Targeting the ATR/CHK1 axis with PARP inhibition results in tumor regression in BRCA mutant models

Abstract

Purpose

PARP inhibition (PARPi) has modest clinical activity in recurrent BRCA mutant (BRCA^MUT) high-grade serous ovarian cancers (HGSOC). We hypothesized that PARPi increases dependence on ATR/CHK1 such that combination PARPi with ATR/CHK1 blockade results in increased cell death and tumor regression.

Experimental Design

Effects of PARPi (olaparib), CHK1 inhibition (CHK1i;MK8776) or ATR inhibition (ATRi;AZD6738) alone or in combination on survival, colony formation, cell-cycle, genome instability and apoptosis were evaluated in BRCA1/2^MUT HGSOC cells. Tumor growth in vivo was evaluated using a BRCA2^MUT patient-derived-xenograft (PDX) model.

Results

PARPi monotherapy resulted in a decrease in BRCA^MUT cell survival, colony formation and suppressed but did not eliminate tumor growth at the maximum-tolerated dose in a BRCA^MUT PDX. PARPi treatment increased pATR and pCHK1 indicating activation of the ATR-CHK1 fork protection pathway is relied upon for genome stability under PARPi. Indeed, combination of ATRi or CHK1i with PARPi synergistically decreased survival and colony formation compared to single agent treatments in BRCA^MUT cells. Notably, PARPi led to G2 phase accumulation, and the addition of ATRi or CHK1i released cells from G2 causing premature mitotic entry with increased chromosomal aberrations and apoptosis. Moreover, the combinations of PARPi with ATRi or CHK1i were synergistic in causing tumor suppression in a BRCA^MUT PDX with the PARPi-ATRi combination inducing tumor regression and in most cases, complete remission.

Conclusions

PARPi causes increased reliance on ATR/CHK1 for genome stability and combination PARPi with ATR/CHK1i is more effective than PARPi alone in reducing tumor burden in BRCA^MUT models.

INTRODUCTION

Ovarian cancer survival has improved minimally over the last decade(1) despite the unprecedented progress in understanding the genetics of ovarian cancer(2) . There is a critical need to develop better therapeutic strategies that exploit the biology and genetics of high grade serous ovarian cancer (HGSOC). Approximately 50% of HGSOCs have defects in genes involved in homologous recombination (HR) repair(2,3) BRCA 1 and 2 (Breast Cancer Susceptibility Gene 1 and 2) mutant HGSOCs have a deficiency in the repair of double strand DNA breaks (DSB) by HR(4). Poly (ADP-ribose) polymerase inhibitors (PARPi) impair the repair of single-stranded DNA breaks leading to DNA DSB which cannot be repaired efficiently in BRCA1/2-mutant (BRCA^MUT) cancers capitalizing on synthetic lethality(5). PARPi, such as olaparib, have demonstrated a 31% overall response rate leading to its FDA approval for recurrent germline BRCA^mut HGSOCs(6). Rare complete responses (3%) are seen with PARPi monotherapy(6-8). Our goal was to optimize PARPi therapy in BRCA^MUT HGSOC by evaluating scientifically rational combinations.

Another approach to modulate DNA repair activity and improve the therapeutic index of PARPi in HR-deficient HGSOCs is to interfere with cell cycle checkpoint signaling. ATR (Ataxia Telengiectasia and Rad3-related) and its downstream kinase CHK1 (Checkpoint Kinase 1), are activated by DNA replication stress and DNA damage thereby arresting cell cycle progression allowing time for appropriate damage repair and completion of replication(9,10). ATR/CHK1 blockade prevents DNA-damage-induced cell cycle arrest, resulting in inappropriate entry into mitosis, chromosome aberrations, unequal partitioning of the genome, and ultimately apoptosis(9). In addition, because the ATR-CHK1 pathway stabilizes replication forks and prevents collapse into DNA double strand breaks (DSBs), inhibition of ATR/CHK1 are expected to increase reliance on HR to reform the replicatoin fork structure and complete replication.

Indeed, ATR inhibition is synthetic lethal with numerous cancer-associated changes, including oncogenic stress (oncogenic RAS mutations, MYC and CCNE1 overexpression), deficiencies in DNA repair (TP53, BRCA1/2, PALB2, and ATM loss), and other defects(9,11-15). CHK1 inhibition similarly, is synthetically lethal with p53 or BRCA 1/ 2 loss(16,17). Almost all HGSOCs harbor a mutation in TP53(2) and thus have lost G1 checkpoint control, significantly increasing reliance on S and G2 checkpoints for survival(11,18). Targeting S and G2 checkpoints by inactivation of the ATR/CHK1 pathway will inhibit the DNA damage induced G2 checkpoint arrest, leading to mitotic catastrophe and tumor cell death in contrast to normal cells, which maintain an intact G1-phase checkpoint(19). A variety of metabolic perturbations in cancers cause a reliance on ATR/CHK1 to facilitate DNA synthesis and prevent the formation of DNA double-stranded breaks (DSBs) at replication forks(20). These breaks can increase to toxic levels in cancer cells when ATR or CHK1 is inhibited(9,11-14,20-22). Thus, ATR or its downstream effector, CHK1, is a reasonable target for treating HGSOCs, all of which have loss of functional TP53 and approximately 50% have defects in HR(2). Drugs targeting ATR (AZD6738, VX-970) and CHK1 (MK8776, SCH 900776, LY2606368, CCT245737) are in early phase I/II clinical trial development (clinicaltrials.gov).

While PARPi is active as monotherapy, it rarely leads to complete tumor responses(6-8,23), emphasizing the need for alternative strategies capitalizing on synthetic lethality. We have developed a BRCA^MUT HGSOC orthotopic PDX platform with over 15 models that is molecularly annotated to strategize synthetic lethal approaches in BRCA^MUT HGSOC(24). Because we observed that PARPi caused ATR-CHK1 pathway activation, we reasoned that PARPi treatment alone may increase dependence on the ATR/CHK1 pathway for survival and that inhibition of ATR or CHK1 would increase DNA replication fork instability and promote cell death in BRCA^MUT HGSOC models. We show that PARPi treatment results in early activation of ATR/CHK1 and that combination PARPi with either CHK1i or ATRi is synergistic in suppressing BRCA^MUT HGSOC growth in culture and in the PDX model.

