimusertib treatment. Consistent with this hypothesis, BrdU pulsation for 2 hours prior to elimusertib treatment resulted in a significant decrease in BrdU uptake in MDA-MB-453 but not in T-47D cells (Fig. 2B). Furthermore, we also observed a significant decrease in the levels of p-CDK2 (Tyr15), an essential regulator of the cell cycle, in MDA-MB-453 and MDA-MB-231 after elimusertib treatment (Fig. 2C). Taken together, these results demonstrate that elimusertib-mediated ATR inhibition exerts anti-proliferative effects by delaying S-phase progression.

Fig. 2.

Inhibition of ataxia telangiectasia and Rad3-related (ATR) causes aberrant cell cycle progression in the S-phase and induces abnormal single-stranded DNA. (A) DNA replication was assessed by flow cytometry with BrdU antibody. (B) Replicating cells were assessed by flow cytometry with BrdU antibody. (C) After elimusertib treatment for 5 days, the phosphorylation of the Tyr15 residue of CDK2 was quantified and normalized using ImageJ. ^*p < 0.05, ^**p < 0.05, ^***p < 0.001.

3. DNA damage is accumulated by elimusertib, with increased ssDNA breaks in sensitive cells which are converted into DSBs

Aberrant DNA replication and subsequent DNA damage results in the generation of ssDNA, and the binding of RPA32 to ssDNA is a critical step in the DNA damage response. Upon binding to ssDNA, RPA32 is phosphorylated at serine residues (Ser4/Ser8) and subsequently recruits enzymes necessary for the repair process. We therefore hypothesized that exposure to elimusertib results in the accumulation of phosphorylated RPA32(Ser4/Ser8) (p-RPA32(Ser4/Ser8)) in sensitive breast cancer cell lines. Indeed, EdU pulsing for 2 hours followed by elimusertib treatment increased p-RPA32(Ser4/Ser8) expression in EdU+ MDA-MB-453 and MDA-MB-231 but not in T-47D cells (Fig. 3A). It was assumed that excessively generated and exposed ssDNA due to prolongation of S-phase in elimusertib sensitive cells would be converted into DSBs during the replication process, and co-positivity of γ-H2AX was actually observed in p-RPA32 positive cells (Fig. 3B). These data were also confirmed by increased γ-H2AX expression in EdU-positive cells after elimusertib treatment only in sensitive cell lines (Fig. 3C). Additionally, western blot revealed a dose-dependent increase in both p-RPA32 and γ-H2AX in MDA-MB-453 and MDA-MB-231 upon exposure to elimusertib (Fig. 3D). The accumulation of DNA damage in sensitive cells in response to ATR inhibition was further confirmed via an alkaline comet assay, where tails were elongated significantly in MDA-MB-453 and MDA-MB-231 cells after elimusertib treatment (Fig. 3E).

Fig. 3.

The aberrant progression of the S-phase induces a DNA damage accumulation. (A) MDA-MB-453, MDA-MB-231 and T-47D cells were treated with elimusertib (0.05 μmol/L) for 2 days, and the nuclei were analyzed by confocal microscopy. Scale bars=10 μm. (B) MDA-MB-453, MDA-MB-231, and T-47D cells were treated with elimusertib (0.05 μmol/L) for 2 days, and protein expression was analyzed by Western blot. ATRi, ataxia telangiectasia and Rad3-related inhibitor. (C) The nuclei of MDA-MB-453, MDA-MB-231, and T-47D cells were stained with Hoechst, and analyzed by confocal microscopy. Scale bars=10 μm. (D) A total of 100 cells were counted for each cell line. Scale bars=10 μm. (E) An alkaline comet assay was conducted on MDA-MB-453, MDA-MB-231, and T-47D and treated with elimusertib (0.05 μmol/L) for 2 days. Scale bars=100 μm. ^*p < 0.05, ^***p < 0.001.

