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. Author manuscript; available in PMC: 2025 Dec 1.
Published in final edited form as: Gynecol Oncol. 2024 Oct 19;191:182–193. doi: 10.1016/j.ygyno.2024.10.009

ATR inhibition increases reliance on PARP-mediated DNA repair revealing an improved therapeutic strategy for cervical cancer

Sugantha Priya Elayapillai a,b, Samrita Dogra a,b, James Lausen a, Madison Parker a, Amy Kennedy a, Doris M Benbrook a,b, Katherine M Moxley c,**, Bethany N Hannafon a,b,*
PMCID: PMC11975412  NIHMSID: NIHMS2054808  PMID: 39427557

Abstract

Objective.

Cervical cancer results from persistent infection with high-risk human papillomavirus (HR-HPV) and the expression of E6 and E7 oncoproteins. E6 and E7 compromise the activity of p53 and Rb, the G1-S cell cycle checkpoint, and ATM-mediated DNA damage repair (DDR), which in turn increases reliance on ATR- and PARP-mediated DDR at the G2 cell cycle checkpoint. This study aimed to determine the effects of an ATR inhibitor (ATRi, AZD6738) and a PARP-inhibitor (PARPi, AZD2281) on HR-HPV+ cervical cancer cell lines.

Methods.

The effects of ATRi and PARPi, alone and in combination, on metabolic viability, cell cycle arrest, apoptosis, and DDR pathways in cervical cancer cell lines were evaluated in vitro, and the in vivo tumor response was evaluated using a xenograft model.

Results.

Cervical cancer cells were sensitive to ATRi and PARPi monotherapy. The combination therapy was only synergistic in reducing metabolic viability when exposed to ATRi first, followed by PARPi, owing to ATRi-mediated upregulation of PARP expression. Combination of ATRi and PARPi induced G2 cell cycle arrest and apoptosis. PARPi induced DNA damage and γH2AX phosphorylation, which was further increased by ATRi treatment. However, PARPi-induced Rad51 foci formation was reduced by ATRi treatment, suggesting the inhibition of homologous recombination repair. ATRi significantly reduced cervical cancer xenograft tumor growth and was not affected by simultaneous PARPi treatment at the doses studied.

Conclusions.

Our findings show that ATRi increased reliance on PARP for metabolic viability, the combination of ATRi and PARPi induced synthetic lethality in cervical cancer in vitro, and reduced tumor burden in vivo.

Keywords: Cervical cancer, ATR, PARP, HPV-related cancer, DNA damage repair

Graphical Abstract

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1. Introduction

Cervical cancer is the fourth most common cancer among women, with over 600,000 cases diagnosed in 2020 (GLOBOCAN) and represents the most lethal gynecological cancer worldwide [1]. Although the incidence of cervical cancer in the United States remains low (13,820 cases projected for 2024), an increase in the rate of cervical cancer is now being reported in women aged 30–40 years. Moreover, in the setting of locally advanced disease, up to 50 % of patients ultimately experience recurrence and progression following primary therapy [2,3]. Despite the development of effective vaccination, improved screening tests, and the evolution of cytotoxic and targeted systemic therapy for recurrent disease, the mortality rate of metastatic cervical carcinoma remains unacceptably high [4,5].

The standard treatment for advanced, recurrent, and metastatic cervical cancer consists of palliative chemotherapy with platinum, paclitaxel, and bevacizumab, with or without immune checkpoint inhibitors. These treatments aim to manage the disease and prolong life; however, their effectiveness is limited, with a median survival time of 2.5 years. Despite the use of biomarker-driven therapies, angiogenesis, and immune checkpoint inhibitors, the efficacy of treatments for metastatic diseases remains limited [6,7]. In addition, there are limited options for second-line therapy after disease progression, with a response rate of 15 % or less and a median time to progression of less than three months [8–12]. However, the FDA has granted expedited approval for tisotumab vedotin, which has a 24 % response rate and a median response of 5–8 months, and is currently the most active agent for second-line treatment of metastatic, platinum-taxane resistant/recurrent cervical cancer [13]. Despite these advances, cervical cancer lags behind other cancers in terms of FDA-approved targeted therapies.

Cervical cancer commonly occurs in patients with persistent high-risk human papillomavirus (HR-HPV) infection. HR-HPV transforms cells by monopolizing the cell cycle replication machinery through the expression of the “early” viral proteins 6 and 7 (E6, E7). E6 and E7 interfere with homologous recombination (HR) DNA repair by allowing it to occur before progression through the cell cycle. E7 binds and inhibits the function of retinoblastoma protein (pRb) and redirects the DNA repair enzyme RAD51 from double-stranded DNA breaks (DSBs), further impairing HR and other standard DNA damage repair (DDR) pathways [14]. E6 binds wild-type p53, causing its degradation and a “p53 deficient” environment. This interference abolishes the G1 checkpoint, increases replication stress, and enhances susceptibility to DDR inhibitors [15]. Reproducible dysregulation of DDR pathways induced by HPV interactions and downregulation of critical tumor suppressor gene expression are potential therapeutic strategies for cervical cancer [16].

