Inhibition of Wee1 sensitizes AML cells to ATR inhibitor VE-822-induced DNA damage and apoptosis

Accepted Manuscript Inhibition of Wee1 sensitizes AML cells to ATR inhibitor VE-822-induced DNA damage and apoptosis Wenxiu Qi, Xiaohao Xu, Manying Wang, Li Xiangyan, Chaonan Wang, Liping Sun, Daqing Zhao, Liwei Sun PII: DOI: Reference:

S0006-2952(19)30157-1 https://doi.org/10.1016/j.bcp.2019.04.022 BCP 13505

To appear in:

Biochemical Pharmacology

Received Date: Accepted Date:

22 February 2019 19 April 2019

Please cite this article as: W. Qi, X. Xu, M. Wang, L. Xiangyan, C. Wang, L. Sun, D. Zhao, L. Sun, Inhibition of Wee1 sensitizes AML cells to ATR inhibitor VE-822-induced DNA damage and apoptosis, Biochemical Pharmacology (2019), doi: https://doi.org/10.1016/j.bcp.2019.04.022

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Inhibition of Wee1 sensitizes AML cells to ATR inhibitor VE-822-induced DNA damage and apoptosis Wenxiu Qi*1, Xiaohao Xu*2, Manying Wang2, Li Xiangyan1, Chaonan Wang1, Liping Sun1, Daqing Zhao#1, Liwei Sun#2

1

Jilin Provincial Key Laboratory of BioMacromolecules of Chinese Medicine, Jilin

Ginseng Academy, Changchun University of Chinese Medicine, Changchun, Jilin, China 2

Research Center of Traditional Chinese Medicine, the Affiliated Hospital to

Changchun University of Chinese Medicine, Changchun, Jilin, China

*Both authors contributed equally to this work.

Corresponding author: #Daqing

Zhao, Jilin Provincial Key Laboratory of BioMacromolecules of Chinese

Medicine, Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun, Jilin, China. Tel: +86-431-86763803, [email protected] Or #Liwei

Sun, Research Center of Traditional Chinese Medicine, the Affiliated Hospital

to Changchun University of Chinese Medicine, Changchun, Jilin, China. Tel: +86431-86177630; E-mail: [email protected] Conflict of interest disclosure: The authors declare no competing financial interests. 1

ABSTRACT Resistance to standard induction therapy and relapse remain the primary challenges for improving therapeutic effects in acute myeloid leukemia (AML); thus, novel therapeutic strategies are urgently required. Ataxia telangiectasia and Rad3related protein (ATR) is a key regulator of different types of DNA damage, which is crucial for the maintenance of genomic integrity. The ATR-selective inhibitor VE-822 has proper solubility, potency, and pharmacokinetic properties. In this study, we investigated the anti-leukemic effects of VE-822 alone or combined with Wee1selective inhibitor AZD1775 in AML cells. Our results showed that VE-822 inhibited AML cell proliferation and induced apoptosis in a dose-dependent manner. AZD1775 significantly promoted VE-822-induced inhibition of AML cell proliferation and led to a decreased number of cells in the G2/M phase. VE-822 and AZD1775 decreased the protein levels of ribonucleotide reductase M1 (RRM1) and M2 (RRM2) subunits, key enzymes in the synthesis of deoxyribonucleoside triphosphate, which increased DNA replication stress. VE-822 combined with AZD1775 synergistically induced AML cell apoptosis and led to replication stress and DNA damage in AML cell lines. Our study demonstrated that AZD1775 synergistically promotes VE-822-induced anti-leukemic activity in AML cell lines and provides support for clinical research on VE-822 in combination with AZD1775 for the treatment of AML patients. Keywords: acute myeloid leukemia, ATR, VE-822, combination treatment, DNA damage 1. Introduction 2