MATERIALS AND METHODS

Cell lines

PEO1 (BRCA2 mut; c.C4965G), and PEO4 (BRCA2 reversion mutation) serous ovarian cancer cell lines were grown in RPMI media with 10% FBS, and penicillin/streptomycin (generous gift from Dr. Andrew Godwin, University of Kansas, Kansas City, KS). JHOS4 (BRCA1; c.5278-1G>A) ovarian cancer cells were grown in DMEM/F12 media with 10% FBS, and penicillin/streptomycin. The WO-20 primary ovarian culture was generated in our laboratory from a patient with HGSOC (UPCC 17909) and the cells were cultured in OCMI-E media (Live Tumor Culture Core at Sylvester Comprehensive Cancer Center, Miller School of Medicine, Miami, FL). Mutation profiles for all cell lines were evaluated using a targeted panel of genes by whole exome sequencing(24). All cell lines were confirmed negative for mycoplasma. Authenticity was confirmed by short tandem repeats by the Wistar Genomics Core.

In vitro cytotoxicity assays

Cells (5×10³) were seeded on 96 well plates and treated with the indicated doses of PARPi (AZD2281), CHK1i (MK8776) and ATRi (AZD6738) for 5 days. At the end of the treatment period, the relative cell viability was determined by an MTT colorimetric assay. Cells were incubated with 10 μl of MTT at 5 mg/ml (Sigma Chemical Co., St Louis, MO) for 2 hr at 37°C. DMSO was added and the absorbance was measured in a microplate reader at a wavelength of 570 nm. IC50s were calculated using GraphPad Prism (GraphPad Software, San Diego, CA).

Colony formation assay

Cells (1-2×10⁴) were plated onto 12-well plates and incubated at 37°C. Cells were treated for 10-14 days. Media and drugs were refreshed every 3-4 days. Colonies were washed with PBS, fixed with 4% paraformaldehyde and then stained with 0.2% crystal violet. Whole well images were scanned and colony forming area was quantitated using ImageJ (National Institutes of Health). For each sample, the results from three replicates were averaged(25).

Patient-derived xenografts (PDX) studies

NSG mice were purchased from the Jackson lab (NOD-SCID IL2Rγ−/−, Bar Harbor, ME). All mice were housed according to the policies of the Institutional Animal Care and Use Committee of the Wistar Institute. Five- eight week old female mice were used for tumor transplantation. Patient-derived xenografts are generated by sectioning of fresh tumor tissue and engrafting pieces (2 ×2 ×2 mm³) orthotopically to the mouse fallopian tube fimbria/ovary. Tumor was obtained from debulking surgeries conducted at the Hospital of University of Pennsylvania (IRB# 702679). Once the transplanted tissue reaches ~700–1000 mm³, it is harvested, analyzed by genomic and proteomic studies, expanded and banked for preclinical studies(24). For preclinical studies, cryopreserved tissue is thawed, washed with Hank's Balanced Salt Solution and re-transplanted to the fallopian tube fimbria/ovary for evaluation of in vivo drug response. Tumor length and width were measured by ultrasound (SonoSite Edge II Ultrasound System) on each mouse and used to calculate tumor volume. Once tumor volume reached 70-100 mm³, animals (n=70) were randomized to 6 treatment groups: vehicle (2-hydroxylpropyl-β-cyclodextrin), MK8776 (50mg/kg IP Q 3 days; Selleckchem), AZD2281 (50mg/kg/day by oral gavage; AstraZeneca), AZD6738 (25mg/kg/day by oral gavage, AstraZeneca), MK8776 + AZD2281 (MK8776 50mg/kg IP Q 3 days and AZD2281 50mg/kg/day) and AZD6738 + AZD2281 (AZD6738 25 mg/kg/day Day 1-3 weekly and AZD2281 50mg/kg/day). Tumor volume and body weight were measured weekly. Animals were euthanized according to Institutional Animal Care and Use Committee guidelines. Tumors were collected and snap frozen for protein analysis and Immunohistochemistry (IHC).

Western blot

Cells and tissues were harvested and lysed in a Laemmli sample buffer (BioRad, Hercules, CA) containing a protease and phosphatase inhibitor cocktail (EMD Millipore, Billerica, MA). Following protein concentration determination (BioRad, Hercules, CA), cell lysates were separated on reducing SDS-PAGE gels and immunoblotted with phospho ATR (Cat. # ABE462, EMD Millipore, Billerica, MA), total ATR (Cat. # sc1887, Santa Cruz Biotechnology, Inc., Dallas, TX), phospho CHK1 (Ser³⁴⁵), (Cat. # 2348, Cell Signaling Technology, Inc., Danvers, MA), total CHK1 (Cat. # sc8408, Santa Cruz Biotechnology, Inc., Dallas, TX),γH2AX (Cat. # 9718, Cell Signaling Technology, Inc., Danvers, MA). The species-appropriate horseradish peroxidase-conjugated secondary antibody was used, followed by detection with chemiluminescent substrate (Thermo Scientific, Rockford, IL). Odyssey Quantitative Fluorescent Imaging Systems (LI-COR Biotechnology, Lincoln, NE) was used for image generation. Anti-β-Actin (Cat. # 3700, Cell Signaling Technology, Inc., Danvers, MA) was used as an internal control. Band intensity was quantitated using ImageJ (National Institutes of Health).

Cell cycle analysis

Cell cycle was analyzed using a FITC-BrdU Flow Kit (BD Biosciences, San Jose, CA). Cells (5×10⁵) were plated on 10 cm dishes. At 48 h after the initial seeding of the cells, the cells were incubated with drugs for an additional 48 hours. Ten μM of BrdU was added to culture medium and incubated for 2 hours before harvest. Cells were fixed and labeled with FITC-conjugated anti-BrdU and PI solution. Cell suspensions were incubated for 15 minutes at room temperature and immediately analyzed in a flow cytometer (BD FACSCalibur, BD Biosciences, San Jose, CA). Data was analyzed by FlowJo (Tree Star, Inc., Ashland, OR).