4. Elimusertib inhibits tumor growth in vivo xenograft models of human breast cancer cell line

We next sought to establish the in vivo efficacy of elimusertib in a cell line-derived xenograft model of MDA-MB-231 cells. Treatment with elimusertib, at a dose 30 mg/kg twice-daily, 3 days on and 4 days off schedule for 4 weeks, slowed tumor growth (Fig. 4A, red line). Treatment with higher dose (50 mg/kg) of elimusertib resulted in decrease in tumor size (Fig. 4A, green line). No significant changes in body weight were observed in either treatment group throughout the treatment period (Fig. 4B). After 60 days of treatment, all but one mouse in the control group exhibited tumor volumes exceeding 2,000 mm³ and were therefore euthanized, whereas only one mouse in the 30 mg/kg elimusertib group and none in the 50 mg/kg treatment group were euthanized due to excessive tumor growth (Fig. 4C and D).

Fig. 4.

Elimusertib induces tumor growth inhibition in vivo human breast cancer cell line xenograft model. (A) The experiment was conducted with elimusertib administered twice a day at dose 50 mg/kg (n=5), 30 mg/kg (n=5), and vehicle alone (n=5). Following a schedule of 3 days on and 4 days off for 4 weeks. (B) The body weight of each mouse was measured three times a week. Error bars, ±standard error. (C) Tumor volume of each mouse was measured three times a week during 8 weeks. (D) The endpoint of survival was defined as the time until tumor size reached 2,000 mm³. This test was conducted during 8 weeks. (E) Tumor tissues were extracted from the mice after 8 weeks and analyzed by immunohistochemistry. Pathological analysis was performed using hematoxylin and eosin (H&E) slides (×400). Ki-67 staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay showed proliferation capacity and apoptosis. The number of Ki-67 and TUNEL positive cells per 100 cells was counted. Scale bars=100 μm (n=3). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

To investigate the in vivo effects of elimusertib on the molecular level, we performed Ki-67 staining via immunohistochemistry and observed significant dose-dependent decreases in the number of Ki-67–positive cells in tumors harvested from elimusertib-treated mice relative to controls (Fig. 4E). Conversely, TUNEL staining showed dose-dependent increases in the frequency of apoptotic cells in tumors harvested from elimusertib-treated mice. Collectively, these results show that ATR inhibition via elimusertib effectively suppresses tumor growth in vivo by both inhibiting cell proliferation and the induction of apoptosis, consistent with our in vitro results.

5. Elimusertib decreases rate of tumor growth in Patient-derived xenograft models

We also investigated the in vivo efficacy of elimusertib in two patient-derived xenograft (PDX) cancer models with intrinsically high rates of replication. X151 was established from a metastatic breast cancer patient with BRCA2 mutation (p.Lys2777*) and IGX-216 was derived from a patient with advanced gastric cancer harboring ATM mutation (p. F2813fs). Relative to vehicle control, administration of 40 mg/kg elimusertib significantly delayed tumor growth in both models (Fig. 5), further supporting the anti-tumor activities of elimusertib.

Fig. 5.

Elimusertib inhibits tumor growth in patient-derived xenograft (PDX) model. (A) In the breast cancer PDX-X151 model, elimusertib was administered twice daily at a dose of 40 mg/kg (n=6) and vehicle alone (n=6). Following a schedule of 3 days on and 4 days off for 4 weeks. Tumor volumes of the mice in each group were measured three times a week and are presented as a graph with standard error (SE) bars. (B) The body weight of each mouse was measured three times a week. There were no significant differences in weight changes between the groups. (C) In the gastric cancer PDX-IGX216 models, elimusertib was administered twice daily at a dose of 40 mg/kg (n=8) and vehicle alone (n=6), following a schedule of 3 days on and 4 days off for 4 weeks. Tumor volumes of the mice in each group were measured three times a week and are presented as a graph with SE bars. (D) The body weight of each mouse was measured three times a week. There were no significant differences in weight changes between the groups. ^*p < 0.05, ^***p < 0.001.

Discussion

ATR is a potential target for cancer therapy, and several ATRis are being developed with promising preliminary early results. We report the anti-tumor effect of elimusertib which is a selective ATRi. Especially, ATR inhibition led to an increase in ssDNA aberrantly and DNA damage accumulation (Fig. 3B). During replication stress, absence of checkpoint signaling causes aberrant replication and induces an increase of ssDNA. Gradually accumulated ssDNA converts into double strand DNA breaks which is known as replication catastrophe [32]. In the absence of ATR, stalled replication forks may collapse, leading to replication stress-associated DNA damage and cell death. This suggests that elimusertib could exert anti-tumor effects through replication catastrophe.