Acute myeloid leukemia (AML) is a type of cancer characterized by uncontrolled clonal proliferation of abnormal myeloid progenitor cells in the bone marrow and blood, accompanied by the disruption of normal hematopoiesis and production of blood cells (1). Overall survival rates are 25% and 65% in the adult and pediatric population, respectively (2, 3). Standard induction therapy for AML consists of cytarabine (Ara-C) and daunorubicin (DNR); however, resistance to these chemotherapeutic agents remains the primary cause of treatment failure (4, 5). Therefore, novel effective and less toxic therapeutic methods are urgently required to improve the curative effects of AML. One mechanism of chemotherapy resistance is the increasing of DNA damage response (DDR) (6-8). Ataxia telangiectasia mutated and Ataxia telangiectasia and Rad3-related protein (ATR) is a key regulator of the cellular response to various types of DNA damage and is crucial for the maintenance of genomic integrity (9-11). Cell survival following DNA damage depends on activating checkpoints to prevent cell proliferation(12). Most cancer cells have dysregulation of the G1 phase checkpoint, making them rely primarily on S and G2/M phase checkpoints, which are activated by ATR/checkpoint kinase 1 (CHK1) signaling (13, 14). Therefore, ATR inhibition is considered as a target for preventing DNA damage repair in cancer cells (15-19). VE822 is a close analog of VE-821 with a marked increase in potency against ATR and good solubility, potency, selectivity, and pharmacokinetic properties (20). Wee1 is an important kinase for G2/M cell cycle checkpoint arrest, as its activation induces cell cycle arrest at the G2/M phase for DNA repair before mitotic 3

entry(21-23). AZD1775, a selective molecular inhibitor of Wee1, induces DNA damage and apoptosis in AML cell lines and other cancer cells (24-28). In clinical trials, AZD1775 was safe and tolerable as a single agent, and AZD1775 in combination with paclitaxel, carboplatin, gemcitabine, or cisplatin was more effective than AZD1775 alone for the treatment of solid tumors (www.clinicaltrials.gov, 29-31). In Allan’s report, they suggested that there was a synergistic interaction between ATR inhibitor VX-821 and RNR loss (by gemcitabine or hydroxyurea) for the treatment of AML (32). In Guan’s article, they showed that they identified the synergistic effect between AZD1775 and the ATR kinase inhibitor AZD6738 is a promising strategy for triple negative breast cancer (33). Thus, we hypothesized that the combination of VE822 and AZD1775 would lead to more DNA damage and synergistic anti-leukemic activity in AML cells. In this study, we used AML cell lines to evaluate the combined effects of VE822 and AZD1775. We found that VE-822 induced inhibition of cell proliferation and apoptosis, and the sensitivity of VE-822 differed in the various cell lines tested. AZD1775 synergistically promoted VE-822-mediated inhibition of AML cell proliferation and led to mitochondrial dysfunction. Furthermore, this combination led to downregulation of Wee1, phosphorylation of Cdc25C (p-CDC25C), ribonucleotide reductase M1 (RRM1), and M2 (RRM2) subunits, and induction of DNA replication stress and apoptosis. In addition, combined treatment with VE-822 and AZD1775 resulted in the increased expression of gamma-histone 2A family member X, demonstrating that treatment with ATR and Wee1 inhibitors cooperatively induced 4

more DNA damage, giving rise to more apoptosis in AML cells. The results of this study provide a potential mechanism underlying the synergistic anti-leukemic activities of VE-822 in combination with AZD1775.

2. Materials and Methods 2.1 Drugs VE-822 and AZD1775 were purchased from Selleck Chemicals (Houston, TX, USA). 2.2 Cell culture The human AML cell lines THP-1 and U937 were purchased from the American Type Culture Collection (Manassas, VA, USA). MOLM-13 cells were purchased from AddexBio (San Diego, CA, USA). These cell lines were cultured in RPMI-1640 medium with 10% fetal bovine serum (CLARK Bioscience, Claymont, DE, USA), 2 mM L-glutamine (Biosharp, Hefei, China), 100 U/mL penicillin (Biosharp), and 100 µg/mL streptomycin (Biosharp). All cells were cultured in a 37°C humidified atmosphere containing 5% CO2 and 95% air. 2.3 Cytotoxicity assays The cytotoxicity of AML cells was measured by the MTT (Sigma-Aldrich, St. Louis, MO, USA) assay. The cells were treated with variable concentrations of VE822, AZD1775, or a combination of both drugs for 72 h. MTT was added at a final concentration of 0.5 mg/mL, and cells were incubated at 37°C for 4 h. The cells were lysed overnight in 10% sodium dodecyl sulfate (SDS) in 10 mM HCl, and plates were 5