Metaphase Spread

Cells were harvested for chromosome preparations using colcemid (50ng/ml for 90 minutes followed by an 18 minute incubation in 0.075 M KCl at 37° and dropwise addition of Carnoy's fixative (3:1methanol:acetic acid). Cells were incubated in fixative for one hour, pelleted at 1000g and fixative was replenished. After cells were incubated at 4° overnight, the fixative was again replenished. Fixed cells were dropped onto uncoated microscope slides and dried for at least 24 hours at room temperature. Dropped slides were stained in Giemsa staining solution (SIGMA GS1L) for 4 minutes. Stained slides analyzed for total gaps and breaks in a blinded fashion using a 100X objective and a Nikon Eclipse 80i microscope. Fifty metaphases were scored for each sample in two independent experiments for a total of 100 metaphases scored for every sample.

Apoptosis Analysis

Cells (5 × 10⁵) were plated on 10cm dishes. At 48 h after the initial seeding, the cells were incubated with drugs for 48 hours. Apoptosis was detected by using an Annexin V flow kit (BD Biosciences) according to the manufacturer's instructions. Annexin V-labeled cells were analyzed in a flow cytometer (FACS Calibur; BD Biosciences). The data was analyzed by FlowJo (Tree Star, Inc., Ashland, OR).

Immunohistochemistry

Tissue samples were fixed in 10% formalin. Tissues were dehydrated in graded ethanol solutions, cleared in xylene, and embedded in paraffin. Paraffin blocks were cut into 4- –6 μm sections, and placed onto slides. After deparaffinization and rehydration, antigen retrieval was done via pressure cooker. Slides were pressure cooked in 1x target retrieval solution at 120°C at 18 to 20 psi. Endogenous hydrogen peroxidase activity was blocked with hydrogen peroxide for 10 minutes followed by rinsing with wash buffer. Slides were incubated with pCHK1 (CST, Cat# 2348) antibody at 1:1000 titer for 40 minutes. Alternatively, slides were incubated with appropriate isotype controls, and diluted similarly. Slides were washed and incubated with anti-rabbit horseradish peroxidase polymer for 30 minutes, followed by a further wash. Slides were developed using 3,3′-diaminobenzidine (DAB)+ chromogen for 5 minutes and washed with water. After staining, slides were counterstained, dehydrated, and mounted with mounting reagent.

Statistical analyses

MTT, Colony Formation Assays (CFA), FACS and Western assays were done at least twice and means ± SEM are displayed in bar graphs. One- or two-way ANOVA was conducted to assess differences among means. Following a significant ANOVA result (p≤0.05) rejecting the null hypothesis that means are the same across the treatment groups, the Tukey's honestly significant difference (HSD) test was used for all pairwise mean comparisons. This multiple comparison procedures ensure actual family-wise error rates no greater than pre-specified 5%. Stata MP Version 14.0 (StataCorp, College Station, TX) or GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego CA) was used for statistical analyses. Tumor growth data was analyzed using two-way analysis of variance (ANOVA) with Tukey's post-test. All other data were analyzed using Student's t-test. To analyze the drug interaction between ATRi and CHK1i and PARPi combined with either agent, the coefficient of drug interaction (CDI) was calculated from in vitro study(26). CDI is defined by the following formula; CDI = AB/(A × B). According to the absorbance of each group, AB is the ratio of the two-drug combination group to the control group, and A or B is the ratio of the single drug group to the control group. CDI < 1 indicates synergism, CDI < 0.7 significant synergism, CDI = 1 additivity and CD > 1 antagonism. Analysis of potential synergy between drug A and drug B on tumor xenograft growth used the combination ratio(27). Fractional tumor volume (FTV), defined as the ratio of mean final tumor volume in drug treated animals divided by the mean final tumor volume in untreated controls. The combination ratio compared the FTV expected if there was no synergy with the observed FTV. The combination ratio was calculated as (FTV of drug A × FTV of drug B)/ observed FTV of combination. Observed and expected FTV are described as follows: Expected FTV = (mean FTV of drug A) × (mean FTV of drug B), Observed FTV= final tumor volume combined therapy/final tumor volume control Combination ratio =Expected FTV/Observed FTV. A combination ratio greater than 1 indicates drug synergy; while a ratio less than 1 indicates a less than additive effect.

RESULTS

PARP inhibition alone is ineffective in killing ovarian cancer in vitro and in vivo and results in activation of the ATR/CHK1 DNA repair pathway

Increasing concentrations of PARPi, olaparib (AZD2281), were more cytotoxic in BRCA^MUT cells (PEO1, JHOS4) compared to HR-proficient cells (PEO4, BRCA2 reversion mutation; WO-20 primary tumor cultures, BRCA wild-type). PARPi did not result in complete cell death even in the BRCA^MUT with 30-45% of cells still viable after 5 days (Figure 1A). Colony forming ability after treatment with increasing concentrations of PARPi similarly decreased more in the BRCA^MUT compared to the HR-proficient cells (Figure 1B). BRCA2^MUT patient-derived xenografts (PDX; BRCA2 8945delAA) were established and when they reached 70-100mm3, were treated with prolonged PARPi (olaparib) at the maximum tolerated dose (100mg/kg/day). Tumor suppression but not regression was seen for 21 weeks (Figure 1C), after which resistance emerged (not shown). Given the lack of complete cell killing and tumor regression in vivo, we investigated ways to improve the anti-tumor effects of PARPi.

Figure 1. PARPi monotherapy decreases cell viability, suppresses tumor growth but increases ATR/CHK1 dependence.

(A) Viability of HR-deficient (PEO1, BRCA2^MUT; JHOS4, BRCA1^MUT), and HR-proficient (PEO4, BRCA2^REV; WO-20 primary tumor culture, BRCA wild-type) after treatment with PARPi (AZD2281) at increasing concentrations as indicated was assessed by MTT assay. Cells were plated (5000 cells/well) in a 96 well plate and incubated in their respective drug concentrations for 5 days (* p<0.0001 control vs PARPi 0.5uM PEO1; ** p=0.004 control vs. PARPi 0.5uM JHOS4; ^ p=0.0002 control vs PARPi 0.5uM PEO4).

(B) Colony formation evaluated after treatment with PARPi at increasing concentrations as indicated in PEO1, JHOS4, and PEO4. Cells (10K cells/well) were seeded into 12 well plates and incubated in their respective drug concentrations for 7-13 days. The relative colony area was calculated using ImageJ (* p=<0.0001 control vs PARPi 1uM PEO1; ** p<0.001 control vs. PARPi 1uM JHOS4; ^ p=n.s. control vs PARPi PEO4 at 1uM).