Poly (ADP-ribose) polymerase (PARP) is a crucial enzyme for the repair of single-stranded DNA breaks (SSBs) through nucleotide base excision repair (NER). Tumor cells typically exhibit higher levels of PARP than normal cells, which is linked to drug resistance and the ability to withstand genotoxic stressors. PARP inhibitors (PARPi) trap PARP at DNA repair sites, resulting in unrepaired single-stranded DNA breaks (SSBs) and double-stranded DNA breaks (DSBs). Breast cancer susceptibility genes 1 and 2 (BRCA1 and 2) mutant high-grade serous ovarian cancers (HGSOCs) with homologous recombination deficiency (HRD) and increase PARPi sensitivity by causing more unrepaired double-stranded DNA breaks [17,18]. This approach led to a 31 % overall response rate and FDA approval of PARPi for recurrent BRCA-mutant HGSOC [19]. Preclinical studies have shown that PARPi induces apoptosis, re-sensitizes cervical cancer cells to cisplatin, and is more effective in the presence of deleterious mutations in the less error-prone HR repair pathways [20]. However, clinical evaluation of PARPi in post-progression metastatic cervical cancer shows limited clinical activity, especially when administered with platinum chemotherapy after disease progression [12,21].

An alternative strategy for targeting the DNA damage response (DDR) in cervical cancer therapy is to disrupt cell cycle checkpoints. Human papillomavirus (HPV) amplifies ataxia telangiectasia and Rad3-related (ATR) protein kinases, which are activated upon single-strand breaks (SSBs). ATR plays a role in both DDR and G2 cell cycle checkpoint regulation, primarily by phosphorylating checkpoint kinase 1 (CHK1) [22–24]. Consequently, HPV infection renders tumors reliant on homologous recombination (HR) for DDR [25], primarily at the G2 checkpoint, which is maintained in cervical cancer [13]. Furthermore, tumors with dysfunctional G1 checkpoints, such as cervical cancer, primarily rely on kinases such as ATR, CHK1, and WEE1 for DDR before cell division [26]. Therefore, inhibition of G2 checkpoint pathways, such as those controlled by ATR, WEE1, and CHK1, is a rational approach for tumors with impaired p53 and dysfunctional G1-M checkpoints [8–11].

Synergistic cell death has been observed when DDR-disrupting agents are combined with DNA-damaging agents, such as platinum-based chemotherapeutics, ionizing radiation, and PARPi [26–28]. Indeed, dual inhibition of PARP and ATR, produces “synthetic lethality” in drug-resistant HGSOC cell lines resulting in a significant decrease in cell survival and an increase in apoptotic cell death [29]. However, whether dual inhibition of PARP and ATR is equally effective in HPV cervical cancer has not yet been demonstrated.

We hypothesized that the dual inhibition of ATR and PARP would increase replication stress and impair the ATR/CHK1 pathway, resulting in increased cervical cancer cell death. This was evaluated using in vitro and in vivo methods, using cervical cancer cell lines and xenograft models. Our results showed that pretreatment with ATRi followed by PARPi treatment resulted in synergistic suppression of cervical cancer cell line growth, indicating activation of PARP and inhibition of the ATR/CHK1 pathway.

2. Materials and methods

2.1. Cell lines

The cervical cancer cell lines SiHa (RRID: CVCL_0032) and CaSki were purchased from the American Type Culture Collection, and authenticity was confirmed by short tandem repeats using the Functional Genomics Core (Stephenson Cancer Center, Oklahoma City, OK, USA). Both cell lines were cultured in RPMI 1640 medium containing 10 % FBS and 1× antibiotic/antimycotic solution at 37 °C with 5 % CO2. All experiments were performed using cell lines free of mycoplasma contamination and tested periodically using a mycoplasma PCR detection kit (abm, G238).

2.2. Cytotoxicity assay

SiHa and CaSki cells (5 × 103 cells/well) were seeded in 96-well plates and treated with various doses of ATRi (Ceralasertib/AZD6738, AstraZeneca) and PARPi (Olaparib/AZD2281, AstraZeneca). Cytotoxicity was measured using CellTiter 96 AQueous One Solution Reagent (Promega, G3580). Average ODs (490 nm) of triplicate treatments normalized to solvent-only controls were plotted against the dose used, and nonlinear regression was performed using GraphPad Prism to determine the half-maximal inhibitory concentrations (IC50). Each experiment was performed in triplicate and repeated three times.

2.3. Combination index

The interaction between ATR and the PARP inhibitor (olaparib) was determined using combination indices (CI) based on isobologram analysis. CI was performed for both simultaneous drug treatment and sequential treatment by pre-treatment with ATRi (pre-ATRi) followed by PARPi or pre-treatment with PARPi (pre-PARPi) followed by ATRi. The drugs were combined at a 1:1 and 2:1 ratio (SiHa and CaSki, respectively) of their IC50 concentrations, and a series of two-fold dilutions were evaluated by MTS assay using the CellTiter 96 AQueous One Solution Reagent. Single drug treatments at doses close to IC50 values were evaluated in parallel. All treatments were performed in triplicate, and averages were used to draw isobolograms and calculate combination indices (CI) and dose-reduction indices (DRIs) using CompuSyn Software according to the Chou and Talalay method [30]. The experiments were performed in triplicate and repeated thrice for each cell line to confirm the results.