read at 590 nm using a microplate reader (Infinite M200pro; Tecan Life Sciences Zurich, Switzerland). IC50 values were calculated as the drug concentrations necessary to inhibit 50% growth compared to control untreated cells. The IC50 values for the cell lines are presented as the mean ± standard error of the mean (SEM) from at least three independent experiments. The combination index (CI) values were calculated using CalcuSyn 2.0 software (Biosoft, Cambridge, United Kingdom). CI < 0.9 indicated synergistic, 0.9 < CI < 1.1 indicated additive, and CI > 1.1 indicated antagonistic antileukemia interactions. 2.4 Lactate dehydrogenase release assay After the AML cells were treated with VE-822 and/or AZD1775 for 24 h, they were centrifuged at 2000 rpm for 5 min, and the supernatant was collected for the subsequent lactate dehydrogenase (LDH) release test using the LDH Release Assay Kit (Beyotime Institute of Biotechnology, Jiangsu, China). According to the manufacturer’s instructions, the indicated agents were added to a 96-well plate and incubated at 37°C for 30 min, after which the plate was read at 490 nm using a microplate reader (Tecan). 2.5 Mitochondrial membrane potential AML cell lines were cultured in a 12-well plate (3×105/mL, 2 mL/well) and treated with VE-822, AZD1775, or a combination of both drugs for 24 h. Then cells were washed twice with phosphate-buffered saline (PBS), stained with 1× JC-1 (Beyotime) in the dark at 37°C for 30 min, washed twice, and analyzed with a flow cytometer (BD Biosciences, San Jose, CA, USA) within 30 min (34). 6

2.6 Annexin V-FITC/propidium iodide staining AML cells were treated with specific drug concentrations in a 12-well plate (3×105/mL, 2 mL/well) for different time periods, washed with PBS, resuspended in 50 µL Annexin V-FITC Binding Buffer (BD Biosciences), incubated with 3 µL Annexin V-FITC for 15 min at room temperature (RT), and then incubated with 6 µL propidium iodide (PI) for another 5 min at RT followed by analysis on a FACS Calibur Flow Cytometer (BD Biosciences). CI values were calculated using CalcuSyn 2.0 software (Biosoft). 2.7 Cell cycle progression AML cells were treated with specific drug concentrations in a 6-well plate (3×105/mL, 4 mL/well) for 24 h. The cells were harvested and fixed in ice-cold 75% (v/v) ethanol overnight. Then they were pelleted, washed with PBS, re-suspended in 300–500 µL PI/RNase Staining Buffer (BD Biosciences), and incubated in the dark for 15 min. DNA content was determined by flow cytometry (BD Biosciences), and cell cycle analysis was performed using FlowJo v7.6.5 software (Tree Star, Ashland, OR, USA). 2.8 Western blot analysis Cells were lysed with RIPA buffer (Beyotime) containing protease and phosphatase inhibitors (Roche Applied Sciences, Shanghai, China). Soluble proteins were subjected to SDS-polyacrylamide gel electrophoresis. The proteins were electrophoretically transferred onto Immobilon-PSQ PVDF membranes (Merck, Darmstadt, Germany). After blocking in 5% non-fat milk, membranes were 7

immunoblotted with the following antibodies: poly (ADP-ribose) polymerase 1 (PARP-1, #9542), p-CDK1 (Y15, #9111), caspase-3 (#9661), and γH2AX (#2577) (all from Cell Signaling Technology, Danvers, MA, USA); RRM1 (ab137114), RRM2 (ab172476), p-CDC25C (S216, ab32051), and p-CDK2 (Y15, ab76146) (all from Abcam, Cambridge, MA, USA); and CDK1 (19532-1-AP), CDK2 (10122-1AP), and GAPDH as a loading control (60004-1-Ig) (all from Proteintech, Wuhan, China). After incubation with appropriate secondary antibodies (Li-Cor, Lincoln, NE, USA) for 1 h at RT, protein bands were visualized using the Odyssey Infrared Imaging System (Li-Cor), as described by the manufacturer. Densitometry measurements were made using Odyssey V2.0 (Li-Cor), normalized to GAPDH, and graphed as fold change compared to the control. 2.9 Comet assay THP-1 and MOLM-13 cells were treated with VE-822 and/or AZD1775 for 8 or 16 h, and then subjected to the alkaline comet assay as previously described (35, 36). Slides were stained with 1 mL SYBR™ Gold Nucleic Acid Gel Stain (Invitrogen™, Carlsbad, CA, USA) for 30 min, and then imaged on an Olympus microscope (Olympus America Inc., Center Valley, PA, USA). At least 50 comets/gel were scored by CometScore software (TriTek Corp., Sumerduck, VA, USA). The median percentage of DNA in the tail was calculated and expressed as the mean ± SEM by GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). 2.10 Chromatin fractionation