(C) To investigate the in vivo impact of prolonged PARPi, tumor [from a patient with a BRCA2^MUT (8945delAA); WO-2-1] was transplanted to the fallopian tube/ovary of 10 NSG mice. Once tumors reached 70-100mm³, mice were treated with PARPi (olaparib 100mg/kg/day by oral gavage). Tumor volume was evaluated by ultrasound weekly. Untreated WO-2-1 tumors were sacrificed within 6-7 weeks due to tumor burden. Mice were treated with PARPi until ~20 weeks of treatment after which resistance developed and mice were sacrificed.

(D) BRCA2^MUT (PEO1) and HR-proficient (PEO4) cells were treated with PARPi 1μM and lysates were collected at 0h, 2h, 6h, 24h over the treatment duration. Western blot for the indicated phospho and total proteins was performed. Densitometry showed pATR increased 3.1 fold in PEO1 (p=0.0039) and 1.7 fold in PEO4 from control to 6 hours (p= 0.02); pCHK1 increased 7.7 fold in PEO1 (p=0.03) and 1.5 fold in PEO4 from control to 6 hrs (p= 0.045). γH2AX increased 2.6 fold in PEO1 and 1.7 fold in PEO4 from control (p= 0.003 in PEO1 vs P=0.04 in PEO4). All data are from 3 biologic assays and graphed as mean±SEM or representative data shown.

In addition to their role of blocking repair of single-strand DNA breaks leading to double-strand breaks(4), PARPi increase G2 arrest(28). We thus sought to evaluate how PARPi affects the ATR/CHK1 cell cycle check point pathway. PARPi treatment at 1uM increased pATR, pCHK1, and γH2AX protein within 2-6 hours in both BRCA^MUT (PEO1, JHOS4 Supplemental Figure 1) and HR-proficient cells (PEO4), but more so in the BRCA^MUT cells at 1uM suggesting activation of ATR/CHK1 for survival (Figure 1D). At higher concentrations of PARPi (5uM), pCHK1 increased within 2 hrs of PARPi treatment in HR-proficient cells (Supplemental Figure 4). DNA damage was increased with PARPi treatment in both the BRCA^MUT and HR-proficient lines but more so with the HR-deficient cells.

CHK1 or ATR inhibition is synergistic with PARP inhibition

Increasing concentrations of CHK1 inhibitor (CHK1i; MK8776) was more cytotoxic in BRCA^MUT cells (PEO1 and JHOS4) compared to the HR-proficient cells (PE04; BRCA^REV). Notably, BRCA2^MUT cell line (PEO1) was more sensitive to CHK1i than BRCA1^MUT cell line (JHOS4). With increasing doses of ATR inhibitor (ATRi; AZD6738), there was a significant decrease in cell viability among both BRCA^MUT (PEO1, JHSO4) and HR-proficient cells (PEO4) beginning at 0.5μM after 5 days of treatment (Figure 2A). Given PARPi increases ATR/CHK1 signaling and both cause replication fork collapse into DSBs using different mechanisms(20,29), we hypothesized that the combination would be more effective in decreasing cell survival.

Figure 2. PARPi in combination with CHK1 or ATR inhibition is synergistic in BRCA mutant cells.

(A) Viability after treatment with CHK1i (MK8776) and ATRi (AZD6738) in PEO1, JHOS4, and PEO4 at increasing concentrations for 5 days assessed by MTT assay (5000 cells/well in a 96 well plate were seeded). CHK1i decreased viability in the PEO1 BRCA^MUT cells at 1μM to 56.60±1.12% (*p<0.0001) and to 63.25±1.43% with 5uM in JHOS4 compared to control (**p<0.0001), whereas PEO4 was more resistant requiring 10uM to decrease viability to 66.51±0.52 (^p<0.0001). With increasing doses of ATRi, there was a significant decrease in viability among all cells at a concentration of 0.5μM compared to control (31.06±0.67 in PEO1 *p<0.0001; 66.09±1.37 in JHOS4 **p<0.0001; 55.07±1.86 in PEO4 ^p<0.0001). (B) The combination effect of CHK1i with PARPi was assessed with both MTT (left) and colony forming assay (CFA; right) in PEO1 and PEO4 cells. Monotherapy with PARPi and CHK1i decreased viability to 32.94±0.96% and 55.46±4.07% from control in PEO1 cells. Combination therapy with PARPi and CHK1i decreased viability to 10% (PARPi vs Both *p<0.0001; CHK1i vs Both **p<0.0001). In PEO1 cells there was synergy for combination compared to either drug alone (CDI=0.50). In PEO4, there was not a significant synergy effect. In PEO4, there was no significant synergy effect.(CDI=0.91). For CFAs (B,C), PEO1 and PEO4 cells were incubated in the indicated drug concentrations for 13 days. Cells were then washed, fixed and stained with 0.2% crystal violet. Whole well images were scanned and colony forming area was quantitated using ImageJ (NIH). For each sample, the results from three replicates were averaged. Monotherapy with PARPi and CHK1i decreased viability to 54.59±3.64% and 82.39±4.32% from control in BRCA^MUT cells. Combination therapy with PARPi and CHK1i decreased colony formation to 16.95±0.78% in BRCA2^MUTcells. (PARPi vs. Both p<0.01, CHK1i vs. Both p<0.001).. In PEO1 cells there was synergy for combination compared to either drug alone (CDI=0.38). In PEO4, there was not a significant synergy effect (CDI=0.92). (C) The combination effect of ATRi with PARPi was assessed with both MTT (left) and colony forming assay (right) in PEO1 and PEO4 cells. Combination therapy decreased viability and colony formation in both the HR-deficient and proficient cells than either drug alone. In PEO1 cells, viability was decreased to 49.94±1.72% with PARPi and 33.39±3.04% with ATRi alone, respectively, compared to control (p<0.0001, P<0.0001). Combination therapy with PARPi and ATRi decreased viability to 5.29±0.19% (PARPi vs Both *p<0.0001, ATRi vs Both **p<0.0001). In PEO4, combination decreased viability more than PARPi and ATRi alone (PARPi vs Both p<0.0001, ATRi vs Both p<0.0001). In both cell lines there was synergy for combination compared to either drug alone (PEO1; CDI=0.32, PEO4;CDI=0.69). CFA (right panel) shows in PEO1 treated cells, both PARPi and ATRi treatment decreased colony formation to 0% compared to PARPi alone (99%; p<0.0001) and ATRi alone (82%; p<0.001). For PEO4 cells, both PARPi and ATRi treatment decreased colony formation to 12% compared to PARPi alone (95%; p<0.001) and ATRi alone (57%; p=0.02). In both cell lines there was synergy for combination compared to either drug alone (PEO1; CDI<0.001, PEO4;CDI=0.22)