2.4. Immunoblot

Whole cell lysates from both SiHa and CaSki cell lines were extracted using RIPA lysis and extraction buffer (Thermo Fisher, 89901) combined with protease and phosphatase inhibitors (Thermo Fisher, 78442). Protein concentration was determined using the BCA method (Thermo Fisher, 23225). Equal volumes and concentrations (30μg) of protein were electrophoresed on 10 % SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with EveryBlot (Bio-Rad, 12010020) and incubated with primary antibodies at 4 °C overnight. The membrane was incubated with the corresponding secondary antibodies for 40–60 min at room temperature, and the bands were visualized using the Clarity Max Western ECL substrate (Bio-Rad, 1705062). Band intensity was quantified using ImageLab, Software (Bio-Rad). The band intensity of the protein of interest was normalized to that of the loading control. Subsequently, the fold change was calculated relative to the controls. The antibody sources and dilutions are listed in Supplemental Table 1.

2.5. Cell cycle analysis

SiHa and CaSki cells were seeded into 6 well plates at a density of 4 × 105 cells/well, treated with both drugs alone and in combination, and pretreated with ATRi for 24 h and followed by PARPi for 72 h at their IC50 concentrations. After 72 h of incubation, cells were labeled with 10μM EDU (5-ethynyl-2′-deoxyuridine) for 1 h before harvest. The cells were fixed and permeabilized, and EDU incorporation was determined by flow cytometry (FACS Calibur, Automated Four-Color Benchtop Flow Cytometer). The data were analyzed using FlowJo (RRID:SCR_008520) and ModFit software.

2.6. Apoptosis assay

The apoptotic cells in response to ATRi and PARPi treatment alone or in combination were determined by using Annexin V assay kit (Biotium # 30061) following manufacturer’s protocol. Breifly, SiHa and CaSki cells were pre-treated with ATRi followed by PARPi alone or in combination for 72 h. cells were harvested and washed with PBS and then stained with 5 μl of Annexin V- FITC and 2 μl of propidium iodide (PI) working solution to quantify the apoptotic (green) and necrotic (red) cells within the same cell population by using Stratedigm 3-laser flow cytometry. Data was then analyzed using ModFit Software (and plotted using GraphPad Prism).

2.7. Immunofluorescence (IF) analysis

Cervical cancer cells were plated in 8 well chamber slides at a density of 30,000 cells/well and then pretreated with ATRi for 24 h followed by PARPi and incubated for 72 h. After drug treatment, the cells were fixed with 100 % methanol for 15 min at 4 °C and washed three times with PBS. The cells were then blocked with a specific blocking buffer containing 1× PBS, 5 % normal serum from the same species as the secondary antibody, and 0.3 % Triton X-100. After blocking, the cells were washed with 1× PBS, probed with RAD51, Geminin, and pH2AX, and incubated at 4 °C overnight. The cells were then incubated with fluorochrome-conjugated secondary antibodies (Alexa Fluor 595 and 488) diluted in an antibody dilution buffer for 1 h. The cells were counterstained with DAPI, mounted using ProLong Diamond Antifade Mountant (Thermo Scientific, P36962), and imaged using a confocal microscope (Olympus Fluoview 1000 laser scanning confocal microscope) at 100× oil immersion. The number of RAD51 foci in geminin-positive cells and pH2AX foci in each cell were quantified using ImageJ software (Version 1.52p; Java 1.8.0_172) (RRID:SCR_003070). The antibody sources and dilutions are listed in Supplemental Table 1.

2.8. Comet assay

A comet assay (single cell gel electrophoresis) was used to determine DNA strand breaks by using both alkaline and neutral conditions, using Enzo comet SCGE assay kit # ADI-900–166 following the manufacturer’s instructions. Briefly, SiHa and CaSki cells were pre-treated with ATRi followed by PARPi for 72 h, 50 μl of cells (1 × 105 cells/ml) was added to 500 μl of molten LM Agarose at 37 °C. After mixing the samples, 75 μl of agarose with cells was pipetted onto an area of the comet slide. The slide was incubated at 4 °C for 10 min. After a clear ring appears at the edge of the comet slide, transferred into prechilled lysis solution for 35 min at 4 °C. For alkaline electrophoresis, a denaturation was performed in alkaline solution (0.3 M NaOH, 1 mM EDTA, pH > 13) at room temperature for 50 min, in the dark. Whereas for the neutral electrophoresis, samples were transferred into the neutral buffer (1×-TBE buffer). The slides were then transferred to horizontal chamber and electrophoresed with appropriate buffer either alkaline or neutral solution at 1v/cm for 15 min. The slide was fixed in ice-cold 70 % ethanol for 5 min and air dried the samples. The samples were stained with CYGREEN nucleic acid dye diluted 1:10,000 in deionized water for 30 min and comets were observed by using EVOS M7000 imaging system (ThermoFisher) with FITC filter (excitation/emission 488/515 nm). The comet tail moment was measured in 35 to 90 comets per condition and was calculated using Open Comet v1.3 plug-in with FIJI software and plotted using GraphPad PRISM.