8

THP-1 and MOLM-13 cells (3×105/mL) were treated with VE-822 or/and AZD1775 at the indicated concentrations for 24 h. Chromatin fractionation was performed as previously described (37, 38). Briefly, AML cells were washed with PBS and resuspended in solution A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM Na2VO3,1 mM DTT, 10 mM NaF and protease inhibitors) with Triton X-100 (final concentration of 0.1%), and then incubated on ice for 5 min. Nuclei were separated from cytoplasmic proteins by centrifugation at 2000 × g for 4 min and then washed with solution A three times. Then nuclei were lysed in 3 mM EDTA, 1 mM DTT, 0.2 mM EGTA, and protease inhibitors (dissolved in water) for 30 min at 4°C. Chromatin was separated from soluble nuclear proteins by centrifugation at 2500 × g for 4 min. Soluble nuclear proteins were combined with cytoplasmic proteins (designated soluble fraction). Chromatin was washed three times with nuclei lysis buffer (centrifugation was conducted at 2500 × g for 4 min). Chromatin was resuspended in 200 µL laemmli sample buffer and sonicated. 2.11 Statistical analysis Differences were compared using the pair-wise two-sample t-test. Statistical analyses were performed using GraphPad Prism 5.0. Error bars represent the SEM. The level of significance was set at p<0.05.

3. Results 3.1 VE-822 induces AML cell proliferation inhibition and apoptosis in a dose9

dependent manner VE-822 is a potent and selective ATR inhibitor with a molecular weight of 463.55. Its chemical structure is shown in Fig. 1A. To investigate the effects of VE822 on the proliferation of AML cell lines, we used MTT assays to determine the sensitivity of VE-822 in a panel of AML cell lines, including MV4-11, HL-60, MOLM-13, THP-1, OCL-AML3, and U937, after 72 h of treatment. As shown in Fig. 1B and C, VE-822 treatment caused growth inhibition in a concentration-dependent manner with IC50 values ranging from 0.2 µM in MV4-11 cells to 1.3 µM in U937 cells. Among these AML cell lines, THP-1 (French-American-British classification, FAB M5; mutated TP53; wild type FLT3 (FMS-like tyrosine kinase 3) and with Rad51, Brca1 and Chk1 expression) and MOLM-13 (FAB M5; wild type TP53; mutated FLT3-ITD (internal tandem duplication, with poor prognosis); with Rad51 and Chk1 expression were chosen as the main cell models for our studies) (35, 39-42). To determine if VE-822 induced cell apoptosis, we treated THP-1 and MOLM-13 cells with VE-822 for up to 4 µM for 24 h. VE-822 treatment induced concentrationdependent apoptosis from 6.9% to 16.1% in THP-1 cells and 8.9% to 46.2% in MOLM-13 cells, as demonstrated by an increase in Annexin V-FITC-positive cells (Fig. 1D). These results showed that VE-822 decreased AML cell proliferation and induced apoptosis in a dose-dependent manner. 3.2 AZD1775 synergistically promotes the effect of VE-822 on inhibiting cell proliferation and leads to mitochondrial dysfunction in AML cells To examine the effects of AZD1775 on VE-822-induced inhibition of AML cell 10