ATR has been reported to play diverse roles in the replication process, including a checkpoint role causing cell cycle arrest and the control of origin firing involved in the replication [33]. In previous studies, it was found that the increase of the population of replicating cells due to indiscriminate origin firings and S-phase or G2/M arrest was induced by ATR inhibition [34]. In line with these previous studies, elimusertib increased the number of cells with DNA contents corresponding to S-phase in the present study and it impaired replication in S-phase. In previous studies, ATR-deleted cells were compared with ATR wild-type cells to confirm that ATR influences S-phase cell cycle progression [35] and that inhibition of ATR induces the disruption of cell proliferation by blocking S-phase progression. Elimusertib induced a delay in the progression of the cell cycle in the S-phase and increased cell population in S-phase with the aberrant cell cycle progression.

Elimusertib (BAY1895344) is an ATR kinase inhibitor that plays a crucial role in the DNA damage response, by inhibiting ATR, it enhances DNA damage in cancer cells, which can lead to their death. On the other hand, topotecan is a chemotherapy drug that works by inhibiting topoisomerase I, leading to DNA strand breaks, thus also inducing cell death, particularly in rapidly dividing cancer cells. The combination of these two drugs is based on the hypothesis that targeting DNA repair pathways (via ATR inhibition) alongside direct DNA damage (via topotecan) could have a synergistic effect, making it more difficult for cancer cells to repair the induced DNA damage. This combination could potentially improve the therapeutic efficacy compared to using each drug alone. In the phase I study, the researchers aimed to assess the safety, tolerability, and initial efficacy of this combination. The study found that the combination of elimusertib and topotecan was generally well tolerated, with no major unexpected toxicities, and showed promising signs of efficacy, suggesting that the dual approach of inhibiting DNA repair (ATR inhibition) and inducing DNA damage (via topotecan) may offer a novel treatment strategy for cancers [8].

ATR regulates cell cycle and origin firing in DNA replication via checkpoint kinase 1 (CHK1) modulation, associated with either S-phase or G2/M arrest with increased replication by unregulated origin firings. In addition, many studies have shown that ATRis also regulate DNA damage response through CHK1. Previous studies have reported that CHK1 is affected also by ATM, which has a complementary role to ATR. Both ATR and mammalian target of rapamycin (mTOR) are phosphoinositide 3-kinase–related protein kinases, but elimusertib has superior selectivity for ATR compared with the PI3K/AKT/mTOR pathway [36].

To study the effects of elimusertib in cancers with intrinsically high replication rates such as breast and gastric cancer, PDX models were used in the present study. PDX models from a gastric cancer patient with ATM mutation (p.F2813fs, kinase domain) and a breast cancer patient with BRCA2 mutation (p.Lys2777*, DNA binding domain) were used. The in vivo test with PDX models showed that elimusertib induced regression of tumor growth in ATM-mutant gastric cancer PDX and decreased tumor growth rate in BRCA2-mutant breast cancer PDX. Moreover, previous studies have shown that AZD6738, another ATRi, increased sensitivity in cells with deficiencies in ATM [31]. These collectively indicate the potential of ATRis in tumors with alterations in DNA damage response or replication stress pathways. Biomarkers predicting clinical response to DNA damage response inhibitors, including ATRis are still scarce. One of the most widely used molecular response predictor used for ATRis is ATM deficiency. Our findings stand in line with current clinical trial data showing that a large fraction of patients with ATM deficiency responds to ATRis. Additionally, this suggests that other factors contribute to ATRi sensitivity.

One of the hurdles in developing ATRis clinically is its toxicities such as hematological toxicities including anemia. Increased vulnerability of erythroid precursors to reactive oxygen species has been related to ATRis and ferroptosis [37]. Various approaches to mitigate this problem, such as optimizing the dosing schedule, have been tried. In addition, optimal patient selection and careful combination strategies would be important. The precise understanding of the characteristics of ATRis in development is fundamental for the patient selection and combination strategies.