2.9. Xenograft studies

Female athymic nude mice (Foxn1nu) (RRID: MGI:5652489) were purchased from Envigo. All mice were housed in accordance with the Institutional Animal Care and Use Committee (IACUC) at OUHSC (temperatures of 68–79 °C with 40–60 % humidity, 12-h light/12-h dark cycle) and acclimatized for 2 weeks. SiHa cells (1 × 107 cells/animal) were implanted subcutaneously with Matrigel and the tumor size was monitored without treatment until it reached 100 mm3. The mice were randomized into four treatment groups (six mice/group) and treated as follows. Group 1, vehicle control (45 % of 2-hydroxypropyl-B- cyclodextrin in water, daily); Group 2, PARPi (Olaparib/AZD2281, AstraZeneca, 50 mg/kg, daily); Group 3, ATRi (Ceralasertib/AZD6738, AstraZeneca, 25 mg/kg day 1,2,3 of each week); Group 4, ATRi + PARPi (AZD6738, 25 mg/kg, Day 1–3 weekly and AZD2281 50 mg/kg, daily). The PARPi and ATRi doses were calculated according to FDA guidelines for cross-species dose conversion. Representative “human equivalent” doses for the in vivo experiments were calculated at the animal equivalent doses (AED) of PARPi 300 mg B.I.D. and ATRi 160 mg p. o. B.I.D. in humans. The drugs were administered through oral gavage for up to 5 weeks. The tumor size was measured three times per week when the tumor reached 100 mm3. After approximately 5 weeks, all animals were euthanized according to the IACUC guidelines. Tumors and other vital organs were collected for further analysis. After measuring the tumors, a portion of the tumor was snap-frozen for protein analysis, and another portion was formalin-fixed for histology and immunohistochemistry (IHC). H&E and IHC staining for pCHK1 and γH2AX was performed on tumor tissues isolated from mice. Quantification was performed using HALO software (Indica Lab, V3.2.1851). The antibody sources and dilutions are listed in Supplemental Table 1.

2.10. Statistical analysis

GraphPad Prism version 10.2 (RRID:SCR_002798) was used for all the statistical analyses. Comparisons between groups were analyzed using ordinary one-way ANOVA followed by Tukeys’/Dunnetťs post hoc test. Statistical significance was set at P < 0.05.

3. Results

3.1. Cervical cancer cells are sensitive to ATRi and PARPi monotherapy

We first evaluated the effectiveness of ATRi and PARPi as single-agent treatments for the HPV-positive cervical cancer cell lines, SiHa and CaSki, using a metabolic viability assay. Both cell lines were sensitive to ATR and PARP inhibitors at micromolar concentrations (Fig. 1A, B), with similar potencies in the 40 μM concentration range for SiHa cells (Fig. 1A). However, CaSki cells showed greater sensitivity to ATRi, with IC50 values of 10 μM and 25 μM for ATRi and PARPi, respectively (Fig. 1B). The IC50 values were used in subsequent experiments.

Fig. 1.

Fig. 1.

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Cervical cancer cells are sensitive to ATR and PARP inhibitors. ATR and PARP inhibitor dose-response curves of SiHa (A) and CaSki (B) cervical cancer cells, C. Heat Map of the combination index values for both ATR and PARP inhibitors calculated using the CompuSyn software. Purple indicates synergism, pink indicates almost additive, orange indicates moderate antagonism, and yellow indicates strong antagonism. Results were obtained from ≥3 independent experiments (mean ± SEM) and analyzed using nonlinear regression. A combination index value (CI) of less than 1 indicates synergy, equal to 1 indicates additive, and greater than 1 indicates antagonism. D. Combination and Dose Reduction Indices (DRI) for ATRi and PARPi combination treatment. A DRI > 1 indicates a favorable dose reduction, <1 indicates a non-favorable dose reduction, and a DRI = 1 indicates no dose reduction.

3.2. Synergy between ATRi and PARPi in cervical cancer cells is dependent on timing

We examined the combinatorial effects of ATR and PARP inhibitors when cervical cancer cells were treated with the two drugs simultaneously or sequentially. To determine the effectiveness of the drug combinations, we performed an isobologram analysis, which measures the combination index (CI) values. CI <1 indicates a synergistic effect, CI = 1 indicates an additive effect, and CI >1 indicates an antagonistic effect. Our results showed that simultaneous treatment with ATRi and PARPi was nearly additive (CI = 1.08 and 1.09, respectively) in both SiHa and CaSki cells. However, pretreatment with PARPi followed by ATRi resulted in a moderate antagonistic effect in CaSki cells (CI = 1.32). Additionally, pre-treatment with ATRi followed by PARPi resulted in a strong synergistic effect in both SiHa and CaSki cells, CI = 0.40 and 0.37, respectively.

PARPi and ATRi have bone marrow suppression as a dose-limiting toxicity, and reducing the dose of one or both agents is clinically meaningful [31,32]. Highly favorable dose-reduction indices (DRI’s) were observed for both PARPi in SiHa and CaSki cells, and for ATRi in SiHa and CaSki cells, when comparing pre-ATRi treatment with pre-PARPi and simultaneous treatments in either cell line (Fig. 1D). Pre-ATRi and sequential PARPi treatment strategies were used in all subsequent experiments.