(D) To study the effects of CHK1i and ATRi in combination with PARPi on ATR/CHK1 pathway, PEO1 and PEO4 cells were treated with PARPi (AZD2281) 1μM, CHK1i (MK8776) 1μM, and ATRi (AZD6738) 1μM as well as with combination PARPi and CHK1i or ATRi. Lysates were collected after 24h and western blot for the indicated phosphor and total proteins. In PEO1, pATR was decreased with ATRi 2.5 fold (Ctrl vs ATRi p=0.04).PARPi increased pATR but combination PARPi + ATRi decreased pATR 4.5 fold ( PARPi vs PARPi+ATRi p=0.04). pCHK1 increased with CHK1i as expected with CHK1i treatment⁵³ and PARPi with ATRi decreased pCHK1 compared to PARPi alone by 2.9 fold (p=0.009). rH2AX was increased approx. 2-3 fold compared to untreated for all treatments (Control vs CHK1i, p=0.03; ATRi, p=0.02; PARPi, p=0.04, CHK1i+PARPi (p=0.03, ATRi+ PARPi, p=0.02). For PEO4, pATR was decreased with ATRi by 4 fold (Ctrl vs ATRi p=0.01). PARPi increased pATR but combination PARPi + ATRi decreased pATR by 3 fold ( PARPi vs PARPi+ATRi p=0.004). rH2AX increased with ATRi treatments but more with PARPi+ATRi by 1.3 fold( ATRi vs PARPi+ ATRi p=n.s.). All data are from 3 biologic assays and graphed as mean±SEM or representative data shown.

Combination therapy with PARPi and CHK1i was significantly more cytotoxic and decreased colony formation ability than either drug alone in the BRCA2^MUT cells compared to wild type. Drug synergy was demonstrated by PARPi-CHK1i combination in BRCA^MUT cells but not in wild-type (Figure 2B; Supplemental Figure 2). ATRi in combination with PARPi was significantly more cytotoxic than either drug alone in both BRCA2 deficient and wild type cells. PARPi-ATRi combination demonstrated synergy (Figure 2C; Supplemental Figure 2). In BRCA^MUT cells, PARPi in combination with ATRi decreased the PARPi upregulation of pATR and pCHK1. pCHK1 increased with CHK1i treatment as expected given inhibition of CHK1 phosphatase(30) site. In HR-proficient cells, addition of ATRi to PARPi decreased PARPi upregulation of pATR and pCHK1 (Figure 2D). There was an increase in p-γH2AX in BRCA2^MUT cells relative to wild type and in the BRCA2^MUT cells there was an increase with combination therapy compared to monotherapy.

PARPi in combination with CHK1 or ATR inhibition releases G2/M arrest and increases DNA damage in BRCA mutant cell model

We reasoned that the function of ATR-CHK1 activation in PARP inhibited cells may be to prevent cell cycle progression in the context of PARPi-induced DNA-damage. Thus, the effects of ATRi/CHK1i added to PARPi treatment on cell cycle were evaluated. In HR-deficient cells, PARPi treatment alone (1uM) increased G2-M phase from 13% to 44%. ATRi and CHK1i each alone increased G2-M but less so than PARPi from 13% untreated to 30% with ATRi and 19% with CHK1i, respectively. When PARPi treated cells were exposed to ATRi or CHK1i, 13% and 22% of the G2-M arrested population was released respectively (Figure 3, and Supplemental figure 3). In HR-proficient cells, a higher dose of PARPi (5uM) was used because 1uM had minimal effects alone or in combination on cell cycle (data not shown). PARPi (5uM) treatment did have an effect on HR-proficient cells but it was different from what was observed in BRCA^MUT cells. PARPi at 5uM increased G2-M from 11% to 17%. ATRi and CHK1i alone had a similar modest effect on G2-M (ATRi: 11% to 15%; CHK1i 11% to 16%). When PARPi treated cells were exposed to CHK1i, G2-M remained at 16%. However, with ATR exposure, a 24% increase in G2-M was noted (Supplemental Figure 4). Interestingly, the increase in G2-M phase cells, as determined by DNA content, could represent either an increase in DNA damage with ATRi/CHK1i addition that activates alternative checkpoint proteins that recognize DSB (ATM), leading to G2 arrest, or aberrant progression into M phase and stalling therein. Either mechanism provides insight into the mechanism of ATRi/CHKi synergy with PARPi.

Figure 3. CHK1i and ATRi override cell cycle arrest induced by PARPi in BRCA mutant cells.

Cell cycle analysis was performed with respective drug treatments and scatter plots of newly synthesized DNA content (FITC-conjugated anti-BrdU antibodies) versus total DNA content (PI) is shown. PEO1 was plated at 50K cells/well in a 6 well plate and incubated with PARPi (AZD2281) 1μM, CHK1i (MK8776) 1μM, and ATRi (AZD6738) 1μM alone and in combination with PARPi for 48 hrs. With PARPi treatment, 44.03±1.02% of cells were arrested at G2-M phase compared to 12.76±0.45% of control (p<0.0001). ATRi also significantly arrested cells at G2-M phase (30.13±0.51%; Ctrl vs ATRi, p<0.0001). CHK1i slightly increased the S phase population (42.67±0.51%; Ctrl vs CHK1i, p=0.0002) compared to control (38.13±0.21%). When PARPi treated cells were also exposed to CHK1i or ATRi, 21.60% and 13.43% of the G2-M arrested population was released, respectively (p<0.0001 PARPi vs PARPi+CHK1; p<0.0001 PARPi vs PARPi+ATRi).