proliferation, we assessed VE-822 and AZD1775 sensitivities using MTT assays after 72 h of treatment. We assessed 0.25, 0.5, 1, and 2 µM VE-822 in THP-1 cells and 0.25, 0.5, and 1 µM VE-822 in MOLM-13 cells. The results showed a decrease in viable cells with increasing concentrations of VE-822 and AZD1775 alone or in combination (Fig. 2A-2B). We also confirmed that VE-822 and AZD1775 showed additive or synergistic anti-leukemia activities in THP-1 and MOLM-13 cells. The combined treatment resulted in a synergistic anti-leukemia interaction, as determined by standard isobologram analyses. Points falling below the line indicate synergism whereas those above the line indicate antagonism (Fig. 2C-2D). To explore the reason of VE-822- and AZD1775-induced cytotoxicity in AML cells, we tested the LDH release rate. LDH is a stable protein in the cytoplasm of normal cells. Once the cell membrane is damaged, it is released into the extracellular environment. Therefore, the leakage rate of LDH can be used to evaluate cell membrane integrity. AZD1775 (500 nM in THP-1 cells, 250 nM in MOLM-13 cells) significantly increased VE-822induced LDH release compared to the control in THP-1 and MOLM-13 cells (1 µM in THP-1 cells, 0.5 µM in MOLM-13 cells; Fig. 2E). We also detected the mitochondrial membrane potential (MMP), which is used to detect early apoptosis. The results showed that compared to VE-822 treatment, VE-822 combined with AZD1775 increased JC-1 monomer-positive cells at 24 h from 12.7% to 30.2% in THP-1 cells and from 25.0% to 49.3% in MOLM-13 cells (Fig. 2F). The scatter diagram of JC-1 monomer-positive cells is shown as quadrant Q1-LR in Fig. 2G. These results indicated that AZD1775 enhanced the cytotoxicity of VE-822 by inducing LDH 11

release and mitochondrial dysfunction. 3.3 VE-822 synergizes with AZD1775 to induce AML cell DNA damage and cell cycle arrest To explore the mechanism of cell proliferation inhibition, we investigated the effects of VE-822 and AZD1775 on cell cycle progression in THP-1 and MOLM-13 cells by PI staining and flow cytometry analyses. Our results showed that in THP-1 cells, the percentage of cells in the S phase was 22.0%. VE-822 and AZD1775 induced S-phase arrest, which increased the percentage of cells to 28.5% and 29.1%, respectively. The combination of VE-822 and AZD1775 abrogated S-phase arrest, reducing the percentage of cells to only 11.5% in THP-1 cells (Fig. 3B). In MOLM13 cells, the percentage of cells in the G0/G1 phase without treatment was 54.7%, whereas VE-822 and AZD1775 induced G0/G1-phase arrest, increasing the percentage up to 69.4% and 58.3%, respectively. However, the combination of VE822 and AZD1775 eliminated G0/G1-phase arrest and decreased the percentage of MOLM-13 cells to 39.7% (Fig. 3C). Both VE-822 and AZD1775 treatment resulted in a significant decrease in the percentage of THP-1 cells in the G2/M phase from about 21.8% to 6.1% and the percentage of MOLM-13 cells from 20.1% to 2.4%. Treatment with AZD1775 also markedly increased VE-822-induced sub-G1 phase THP-1 cells from 9.0% to 50.5% and MOLM-13 cells from 12.4% to 54.8%, indicating that this drug combination induced apoptosis (Fig. 3A–3C). These cell cycle results demonstrated that the combined treatment resulted in cell cycle arrest, abrogation of the G2/M cell cycle checkpoint, and induction of apoptosis in AML 12

cells. To confirm the hypothesis that the combination of VE-822 and AZD1775 induced apoptosis in AML cells, we assessed Annexin V-FITC/PI staining in THP-1 and MOLM-13 cell lines. Compared to control or single drug treatment, the combined treatment of VE-822 and AZD1775 markedly increased Annexin V-positive cells to about 55.0% and 60.0% in THP-1 and MOLM-13 cells, respectively (Fig. 3D, 3E). The combination also resulted in synergistically induced apoptosis in THP-1 cells (CI<0.288) and MOLM-13 cells (CI<0.707) (CI value less than 0.9 indicates synergism). According to the detected apoptosis pathway-related proteins, we found that the combination of VE-822 and AZD1775 caused a significant increase in cleaved PARP and caspase-3 in THP-1 and MOLM-13 cells (Fig. 3F). These results indicated that AZD1775 promoted VE-822-induced apoptosis synergistically. 3.4 AZD1775 increases VE-822-induced DNA double-strand breaks γH2AX is a biomarker of double-strand breaks (DSBs). We found that AZD1775 (γH2AX protein expression after AZD1775 treatment was 1.30-fold in THP-1 cells and 1.28-fold in MOLM-13 cells) increased VE-822-induced γH2AX protein expression (1.77-fold in THP-1 cells and 1.46-fold in MOLM-13 cells), and the combination increased expression by 1.86-fold in THP-1 cells and 1.60-fold in MOLM-13 cells (Fig. 3F). To estimate the time sequence of apoptosis and DNA damage occurrence, time course experiments were performed in AML cell lines. Drug-treated cells were harvested at 4, 8, 12, 16, and 24 h and subjected to flow cytometry and western blot analysis (Data not shown the results of WB from 16 h to 24 h). Annexin V-FITC/PI staining revealed that VE-822 combined with AZD1775 13