3.3. Combined ATR and PARP inhibition modulates the DNA damage response pathway

ATR and PARP play crucial roles in DNA repair and cell cycle regulation through HR and error-prone non-homologous end-joining (NHEJ), respectively [22–24]. Therefore, we aimed to investigate the downstream signaling events in the DDR pathway modulated by ATR and PARP inhibitors, both alone and in combination. SiHa and CaSki cells were treated with ATR inhibitor (IC50) alone, PARP inhibitor (IC10, IC25, IC50) alone, or pretreated with ATR inhibitor (IC50) for 24 h, followed by PARP inhibitor treatment for 72 h at their respective IC50 concentrations. The effects of ATR and PARP inhibitors, along with other DDR effector proteins such as ATM, CHK1, pCHK1, and pCHK2, were measured. ATR inhibition alone decreased ATR expression and subsequently repressed pCHK1 levels in both cervical cancer cell lines. However, PARP inhibition had little effect on ATR expression, and pCHK1 levels were only moderately reduced in CaSki cells. In cells treated with both inhibitors, ATR levels were slightly reduced, with increased levels of PARP inhibition. Furthermore, combined treatment with ATR and PARP inhibitors led to decreased CHK1 and pCHK1 levels (relative to total CHK1) in both cell lines (Fig. 2A, B). These findings indicate that the combination of ATR and PARP inhibitors effectively represses ATR signaling through CHK1 in cervical cancer cell lines.

Fig. 2.

Fig. 2.

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Combination of ATR and PARP inhibition targeting the DDR pathway. Representative western blots and fold change values of ATR, CHK1, and pCHK1 in whole-cell lysates (WCL) derived from SiHa (A) and CaSki (B) cervical cancer cells. Representative western blots and fold-change values of ATM and pCHK2 in whole-cell lysates (WCL) derived from SiHa (C) and CaSki (D) cervical cancer cells. One membrane was re-probed for all proteins and normalized to the GAPDH loading control. ATR and ATM levels were normalized to GAPDH levels, whereas pCHK1 levels were relative to total CHK1 and normalized to GAPDH levels. Representative western blots and quantification of Cleaved PARP in whole-cell lysates (WCL) derived from both cervical cancer cell lines (E, F). Results were obtained from two independent experiments (mean ± SEM).

To evaluate the activation of the ATM/p-CHK2 checkpoint by ATRi and PARPi, alone and in combination, the response of HPV-positive cervical cancer cells to these treatments was assessed. ATRi treatment resulted in increased ATM and pCHK2 levels in SiHa cells, while an increase was not observed in CaSki cells. While PARPi treatment consistently caused an increase in ATM and pCHK2 levels in both SiHa and CaSki cells. However, when the two treatments were combined, checkpoint activation was repressed (Fig. 2C, D).

We examined PARP, which is involved in NHEJ DNA repair and is an indicator of apoptosis. Our findings showed that PARPi treatment alone decreased PARP and cleaved PARP levels in both cell lines. Interestingly, ATRi monotherapy increased PARP levels in CaSki cells and cleaved PARP levels in both cell lines relative to PARPi treatment alone and in untreated control cells. Additionally, ATRi and PARPi treatment resulted in increased cleaved PARP levels in both SiHa and CaSki cell lines compared with PARPi treatment alone and in untreated control cells. Together, these results suggest that ATRi treatment increases PARP expression, resulting in increased PARP cleavage following sequential treatment with PARPi (Fig. 2E, F). The combination of ATRi and PARPi is synthetically lethal in CC cells by blocking the compensatory DDR pathways.

3.4. ATR and PARP inhibition causes G2/M cell cycle arrest and apoptosis in cervical cancer cells

ATR/CHK1 regulates the G2/M cell cycle checkpoint, and our immunoblot data showed that ATRi monotherapy and ATRi + PARPi combination therapy decreased ATR/pCHK1 (Figs. 2A–B), which could lead to G2 cell cycle checkpoint arrest. To confirm this, we analyzed cell cycle profiles after ATRi, PARPi, and ATRi + PARPi treatments, which revealed a significantly higher population of cells in the G2/M phase in both cell lines when treated with the combination therapy compared to monotherapy or the control (Fig. 3A–3C). Additionally, the thymidine analog, 5-Ethynyl-2′-deoxyuridine (EDU), was used to label replicating DNA in proliferating cells to evaluate the effects of PARPi and ATRi on cell proliferation and cell cycle progression. A significant reduction in the percentage of EDU-positive (S-phase) SiHa cells in both single-agent groups was observed, while no significant change was measured in CaSki cells (Fig. 3D, upper panel). The combination of ATRi and PARPi resulted in a greater reduction in the percentage of EDU cells (S-phase) in SiHa and Caski cells compared to monotherapy and control, although this difference was not statistically significant in Caski cells (Fig. 3D, lower). However, a significant increase in G2/M phase cells was observed relative to control and single agent ATRi and PARPi treatment in both SiHa and CaSki cells, suggesting a G2/M cell cycle arrest. Next, we sought to determine whether the ATRi and PARPi alone or in combination could induce cell death via apoptosis. A significant increase in early and late-apoptotic cells were observed with single agent PARPi treatment in SiHa cells and not in CaSki cells (Fig. 3E, F). Single agent ATRi treatment resulted in significantly increased late apoptotic and necrotic cells in both SiHa and CaSki lines (Fig. 3F, G). The combination of ATRi and PARPi resulted in a significant increase in both early- and late-apoptosis in both SiHa and CaSki cells (Fig. 3E, F). Overall, these results suggest that the combined ATR and PARP inhibition induces G2/M cell cycle arrest and apoptosis in CC cell lines.

Fig. 3.

Fig. 3.