We hypothesized that cells treated with the ATRi/CHK1i-PARPi combination sustain significant DNA damage and some of these cells were permitted to progress through G2 and M phase due to ATR-CHK1 pathway inhibition. Indeed, the mechanism of synergy may at least partly involve progression into M phase with chromosome breaks. If so, then a synergistic increase in breaks and chromosome abnormalities in mitosis should be observed when ATRi/CHK1i treatments are added to PARPi treatment. Thus, we tested the effects of these drugs singly and combined on chromosomal breaks, gaps and aberrations by metaphase chromosome spreads in BRCA^MUT cells (Figure 4).

Figure 4. CHKi and ATRi synergize with PARPi to cause chromatid breaks chromosome aberrations.

Drug effects on DNA damage was measured by metaphase chromosome spread as shown by chromosomal gaps and breaks and aberations scoring. PEO1 cells were plated at 500K cells and incubated with 1μM PARPi (AZD2281) , 1μM CHK1i (MK8776), or 1μM ATRi (AZD6738) as well as with both PARPi and CHK1i or PARPi and ATRi for 14 hr. Nocodazole (0.5 μM) was added for 3hr prior to harvest. Cells were incubated in KCl, then fixed, dropped on glass slides, and stained with Giemsa. (A) Green arrows show either gaps and breaks (see inserted image in CHK1i box) or chromosomal aberrations (including interchanges [see inserted image in PARPi box] and inter-arm interchanges [see inserted image in ATRi box]).

(B) Gaps and breaks of chromosomes were counted (50 metaphase spreads in each group were counted and average number of gaps and breaks/cell was calculated, Graph depicts two independent experiments. Treatment with ATRi increased chromosomal gaps and breaks from an average 0.84±0.76/cell (untreated) to 4.91±0.57/cell with ATRi (p=0.0132). Combination therapy with PARPi and ATRi increased gaps and breaks (15.67±0.72) more than ATRi alone 4.91±0.57/cell (p=0.019). Chromosome aberrations were increased specifically by PARPi compared to control group (PARPi 0.1±0.028 vs Control 0.01±0.007, P=0.019). Both ATRi and CHK1i increased PARPi induced chromosome aberrations (PARPi+CHK1i 0.27±0.049 vs PARPi alone 0.1±0.028, p=0.032; PARPi+ATRi 0.19±0.028 vs PARPi alone 0.1±0.028, p=0.07).

ATRi treatment alone significantly increased gaps and breaks (5/cell) relative to untreated BRCA^MUT cells (2/cell), consistent with prior reports of the effect of ATR suppression(15,31,32). PARPi or CHK1i alone had minimal effects at the doses tested. However, chromosomal aberrations, in which the DSBs have been incorrectly repaired, were increased with both ATRi-PARPi and CHK1i-PARPi combinations (Figure 4B). Moreover, the combination PARPi–ATRi treatment caused 3x more gaps and breaks than ATRi monotherapy (Figure 4B), and such breaks appearing in mitosis is indicative of unrepaired DNA DSBs entering inappropriately into M phase. Therefore, particularly in the case of ATRi, PARPI treatment in combination with checkpoint abrogation increases the incidence of chromosome damage in metaphase, which causes cell lethality through abnormal partitioning of damaged and under-replicated DNA into daughter cells, a process known as mitotic catastrophe(33).

Targeting ATR/CHK1 with PARPi increases apoptosis

Given that combination therapy resulted in increased DNA damage compared to monotherapy in BRCA^MUT cells, the effects on apoptosis using Annexin V, propidium iodide (PI) and cleaved caspase 3 were then evaluated. PARPi and CHK1i each alone increased early (Annexin V positive) and late apoptosis (PI positive) by ~2-fold in the BRCA^MUTcells without an additional increase when in combination (Figure 5B). This combination did not induce apoptosis (by Annexin V or PI) in HR-proficient cells. PARPi and ATRi each alone increased early/late apoptosis ~2 and 4-fold from control, respectively in BRCA^MUT cells (Figure 5C). Combination PARPi-ATRi treatment, increased apoptosis 2-fold from PARPi alone in BRCA^MUT cells. When similar drug concentrations were tested in HR-proficient cells, apoptosis increased minimally with monotherapy but 2-fold with combination PARPi-ATRi (Figure 5C). When higher concentrations of PARPi was tested (5uM), apoptosis increased 3-fold to 64% with the addition of ATRi to PARPi compared to PARPi alone (23%) and ATRi alone (19%; Supplemental Figure 4) correlating with cell cycle findings where G2/M is increased suggesting cells are unable to repair DNA. Caspase-3, a protein activated in the apoptotic cell both by extrinsic (death ligand) and intrinsic (mitochondrial) pathways, is another marker that was evaluated(34,35). In BRCA^MUT and HR-proficient cells, treatment with CHK1i and ATRi increased cleaved caspase 3 compared to control (Figure 5D). Combination treatments did not substantially increase this protein compared monotherapy.

Figure 5.

(A) Annexin-V staining was used to identify cells in early apoptosis and propidium iodide (PI) was used to identify cells in late apoptosis. Results of apoptosis analysis highlighting number of cells in both early and late apoptosis are shown in PEO1 and PEO4. Cells were plated at 50K cells/well in 6 well plates and incubated in their respective drug concentrations for 48 hrs. Only Annexin-V positive cells with either both high and low PI signals were counted and shown. (B) PEO1 (BRCA2^MUT) cells demonstrated increased apoptosis by PARPi (9.74±0.49) and CHK1i (8.89±0.97) treatment compared to controls (4.23±0.55) (Ctrl vs PARPi, p=0.001, Ctrl vs CHK1i p=0.001, control vs both p=0.001). Relative to either agent alone, there was no difference in number of apoptotic cells with combination treatment (9.01±0.18). At concentrations tested in PEO4, there was not an increase in apoptosis. (C) In PEO1, apoptosis was increased by ATRi (15.93±1.62) compared to control (4.23±0.55; ctrl vs ATRi p=0.001) and when used in combination with PARPi there was about a 10% increase (20.67±1.67) compared to PARPi alone (9.74±0.49; *p=0.008) and about a 5% increase compared to ATRi alone (15.93±1.62; **p=0.03). For PEO4, there was only a significant increase in apoptosis with combination compared to control (^ p= 0.001). (D) BRCA2^MUT (PEO1) and BRCA2^REV (PEO4) cells were treated with PARPi 1μM, CHK1i 1μM, and ATRi 1μM and lysates were collected after 48-hours of treatment. Cleaved-Caspase3, apoptosis marker was evaluated by western blot. In PEO1 (BRCA2^MUT) cells apoptosis was increased by both ATRi alone (2.48±0.02 fold; p=0.01) and ATRi combination with PARPi (2.78±0.18 fold; p=0.01). Cleaved-Caspase3 was significantly increased by CHK1 alone (1.41±0.07 fold; p=0.05) and CHK1 combination with PARPi (1.43±0.03 fold; p=0.05). PEO4 cells showed an increase in cleaved caspase 3 protein with ATRi by 1.7 fold (P=0.0046) and 1.6 fold with combination of ATRi and PARPi (p=0.0076). Cleaved-Caspase3 was not significantly increased by PARPi or CHK1 alone.