ATRis, such as elimusertib, are being developed both as monotherapies and in combination with various agents, including chemotherapy or targeted agents such as PARP inhibitors. These combinations are based upon the findings that ATR pathway protects the genome against DNA damage and replication stress and that ATRis are a potential therapy for cancer that can increase the effects of genotoxic therapies. We tested elimusertib only as a monotherapy in the present investigation, which is a caveat of the study. Combinations strategies using elimusertib could be investigated in further preclinical and clinical studies. Moreover, more detailed analyses in the mechanisms of the replication stress and the repair could give insights in the monotherapy and combination strategies.

Additionally, elimusertib has been shown to be active in vivo against ATRX mutated uterine leiomyosarcoma. These results, as well as the results of the present study, collectively suggest the promise of ATR inhibition as a strategy for cancer treatment and the potential of clinical development of elimusertib.

Electronic Supplementary Material

Supplementary materials are available at Cancer Research and Treatment website (https://www.e-crt.org).

crt-2024-1105_S1_Table.pdf

crt-2024-1105_S2_Fig.pdf

Notes

Ethical Statement

This study was not carried out in humans and informed consent was not applicable. Animal experiments were approved by the Institutional Animal Care and Use Committee in Seoul National University Hospital (SNUH-IACUC No. 19-0176) and animals were maintained in the facility accredited AAALAC International (#001169) in accordance with Guide for the Care and Use of Laboratory Animals 8th edition, NRC (2010).

Author Contributions

Conceived and designed the analysis: Kim M, Min A, Lee KH, Im SA.

Collected the data: Kim M.

Contributed data or analysis tools: Kim M, Min A, Kim S (Sohyeon Kim), Kim S (Seongyeong Kim), Kim YJ, Ham SJ, Lee MS, Kim JY.

Performed the analysis: Kim M.

Wrote the paper: Kim M, Min A, Lee KH, Lee EY, Lee DW, Im SA.

Conflicts of Interest

Seock-Ah Im reported advisory role for AstraZeneca, Eisai, GSK, Hanmi, Lilly, MSD, Novartis, Roche, and Pfizer outside of this work. Seock-Ah Im received research funding from AstraZeneca, Boryung Pharm, Daewoong Pharm, Roche, Pfizer, and Eisai outside of this work. Kyung-Hun Lee reported advisory role for Surface oncology outside of this work. Kyung-Hun Lee received research funding from Merck outside of this work. Kyung-Hun Lee reported advisory role for AstraZeneca, Eisai, Lilly, Novartis, Roche, and Pfizer outside of this work. Elimusertib was provided by Bayer AG (Leverkusen, Germany). None of the other authors have any potential conflicts of interest.

Funding

This study was partly supported by National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT; No. NRF2020 R1A2C301088311) to Seock-Ah Im at Seoul National University.

References

1. Hanahan D. Hallmarks of cancer: new dimensions. Cancer Discov 2022;12:31–46.

2. Liu S, Opiyo SO, Manthey K, Glanzer JG, Ashley AK, Amerin C, et al. Distinct roles for DNA-PK, ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress. Nucleic Acids Res 2012;40:10780–94.

3. Saldivar JC, Cortez D, Cimprich KA. The essential kinase ATR: ensuring faithful duplication of a challenging genome. Nat Rev Mol Cell Biol 2017;18:622–36.

4. Flynn RL, Zou L. ATR: a master conductor of cellular responses to DNA replication stress. Trends Biochem Sci 2011;36:133–40.

5. Kim HJ, Min A, Im SA, Jang H, Lee KH, Lau A, et al. Anti-tumor activity of the ATR inhibitor AZD6738 in HER2 positive breast cancer cells. Int J Cancer 2017;140:109–19.

6. Sartori G, Tarantelli C, Spriano F, Gaudio E, Cascione L, Mascia M, et al. The ATR inhibitor elimusertib exhibits anti-lymphoma activity and synergizes with the PI3K inhibitor copanlisib. Br J Haematol 2024;204:191–205.