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ATR and PARP inhibition caused G2/M cell cycle arrest and apoptosis in cervical cancer cells. (A, B) Cell cycle analysis of SiHa and CaSki cells pretreated with ATRi for 24 h followed by PARPi with their respective IC50 concentrations for 72 h. (A). The plot shows the newly synthesized DNA content (labeled with EDU) versus the total DNA content (PI). (B) Cell cycle distribution, (C) percentage of G2-M phase cells, and (D) EDU-positive cells among SiHa and CaSki cell lines. Results were obtained from three independent experiments (mean ± SEM). Early apoptosis (E), late apoptosis (F) and necrosis (G) were assessed with Annexin V-FITC staining. The data were analyzed by flow Jo and ModFit software and plated graph using Graph Pad prism with ordinary one-way ANOVA followed by Tukey’s/Dunnetťs multiple comparison test. *P < 0.05; **P < 0.01; ****P < 0.0001.

3.5. Combined ATR and PARP inhibition increases DNA damage and alters homologous recombination and DNA double-strand break repair

DNA double-strand breaks (DSBs) often occur through homologous recombination (HR) and require ATM activity. ATR is required for the repair of single-strand breaks (SDBs) that can result from stalled replication forks or the processing of DSBs. Hence, the responses of HR and DSB repair to ATR and PARP inhibition were evaluated. HR occurs during the S and G2 phases of the cell cycle; therefore, geminin, a marker of the G2/S phase, was used to identify cells in these phases, and RAD51, a marker of HR repair, nuclear foci (prominence > 5), were measured in geminin-positive (G2/S phase) cells (Fig. 4A, B). PARPi monotherapy significantly increased RAD51 foci (P < 0.0001) in both SiHa and CaSki cell lines. The increase in RAD51 foci was blocked or reduced by ATRi treatment (Fig. 4C,D), suggesting that ATR inhibition results in an HR-deficient phenotype in cervical cancer cells, which can be restored by the administration of PARPi This restoration of HR could allow for DSB repair. DSBs were approximated by measuring p-γH2AX nuclear foci (prominence > 10) in cervical cancer cells treated with ATRi and PARPi monotherapy or ATRi + PARPi combination (Fig. 4E, F). PARPi monotherapy significantly increased the number of p-γH2AX foci (P < 0.0001) in both cell lines, whereas ATRi treatment increased p-γH2AX foci (P < 0.0001) only in CaSki cells (Fig. 4G, H). The combination of ATRi + PARPi significantly (P < 0.0001) increased pH2AX foci compared to PARPi alone, ATRi alone, and control SiHa cells, whereas the combination of ATRi + PARPi significantly (P < 0.0001) increased p-γH2AX foci compared to the control in CaSki cells.To further assess the amount and type of DNA damage occurring with ATRi and PARPi treatment, we utilized the Comet assay, under both neutral conditions (to measure only DNA double strand breaks (DSBs)) and alkaline conditions (to measure both DNA single-stranded breaks (SSBs) and DSBs). Under neutral conditions, the amount of DSBs (% Tail moment) with single agents was significantly higher in ATRi alone treated SiHa (P < 0.01) and CaSki (P < 0.05) cells relative to control and in PARPi alone treated CaSki (P < 0.001) cells relative to control. The combination of ATRi and PARPi resulted in significantly increased DSBs relative to control (P < 0.0001) and PARPi treated SiHa (P < 0.05) and CaSki (P < 0.0001) cells, while the combination did not result in significantly more DSBs relative to ATRi alone in SiHa cells (Fig. 4I and K). Under alkaline conditions, the amount of DSBs and SSBs was significantly increased with ATRi and PARPi alone treated SiHa cells (P < 0.0001) and with ATRi alone in CaSki (P < 0.0001) cells relative to control. In the combination ATRi+PARPi treated cells there was a significant increase in DSBs and SSBs compared to control and ATRi and PARPi single agent treatment in CaSki cells (P < 0.0001), and PARPi alone treatment in SiHa cells (P < 0.05) (Fig. 4J and L). Collectively, these results demonstrate that ATR inhibition impedes HR, whereas PARP inhibition induces synthetic lethality by enhancing HR deficiency and inducing DNA damage through both SSBs and DSBs, leading to increased apoptotic cell death in CC cells in vitro.

Fig. 4.

Fig. 4.

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Combination of ATR and PARP inhibition increases DNA damage and alters homologous recombination and DNA double-strand break repair. SiHa and CaSki cell lines were pretreated with ATRi for 24 h followed by PARPi at their respective IC50 concentrations for 72 h. (A, C) Representative images of RAD51(red) nuclear foci in geminin (green)-positive (cells in G2/S phase) SiHa and CaSki cells, respectively. (B, D) Quantification of RAD51 nuclear foci counts in both CC cell lines. Cells with >5 foci in the nucleus were counted as positive RAD51 cells. (E, F) Representative immunofluorescence images of pH2AX foci (green) in the SiHa (E) and CaSki (F) cells. Quantification of pH2AX nuclear foci in SiHa (G) and CaSki (H) cells. Prominence was set at >10 to count the pH2AX nuclear foci. Images were taken using a confocal microscope at 100× magnification, scale bar = 20 μm). Results were obtained from two independent experiments (mean ± SD). Representative comet tail images in SiHa and CaSki in neutral (I) and alkaline (J) conditions. Quantification of % Tail moment in SiHa and CaSki in neutral (K) and alkaline (L) conditions. Results are obtained from counting 35–90 comets per condition. Data were analyzed using one-way ANOVA followed by Dunnetťs multiple comparison test. *P < 0.05; ****P < 0.0001.