significantly induced Annexin V-positive cells at 4 h in THP-1 cells and 8 h in MOLM-13 cells. The percentage of Annexin V-positive cells was increased over time. The scatter diagram of Annexin V-FITC/PI staining is shown in Fig. 4A and 4C. The combination of VE-822 and AZD1775 induced 49.1% Annexin V-positive cells in THP-1 cells and 74.2% Annexin V-positive cells in MOLM-13 cells at the 24 h time point (Fig. 4B and 4D). Western blot analysis was used to confirm the time sequence that apoptosis and DNA damage occurred. After treatment with VE-822 combined with AZD1775, an increase in cleaved PARP was detected at 4 h in THP-1 cells (4.78-fold) and at 8 h in MOLM-13 cells (4.55-fold), and increased γH2AX expression was found at 4 h in THP-1 cells (3.83-fold) and at 8 h in MOLM-13 cells (5.10-fold) (Fig. 4E). A decrease in RRM2 levels was detected at 8 h (0.58-fold), whereas a decrease in p-CDK1 (0.34-fold) and p-CDK2 (0.76-fold) was detected as early as 4 h in THP-1 cells. Similar protein level changes were detected after 4 h of treatment in MOLM-13 cells (Fig. 4E). Taken together, the combination of VE-822 and AZD1775 resulted in the increase in γH2AX expression, these results indicated that DNA damage and apoptosis occurred simultaneously. To confirm the effects of VE-822 and AZD1775-induced DNA damage, after combination treatment, we isolated chromatin from nuclei, and then subjected the cells to sonication and western blot analysis. In THP-1 cells, there was a substantial increase of chromatin-bound replication protein A 32 (RPA32, 14.09-fold) and γH2AX (8.74-fold) after combined treatment compared to treatment with AZD1775 alone (RPA32, 2.98-fold; γH2AX, 3.47-fold) or VE-822 alone (RPA32, 5.15-fold; 14

γH2AX, 3.08-fold), demonstrating that the combination treatment increased DNA replication stress and DNA damage (Fig. 5A). Similar results were observed in MOLM-13 cells, as compared to single drug treatment, the combination of VE-822 and AZD1775 induced an increase in RPA32 (17.37-fold) and γH2AX (19.40-fold) protein expression (Fig. 5B). To confirm that VE-822 and AZD1775 caused DNA damage, the comet assay was performed at 16 h. AZD1775 markedly promoted VE822-induced DSBs, as indicated by the increased percentage of DNA in the tail of THP-1 and MOLM-13 cells (Fig. 5C, 5D). After 16 h of treatment with VE-822 and/or AZD1775 in THP-1 cells, the percentage of DNA in the tail of the cells was 13.5% and 10.1%, respectively, whereas the combined treatment increased the percentage to 34.6% (Fig. 5E). After treatment with VE-822 and/or AZD1775 for 16 h in MOLM-13 cells, the percentage of DNA in the tail was 14.92% and 8.54%, respectively, and combination treatment significantly increased the percentage to 23.96% (Fig. 5F). These results showed that the combination of VE-822 with AZD1775 induced more DNA replication stress and DSBs in AML cells. 3.5 AZD1775 enhances VE-822-induced cell apoptosis primarily through downregulation of Wee1 and ribonucleotide reductase in AML cells The above-mentioned results showed that AZD1775 promoted VE-822-induced expression of γH2AX. Thus, next we investigated the effects of the AZD1775 and VE-822 combination on the downstream signaling of DNA damage repair. In THP-1 cells, VE-822 caused decreased phosphorylation of Cdc25C at serine 216 (p-Cdc25C, 0.79-fold) and CDK1 at tyrosine 15 (0.95-fold), confirming the inhibition of ATR. In 15