7. Haciefendi A, Guney Eskiler G. The suppression of ATR/Chk1 pathway by Elimusertib ATR inhibitor in triple negative breast cancer cells. Am J Transl Res 2023;15:4902–11.

8. Stockton S, Shyr C, Cecchini M, Aljumaily R, Halfdanarson TR, Sonbol MB, et al. A phase I study of ATR inhibitor BAY1895344 (elimusertib) plus topotecan (ETCTN 10402): results of dose escalation. J Clin Oncol 2024;42(16 Suppl):3076.

9. Pusch FF, Dorado Garcia H, Xu R, Gurgen D, Bei Y, Bruckner L, et al. Elimusertib has antitumor activity in preclinical patient-derived pediatric solid tumor models. Mol Cancer Ther 2024;23:507–19.

10. Barnieh FM, Loadman PM, Falconer RA. Progress towards a clinically-successful ATR inhibitor for cancer therapy. Curr Res Pharmacol Drug Discov 2021;2:100017.

11. Lin SF, Hsueh C, Chen WY, Chou TC, Wong RJ. Targeting ataxia telangiectasia-mutated and Rad3-related for anaplastic thyroid cancer. Cancers (Basel) 2025;17:359.

12. Middleton MR, Dean E, Evans TRJ, Shapiro GI, Pollard J, Hendriks BS, et al. Phase 1 study of the ATR inhibitor berzosertib (formerly M6620, VX-970) combined with gemcitabine +/- cisplatin in patients with advanced solid tumours. Br J Cancer 2021;125:510–9.

13. Telli ML, Tolaney SM, Shapiro GI, Middleton M, Lord SR, Arkenau HT, et al. Phase 1b study of berzosertib and cisplatin in patients with advanced triple-negative breast cancer. NPJ Breast Cancer 2022;8:45.

14. Baschnagel AM, Elnaggar JH, VanBeek HJ, Kromke AC, Skiba JH, Kaushik S, et al. ATR inhibitor M6620 (VX-970) enhances the effect of radiation in non-small cell lung cancer brain metastasis patient-derived xenografts. Mol Cancer Ther 2021;20:2129–39.

15. Yap TA, O’Carrigan B, Penney MS, Lim JS, Brown JS, de Miguel Luken MJ, et al. Phase I trial of first-in-class ATR inhibitor M6620 (VX-970) as monotherapy or in combination with carboplatin in patients with advanced solid tumors. J Clin Oncol 2020;38:3195–204.

16. Reichert ZR, Devitt ME, Alumkal JJ, Smith DC, Caram MV, Palmbos P, et al. Targeting resistant prostate cancer, with or without DNA repair defects, using the combination of ceralasertib (ATR inhibitor) and olaparib (the TRAP trial). J Clin Oncol 2022;40(6 Suppl):88.

17. George E, Kim H, Butler LR, Morgan MA, Gilad O, Brown EJ, et al. Improving the efficacy of PARP inhibition with a novel ATR inhibitor, ATRN119, in ovarian high-grade serous cancers. Gynecol Oncol 2018;149(Suppl 1):45.

18. Mahdi H, Hafez N, Doroshow D, Sohal D, Keedy V, Do KT, et al. Ceralasertib-mediated ATR inhibition combined with olaparib in advanced cancers harboring DNA damage response and repair alterations (olaparib combinations). JCO Precis Oncol 2021;5:PO.20.00439.

19. Shah PD, Wethington SL, Pagan C, Latif N, Tanyi J, Martin LP, et al. Combination ATR and PARP inhibitor (CAPRI): a phase 2 study of ceralasertib plus olaparib in patients with recurrent, platinum-resistant epithelial ovarian cancer. Gynecol Oncol 2021;163:246–53.

20. Yap TA, Tolcher AW, Plummer R, Mukker J, Enderlin M, Hicking C, et al. A phase I study of ATR inhibitor M1774 in patients with solid tumours (DDRiver Solid Tumours 301): part A1 results. Ann Oncol 2022;33(Suppl 7):S747–8.

21. Yap TA, Tan DS, Stathis A, Shapiro GI, Iwasa S, Joerger M, et al. Phase Ib expansion trial of the safety and efficacy of the oral ataxia telangiectasia and Rad3-related (ATR) inhibitor elimusertib in advanced solid tumors with DNA damage response (DDR) defects. Cancer Res 2022;82(12 Suppl):CT006.