3.6. The combination of ATR and PARP inhibition was effective in reducing cervical cancer tumor growth in vivo

The SiHa xenograft model was used to evaluate the effects of ATRi and PARPi, alone and in combination. ATRi pre-treatment showed a synergistic combinatorial effect and favorable DRI in the combination therapy group; therefore, ATRi was administered on days 1,2, and 3 of each week, whereas ATRi and PARPi monotherapies were administered daily (Fig. 5A). ATRi monotherapy and combined drug treatment significantly reduced SiHa xenograft tumor volume compared to the control group (P = 0.0009 and P = 0.0006, respectively), whereas PARPi monotherapy yielded a non-significant decrease in tumor volume (Fig. 5B).

Fig. 5.

Fig. 5.

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In vivo validation of ATR and PARP inhibitor combinations in cervical xenografts. (A) Schematic diagram of the SiHa xenograft establishment and drug treatment. SiHa cells (1 × 107 cells/mouse) in matrigel were injected subcutaneously into 6-week-old athymic nude mice. (B) Tumor volume. (C) Body weight during drug treatment. (D) Representative IHC images of pH2AX and pCHK-1 in SiHa xenografts at a magnification of 20× and a scale bar of = 50 μm. (E) Quantification of the pH2AX and pCHK-1 levels. (F, G) Representative western blot and quantification of ATR, p-CHK1, and cleaved PARP in SiHa xenograft tissues. Results were obtained from three independent biological animals, mean ± SEM (E), and from two independent experiments with three biologically independent animals per group, mean ± SD (F and G). Data were analyzed using one-way ANOVA followed by Dunnetťs multiple comparison test (G). *P < 0.05; ***P < 0.001.

Drug toxicity was assessed by measuring body and organ weights. No significant differences in body weight were observed between the monotherapy, combined drug treatment, and control groups (Fig. 5C). Additionally, no significant differences in the organs (liver, kidney, and spleen) were observed among the treatment groups (Supplementary Fig. S1). The tumor tissue was evaluated by IHC analysis, which demonstrated that combination therapy with ATRi and PARPi significantly increased DSBs, as evidenced by the increased pH2AX staining compared to that in the control group (Fig. 5D, E). Additional IHC evaluation demonstrated that pCHK1 levels were downregulated by ATRi monotherapy and ATRi + PARPi combination therapy and were unchanged by PARPi alone. However, these values did not differ significantly between groups (Fig. 5D, E). ATR/pCHK-1 was significantly decreased in the xenograft tumors of ATRi + PARPi and PARPi alone compared to that in the control (Fig. 5F, G). PARP cleavage was not significantly increased in either the ATRi or PARPi monotherapy groups but was significantly increased in the ATRi + PARPi combination group compared to that in the control group (Fig. 5F, G). Taken together, these data suggest that the combination of PARPi and ATRi in a toxicity-limiting dose-reduction strategy is effective in inhibiting cervical cancer tumor growth in vivo without substantial organ toxicity.

4. Discussion

Cervical cancer is the most lethal gynecological cancer globally, mainly because of ineffective first-line treatment in advanced stages and recurrent metastatic disease. Despite efforts to identify molecular drivers through TCGA, the results did not provide immediate actionable targets, possibly due to a bias towards early-stage primary cervical cancer samples [33]. However, dysregulation of DDR pathways owing to HR-HPV integration is a consistent theme. Abrogation of G1 (PARP-dependent) DNA repair mechanisms coupled with HR deficiency make cervical cancers highly sensitive to DNA-damaging therapies. The degradation of p53 and dysregulation of the cellular proliferation machinery, which are hallmarks of HR-HPV-induced carcinogenesis, provide a rationale for the more specific targeting of DDR. Therefore, it is crucial to identify targeted combination treatment strategies directed at the DDR to improve outcomes in patients with cervical cancer.

Phase I/II clinical trials are underway for therapeutics that inhibit DDR kinases in all solid tumors [34]. Preclinical studies have indicated that combining PARP and ATR inhibitors can be effective in HR-deficient solid tumors, leading to improved tumor regression [35–38]. For example, in HR-deficient BRCA1-mutant high-grade serous ovarian cancer, PARP inhibition activates the ATR-CHK1 pathway and causes G2 phase accumulation. When ATR or CHK1 inhibitors are added to PARPi, there is a synergistic decrease in cell survival and tumor suppression [39]. Additionally, combining ATRi with PARPi can re-sensitize PARPi-resistant cells and synergistically induce cell death [29]. Overall, these studies suggest that combined PARP and ATR inhibition is a promising strategy for treating tumors with impaired DDR.

HR-HPV-mediated carcinomas typically depend on the HR pathway for DNA repair, which leads to ATR amplification to maintain DDR through the G2 checkpoint [14]. Phase I clinical trials in advanced solid malignancies have shown acceptable toxicity of ATR inhibitors, both alone and in combination with PARP inhibitors, with side effects mainly related to bone marrow suppression [34,38]. Although the use of PARP inhibitors with cytotoxic chemotherapy does not improve response rates or disease-specific survival in women with metastatic disease [40,41], there is limited published data on the effects of combining DDR therapy specifically targeting the G2 checkpoint in cervical cancer treatment.