THP-1 cells, AZD1775 reduced the level of Wee1 (0.82-fold), RRM1 (0.82-fold), RRM2 (0.74-fold), p-CDK1 at tyrosine 15 (0.97-fold), and p-CDK2 at tyrosine-15 (0.76-fold). It was recently reported that inhibition of ATR causes a decrease in RRM2 expression (37, 43). In our study, both VE-822 and/or AZD1775 treatment caused downregulation both of RRM1 and RRM2. These results suggest that VE-822 and AZD1775 can affect the synthesis of dNTP by regulating ribonucleotide reductase (RNR), the key enzyme in nucleotide synthesis. The combination of VE822 and AZD1775 led to a further decrease of Wee1 (0.75-fold), p-Cdc25C (0.38fold), p-CDK1 (0.76-fold), p-CDK2 (0.38-fold), RRM1 (0.76-fold), and RRM2 (0.55fold) in THP-1 cells; similar results were found in MOLM-13 cells (Fig. 6A). These results demonstrated that the combination of VE-822 and AZD1775 caused DNA damage mainly through downregulation of Wee1, p-CDK1/2, RRM1, and RRM2 in AML cells.

4. Discussion Resistance to standard chemotherapy agents remains a major course of AML treatment failure, therefore, new therapeutic strategies should be found for improving treatment outcomes for AML patients. AML mainly affects the elderly population (44, 45). The prognosis of patients aged > 65 years is dismal, with 1-year overall survival rates of only 10% after traditional therapy method (46). Thus, novel therapies of rationally designed combination therapies are needed to enhance therapeutic effect for AML patients. Preclinical trials have shown that ATR inhibitors effectively sensitive 16

various cancer cells to gemcitabine, cisplatin, cytarabine, etoposide, PARP inhibitors, and ionizing radiation (38, 43, 47-51). Thus, suppression of ATR may have synergistic effects with DNA damaging agents in AML cell lines. Based on these studies, our results also showed that the combination of the ATR inhibitor VE-822 and of the Wee1 inhibitor AZD1775 was more effective than treatment with each drug alone in improving anti-AML cell activity. Previous studies have mainly focused on the role of ATR in cell cycle checkpoints (49, 52, 53). In contrast to those reports, we found that the novel ATR inhibitor VE-822 enhanced the anti-leukemic activity of AZD1775 primarily by abrogating the G2/M cell cycle checkpoint (Fig. 3A–3C) and by elevating AZD1775-induced DNA DSBs (Fig. 3F, 5A–5D). RNR is a key enzyme in dNTP synthesis and consists of subunits RRM1 and RRM2 (37, 54). In our study, we demonstrated that VE-822 caused AML cell apoptosis in a dose-dependent manner (Fig. 1D), and ATR inhibition caused more downregulation of RRM2 than of RRM1 (Fig. 4E). Pfister et al. showed that Wee1 inhibition selectively killed H3K36me3-deficient cancer cells through dNTP starvation resulting from a decrease in RRM2 levels (55). Buisson et al. showed that ATR suppression attenuated the accumulation of RRM2 in the S phase, suggesting that ATR-CHK1 pathway promotes RRM2 accumulation in cycling cells (37). Our results confirmed their conclusion that inhibition of ATR decreased the expression of RRM2. Based on our results and those previously reported, we proposed a mechanism for the cooperative anti-leukemic activity of VE-822 and AZD1775 in Fig. 6B. 17

AZD1775 inhibits Wee1, inhibiting the phosphorylation of CDK1/2, leading to aberrant CDK activity in the S phase, resulting in aberrant origin firing, reducing dNTP pool levels, and finally, inhibition of DNA replication and increasing replication pressure. As a result, exposure to the combination of VE-822 and AZD1775 significantly increases single-stranded DNA coated by RPA (55-57). However, overactive CDK1/2 ultimately leads to DNA DSBs, which triggers the activation of the DNA damage repair-ATR-CHK1 pathway. Activated ATR phosphorylates CHK1 and then CHK1 phosphorylates Cdc25C, leading to reduced levels of active CDK1/2, thereby inhibiting the cell cycle and allowing DNA damage repair. However, VE-822 is an ATR-selective inhibitor that arrests this process, causing DNA damage to continue and ultimately inducing AML cell death (Fig. 6B). In conclusion, our results provide insight into the mechanism of action underlying the synergistic anti-leukemic activities of the ATR inhibitor VE-822 in combination with AZD1775 in AML cell in vitro. These findings provide a clear understanding of the molecular mechanisms about VE-822 and AZD1775 activity in AML cell lines. These two kinase inhibitors decreased RRM1/2 expression, abrogated G2/M cell cycle checkpoint, and induced DNA replication stress and DNA damage, displaying synergistic anti-leukemic activity. These results support the further development of ATR inhibitors in combination with AZD1775 for the treatment of AML patients in the clinical setting.