Combination therapy is more effective than PARPi alone in a BRCA2 mutant PDX modes

We next tested whether the synergistic increases in genomic instability and cell death resulting from ATRi/CHK1i combinations with PARPI would be reflected in increased therapeutic efficacy. To test this, we utilized the best known animal model of human ovarian cancer progression, genetics, and response to therapy: the orthotopic PDXs.(24,36,37) While some tumor growth suppression was observed with PARPi and CHKi as single agents, the addition of ATRi/CHK1i to PARPi in a BRCA2-mutant PDX model led to a statistically significant decrease in tumor volume relative to single agent therapies (Figure 6A). Notably, significant differences were observed in responsiveness to the PARPi-ATRi and PARPi-CHK1i combinations. Although PARPi-CHK1i combination indeed led to significantly increased tumor suppression over single agent treatments, PARPi-ATRi led to a significant increase in the incidence of tumor regression When looking at individual responses in each group using the RECIST 1.1 score(38), 57 % of mice had a complete response (CR) in the PARPi and ATRi combination group compared to only 14% (1 mouse) in the PARPi and CHK1i combination group (Figure 6C). There were no CRs using single agent therapy (Figure 6B). Toxicity was acceptable as mouse weights were comparable in the PARPi-ATRi and PARPi-CHK1i treatment arms to the control vehicle arm. Although not associated with obvious gastrointestinal symptoms such as weight loss, abdominal distension or death, we did observe increased bowel dilatation at necropsy for the PARPi-CHK1i group compared to the control arm. These findings indicate that checkpoint abrogation, particularly ATRi, synergizes with PARPi to promote tumor suppression and regression in BRCA1^MUT tumors in an orthotopic PDX model.

Figure 6. Combination ATRi or CHK1i with PARPi is more effective than PARPi alone in a BRCA mutant PDX model (A, B).

To investigate the in vivo impact of drugs, tumor was orthotopically transplanted onto the fallopian tube/ovary of 5-8wk NSG mice and monitored weekly until tumor volume reached 70-100mm³. Mice were randomized into the following treatment groups: control, PARPi, CHK1i, ATRi, PARPi+CHK1i, PARPi+ATRi. Treatment with PARPi (AZD2281) 50mg/kg by oral gavage daily, CHK1i (MK8776) 50mg/kg IP every 3 days, ATRi (AZD6738) 25mg/kg D1-3 Q week continued for 6-7 weeks and tumor volume was measure with weekly ultrasound. (A) The addition of CHK1i to PARPi lead to a significant decrease in tumor volume relative to single agent therapy (p=0.02 for PARPi vs PARPi and CHK1i) There was synergy for combination compared to CHK1i alone (Combination Ratio=1.76). (B) The results with the addition of ATRi to PARPi was also synergistic (p=0.003 for PARPi vs PARPi and ATRi; Combination Ratio=3.20) (C) The RECIST score as calculated by percent change in tumor volume at the end of treatment compared to the starting tumor volume. A change of −100% was complete remission (CR), between −100% and −30% was a partial remission (PR), between −30% and +20% was stable disease (SD), and over +20% was progressive disease (PD). When looking at individual responses in each group, 57.1% of mice had a CR in the PARPi and ATRi combination group compared to only 14.3% (1 mouse) in the PARPi and CHK1i combination group. There was no CR with single agent therapy. (D) To study the effects of single agent and combination therapy in vivo on the ATR/CHK1 axis, mice from each group were sacrificed after 1 wk of treatment approximately 6h after drug treatment. Lysates were immunoblotted for the indicated proteins and phosphoproteins. There was an increase in p-CHK1 in the CHK1i group confirming drug target. There was also a slight increase in pCHK1 in the PARPi group which was overcome with the addition of ATRi by 3 fold (p=.0004 PARPi vs PARPi and CHK1i). There was not an increase in rH2AX noted at treatment after 7 days.

Consistent with our observations (Figs. 1-5) and prior reports of the stimulatory effect of CHK1i on CHK1 phosphorylation(10,30). PDX tumors, evaluated after 1 week of treatment, exhibited an increase in p-CHK1 in mice treated with PARPi and CHK1i as single agents. Furthermore, PARPi-ATRi, decreased pCHK1 compared to PARPi monotherapy (Figure 6D; Supplemental Figure 5). Thus, the drugs recapitulated our cell culture observations and expected effects, indicating that they maintained access to orthotopic tumors in vivo.

DISCUSSION

Capitalizing on synthetic lethality, PARP inhibitors (PARPi) have proven their clinical potential in treating cancer, both in BRCA mutated and wild-types. Olaparib, currently the only FDA approved PARPi, results in a 40% and 30% response rate for recurrent BRCA^MUT and wild-type HGSOC, respectively, after first-line carboplatin-taxane standard of care(7,39). Unfortunately, such responses are short lived, most lasting only 5-7 months with complete responses occurring rarely (2%)(7,39). Using our orthotopic mouse model, we have demonstrated that PARPi treatment alone can suppress tumor growth at the maximum tolerated dose in a BRCA mutant PDX model, but, similar to the clinical setting, it does not completely eliminate tumor burden despite prolonged treatment (Figure 1C). Thus, strategies to optimize PARPi therapies for ovarian cancer are needed. The purpose of our study was to increase the efficacy of PARP inhibition by targeting critical cell-cycle checkpoints that are relied upon for cell survival during PARPi treatment.