In this study, we demonstrated that HPV+ cervical cancer cells are sensitive to both ATR and PARP inhibitors when used as single agents. When tested in combination, pretreatment with an ATRi inhibitor followed by PARPi resulted in synergistic inhibition of the metabolic viability of cervical cancer cell lines. This synergistic effect was likely due to ATRi-mediated upregulation of PARP, which increased the availability of the target. We also found that PARPi treatment alone resulted in G2-M cell cycle arrest and apoptosis (in SiHa cells), which was increased by the combination of ATRi and PARPi. While induction of necrosis was only seen with ATRi alone. Increased HR DNA damage repair, as measured by the formation of RAD51 foci [42], was observed with PARPi treatment alone, indicating an active HR and response to PARPi. However, the addition of ATRi decreased active HR and reduced sensitivity to PARPi. These findings suggest that the combination of ATRi and PARPi further impairs HRR in cervical cancer cells, resulting in an increased sensitivity to PARPi treatment. This was further supported by the observed increase in pH2AX nuclear foci, a marker of DSBs) and the increase in both DSBs and SSBs following combined ATRi and PARPi treatment. The results of this study indicate that the combination therapy of ATRi and PARPi leads to an upregulation of cleaved PARP levels, while the levels decline when treated with PARPi alone. This suggests that the re-engagement of PARP-driven DNA damage repair when the ATR-dependent G2 checkpoint is inhibited may be one of the underlying mechanisms of synergy between the two treatments. The effectiveness of the combination therapy was also observed in xenograft experiments, in which the inhibitors were administered with intermittent ATRi and continuous PARPi dosing. The reduction in cervical cancer tumor volume was significant and there was no significant organ toxicity, as measured by body weight over time. The increase in total PARP levels with inhibition of the G2 checkpoint by ATRi provides a strong biological rationale for sequencing and de-escalating the dose of PARPi in vivo.

This study demonstrated that combining ATR inhibitors and PARP inhibitors in vivo resulted in DNA double-strand breaks, inhibition of ATR/pCHK1, and increased levels of cleaved PARP, which was consistent with the in vitro effects of the combined treatment. However, the in vivo model had some limitations, such as PARP inhibitor treatment not having a significant effect on tumor growth and the combination treatment resulting in significant tumor reduction, but not greater than ATRi treatment alone. These limitations were likely due to the necrotic nature of cervical cancer subcutaneous xenografts and the chosen dosing schedule to reduce toxicity, which may have reduced the efficacy of the combination therapy compared to the single-agent treatment groups.

In summary, we demonstrated that ATR inhibition prevents DDR through the preferred G2 checkpoint in HR-HPV+ cervical cancer cells by increasing replication fork stress, which leads to an increased reliance on PARP-mediated repair pathways for DNA repair. Combining ATRi pretreatment with PARPi treatment yields synthetic lethality, resulting in apoptotic cell death and increased cytotoxicity, providing a promising treatment option for HR-HPV+ metastatic cervical cancer.

Supplementary Material

Suppl 1
Suppl 2

HIGHLIGHTS.

  • Cervical cancer cells are sensitive to both ATR and PARP inhibitors.

  • ATR inhibition up-regulates PARP expression and when followed by PARP inhibition synergistically reduces cell viability.

  • ATR and PARP inhibition causes cell cycle arrest, apoptosis, and inhibits DNA repair and cervical tumor growth in vivo.

  • The dual targeting of PARP and ATR is a promising therapeutic strategy for advanced cervical cancer.

Acknowledgments

We are grateful for the cell-line authentication services provided by the SCC MTCRO-COBRE cell-line authentication unit. Histology and IHC services were provided by the Stephenson Cancer Tissue Pathology Core, which was supported by the National Institute of General Medical Sciences Grant P20GM103639, the National Institutes of Health Award (1S10OD026744), and the National Cancer Institute Grant P30CA225520 from the National Institutes of Health. We thank the Institutional Research Core Facility at OUHSC for using the FACSCalibur and Stratedigm 3-laser flow cytometer.

Funding

This research was funded by the Memorial Sloan Kettering Cancer Center pilot grant (K. M.) and Oklahoma Tobacco Settlement Endowment Trust awarded to the University of Oklahoma Stephenson Cancer Center (B.H.).

Footnotes

Declaration of competing interest

Author K.M.M serves on the American Board of Obstetrics and Gynecology, and is a member of the NRG Oncology Cervix and Diversity Equity and Inclusion committees. The remaining authors declare that they have no conflict of interest.

Ethical approval and consent to participate

Animal studies were conducted using an IACUC-approved protocol (19–093-CHI).

Consent for publication

All the authors provided consent to participate in the study.

CRediT authorship contribution statement

Sugantha Priya Elayapillai: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. Samrita Dogra: Writing – review & editing, Methodology, Data curation. James Lausen: Methodology, Data curation. Madison Parker: Methodology, Data curation. Amy Kennedy: Methodology, Data curation. Doris M. Benbrook: Writing – review & editing, Supervision, Conceptualization. Katherine M. Moxley: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. Bethany N. Hannafon: Writing – review & editing, Writing – original draft, Supervision, Funding acquisition, Formal analysis, Conceptualization.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ygyno.2024.10.009.

Availability of data and materials

Data generated by the authors are available upon request.

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Supplementary Materials

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Data Availability Statement

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