22. Roulston A, Zimmermann M, Papp R, Skeldon A, Pellerin C, Dumas-Berube E, et al. RP-3500: a novel, potent, and selective ATR inhibitor that is effective in preclinical models as a monotherapy and in combination with PARP inhibitors. Mol Cancer Ther 2022;21:245–56.

23. Jo U, Senatorov IS, Zimmermann A, Saha LK, Murai Y, Kim SH, et al. Novel and highly potent ATR inhibitor M4344 kills cancer cells with replication stress, and enhances the chemotherapeutic activity of widely used DNA damaging agents. Mol Cancer Ther 2021;20:1431–41.

24. Burris HA, Berlin J, Arkenau T, Cote GM, Lolkema MP, Ferrer-Playan J, et al. A phase I study of ATR inhibitor gartisertib (M4344) as a single agent and in combination with carboplatin in patients with advanced solid tumours. Br J Cancer 2024;130:1131–40.

25. Wethington SL, Shah PD, Martin L, Tanyi JL, Latif N, Morgan M, et al. Combination ATR (ceralasertib) and PARP (olaparib) inhibitor (CAPRI) trial in acquired PARP inhibitor-resistant homologous recombination-deficient ovarian cancer. Clin Cancer Res 2023;29:2800–7.

26. Wilson Z, Odedra R, Wallez Y, Wijnhoven PW, Hughes AM, Gerrard J, et al. ATR inhibitor AZD6738 (ceralasertib) exerts antitumor activity as a monotherapy and in combination with chemotherapy and the PARP inhibitor olaparib. Cancer Res 2022;82:1140–52.

27. Yano K, Shiotani B. Emerging strategies for cancer therapy by ATR inhibitors. Cancer Sci 2023;114:2709–21.

28. Wengner AM, Siemeister G, Lucking U, Lefranc J, Wortmann L, Lienau P, et al. The novel ATR inhibitor BAY 1895344 is efficacious as monotherapy and combined with DNA damage-inducing or repair-compromising therapies in preclinical cancer models. Mol Cancer Ther 2020;19:26–38.

29. Yap TA, Tan DS, Terbuch A, Caldwell R, Guo C, Goh BC, et al. First-in-human trial of the oral ataxia telangiectasia and RAD3-related (ATR) inhibitor BAY 1895344 in patients with advanced solid tumors. Cancer Discov 2021;11:80–91.

30. Ngoi NY, Pilie PG, McGrail DJ, Zimmermann M, Schlacher K, Yap TA. Targeting ATR in patients with cancer. Nat Rev Clin Oncol 2024;21:278–93.

31. 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;16:566–77.

32. Toledo LI, Altmeyer M, Rask MB, Lukas C, Larsen DH, Povlsen LK, et al. ATR prohibits replication catastrophe by preventing global exhaustion of RPA. Cell 2013;155:1088–103.

33. Toledo LI, Murga M, Fernandez-Capetillo O. Targeting ATR and Chk1 kinases for cancer treatment: a new model for new (and old) drugs. Mol Oncol 2011;5:368–73.

34. Vassin VM, Anantha RW, Sokolova E, Kanner S, Borowiec JA. Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress. J Cell Sci 2009;122:4070–80.

35. Hurley PJ, Wilsker D, Bunz F. Human cancer cells require ATR for cell cycle progression following exposure to ionizing radiation. Oncogene 2007;26:2535–42.

36. Harold J, Bellone S, Manavella DD, Mutlu L, McNamara B, Hartwich TMP, et al. Elimusertib (BAY1895344), a novel ATR inhibitor, demonstrates in vivo activity in ATRX mutated models of uterine leiomyosarcoma. Gynecol Oncol 2023;168:157–65.

37. Levy M, Ferraro G, Li L, Han Y, Varricchio L, Fournier S, et al. ATR inhibitor camonsertib (RP-3500) suppresses early stage erythroblasts by mediating ferroptosis. Blood 2022;140(Suppl 1):8194–5.

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