Herein, we demonstrate that PARPi treatment increases reliance on the ATR-CHK1 pathway for genome stabilization and survival of BRCA mutant cells. Indeed, combination of PARPi with ATRi or CHK1i treatment synergistically decreases cell viability and colony formation of BRCA mutant cells, and to a less extent, HR-proficient cells. We propose that the synergistic effect of CHK1i or ATRi when combined with PARPi results both from 1) an increase in replication fork collapse by loss of two independent fork-stabilizing mechanisms that are controlled by CHK1/ATR and PARPi, and 2) loss of the G2-M phase checkpoint, which permits cells with this high level of DSBs to enter mitosis prematurely. The inability to appropriately partition broken chromatid fragments symmetrically dramatically increases cell death, a process known as mitotic catastrophe (Supplemental Figure 6). Apoptosis either in G2 or after mitotic catastrophe can be activated by a variety of DSB-sensing mechanisms, including those regulated by ATM.

However, key differences were observed between the PARPi-ATRi and PARPi-CHKi combinations on genome stability and survival of BRCA mutant tumor cells and PDX tumors. While the PARPi-CHKi combination was well tolerated PDX mice and resulted in tumor suppression in BRCA2-mutant orthotopic transplant model (Fig. 6), this combination did not lead to tumor regression. In contrast, the PARPi -ATRi combination resulted in tumor regression and eradication of BRCA^MUT ovarian cancer PDX tumors. The dosing regimen studied, continuous PARPi with Day 1-3 ATRi, was well tolerated in vivo, as evidenced by weight stability over the treatment course. In contrast, apoptosis was significantly increased with the ATRi-PARP1i combination compared to monotherapy in both BRCA mutant and HR-proficient cell models (Figure 5 and Supplemental Figure 4).

The underlying causes of this clinically relevant difference may be best surmised from the distinct signaling roles of these kinases and there effects on genome stabilization when combined with PARPi. The ATR kinase lies upstream of CHK1 and phosphorylates numerous factors that may help preserve replication fork stability and control cell cycle progression. The direct substrates of ATR include RPA, CLSPN, MCM2, p53 and many other factors that play roles in replication fork progression, DNA repair, and the cell cycle(19,20). Thus, ATR may be able to stabilize replication forks independent of CHK1(40), and permit cell survival when CHK1 is inhibited(41). Additionally, ATR can suppress origin firing and the intra-S checkpoint independent of CHK1(42,43). Consistent with these interpretations, ATRi in combination with PARPi caused a substantial increase in chromatid breaks in M phase, a phenotype that represent unrepaired DSBs being permitted to enter M phase inappropriately (Figure 4). In contrast, the appearance of chromosome aberrations in either PARPi-ATRi or PARPi-CHKi implies inappropriate repair of DSBs before entry into mitosis, and such capping of DSB ends would be expected to suppress alternative DSB-stimulated checkpoint pathways. Therefore, the more substantial effects of the PARPi-ATRi combination on tumor progression likely results from the sum combination of increased replication fork collapse and abrogation of the G2-M phase pathways, as described in more detail in the following paragraph. Additional research is required to further dissect the effect of PARPi-ATRi on genome stability and cancer cell survival, which may also depend on the genetics of the tumor.

Although differences in the efficacy of PARPi-ATRi and PARPi-CHKi were observed, each of these combinations demonstrated significantly improved treatment efficacy over the application of any single agent. The mechanism behind this improvement is likely rooted in the distinct functions of PARP and ATR-CHK1 in preserving genome integrity. PARP helps ligate single-stranded breaks (SSBs), which occur spontaneously at 20,000-50,000 sites per genome per day(5,29). When left unrepaired because of PARPi treatment, these SSBs are converted into DSBs during DNA replication(5,29). In contrast, ATR prevents DSB formation by making the replication fork less vulnerable to endonuclease attack(20,42,44). The additive, or possibly synergistic, effects of inhibiting these distinct pathways are further exacerbated by suppression of G2-M phase cell cycle control by ATR-CHK1 pathway inhibition, leading to mitotic catastrophe (Figure 4,5).Therefore, inhibition of ATR-CHK1 and PARP together increases DSB generation from fork collapse, which results either in elevated apoptosis in S/G2 phase from other DSB-sensing mechanisms, or mitotic catastrophe through cell cycle checkpoint abrogation through ATR-CHK1 suppression. These mechanisms help explain the effects of PARPi-CHK1i and PARPi-ATRi combinations on tumor suppression, and in the case of PARP-ATRi, tumor regression.

In summary, we have shown that PARPi increases reliance on ATR/CHK1 for genome stability and that the combination of PARPi with ATRi leads to complete ovarian tumor regression in an HR-deficient PDX model. Such responsiveness is not achievable with the maximum dose of PARPi alone, which is in accord with response rates to PARPi single-agent therapy in the clinic. Our goal is to convert the partial tumor responses typically seen with PARPi monotherapy into durable complete regressions using the combination of PARPi plus ATRi. AZD6738, a selective and bio-available ATRi, is being investigated in early phase clinical trials as monotherapy or in combination with chemotherapy or radiation therapy (clinicaltrials.gov). Preliminary studies investigating AZD6738 as a monotherapy in the clinic show it is tolerable and demonstrates anti-tumor efficacy(45). PARPi in combination with ATRi will be evaluated in ovarian cancer patients in the near future.

Statement of Translational Relevance.

Strategies to increase the efficacy of PARP inhibitors (PARPi) are needed given the rare complete tumor responses demonstrated in ovarian cancer. We describe the pre-clinical efficacy of a novel therapeutic combination of PARPi with ATR/CHK1 blockade using an orthotopic ovarian cancer patient-derived xenograft (PDX) model. Our study shows that PARPi treatment increases reliance on ATR/CHK1 for survival and ATRi or CHK1i in combination with PARPi is synergistic in decreasing survival and colony formation compared to PARPi alone in BRCA mutant and wild-type cells. The addition of ATRi or CHK1i to PARPi resulted in a G2 release with increased chromosomal aberrations, and apoptosis in BRCA mutant cells. PARPi with CHK1i caused tumor suppression however, PARPi with ATRi caused tumor regression and, in most cases, complete remission in a BRCA mutant PDX. This study supports evaluation of ATR/CHK1i with PARPi in the clinic.