Acknowledgments 18

This study was supported by a grant from the National Natural Science Foundation of China (No. 81700136), Administration of Traditional Chinese Medicine of Jilin Province, China (No.2017158), The National Key Research and Development Program of China (No.2017YFC1703200 and No.2017YFC1702103), The Science and Technology Development Plan Project of Jilin Province (No. 20190101010JH). The funders had no role in the study design, data collection, analysis, data interpretation, decision to publish, or preparation of the manuscript. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

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Figure Legends Figure 1. VE-822 inhibits cell proliferation and induces apoptosis in AML cell lines in a dose-dependent manner. (A) The chemical construction of the ATRselective inhibitor VE-822 (molecular weight of 463.55) is shown. (B and C) AML cells were cultured in 96-well plates with 0, 0.125, 0.25, 0.5, 1, and 2 µM VE-822 for 72 h and cells were incubated with MTT. The IC50 was calculated as the drug concentration necessary to inhibit 50% at OD590 compared to untreated control cells. The data for panels B and C are presented as mean value ± SEM from at least three independent experiments. (D) THP-1 and MOLM-13 cells were treated with a series of concentrations of VE-822 for 24 h. Cells apoptosis was determined by Annexin-V FITC/PI staining and flow cytometry analysis. Mean percentage of Annexin Vpositive cells ± SEM was shown in THP-1 and MOLM-13 cells. Results were analyzed with one-way analysis of variance (ANOVA); n = 3. ***indicates p<0.001.

Figure 2. AZD1775 synergistically promotes VE-822 to inhibit AML cell proliferation and leads to mitochondrial dysfunction. (A and B) THP-1 (Panel A) and MOLM-13 (Panel B) cells were treated with VE-822 (VE) and AZD1775 (AZD) simultaneously, alone, or in combination for 72 h, and then viable cells were detected using MTT reagent. (C and D) Anti-leukemic interactions between VE-822 and AZD1775 were determined by standard isobologram analyses. The IC50 value of each drug is plotted on the axes. The solid line represents the additive effect, while the points represent the concentrations of each drug resulting in 50% inhibition of 23

proliferation. Points falling below the line indicate synergism whereas those above the line indicate antagonism. MTT assays were repeated at least three times and the data are presented as the mean ± SEM. CI < 0.9 indicates synergistic, 0.9 < CI <1.1 indicates additive, and CI > 1.1 indicates antagonistic anti-leukemia interactions, respectively. (E) THP-1 and MOLM-13 cells were exposed to the indicated concentration of VE-822 and AZD1775 alone or in combination for 24 h in a 96-well plate. The LDH assay was used to examine the integrity of cell membrane in AML cells. Data are expressed as the mean ± SEM. (F) Effects of VE-822 and AZD1775 on the MMP. THP-1 and MOLM-13 cells were seeded in 12-well plates for 24 h. After treatment with the indicated concentrations of VE-822 and AZD1775 alone or in combination, cells were harvested, washed with PBS, and stained with JC-1 for 20 min at 37°C. Representative dot plots are shown (Panel G). Results were analyzed with one-way ANOVA; n = 3. ***indicates p<0.001.

Figure 3. AZD1775 synergistically enhances VE-822-induced cell apoptosis and DNA damage in AML cells. (A) THP-1 and MOLM-13 cells were treated with the indicated concentrations of VE-822 and AZD1775, alone or in combination for 24 h. Then the AML cells were fixed in 80% ethanol, stained with PI, and subjected to flow cytometry analysis. Dead cells are expressed as the percentage of PI-positive cells with sub-G1 DNA content. (B and C) The percentage of cells in each cell cycle phase after drug treatment is presented as the mean ± SEM from one representative experiment. (D and E) THP-1 (Panel D) and MOLM-13 (Panel E) cells were treated 24

with VE-822, AZD1775, or the combination for 24 h and subjected to Annexin VFITC/PI staining and flow cytometry analysis. The mean percentage of Annexin Vpositive cells ± SEM is shown in AML cell lines. CI values were calculated using CalcuSyn software. CI < 0.9 indicates synergistic, 0.9 < CI <1.1 indicates additive, and CI > 1.1 indicates antagonistic anti-leukemia interactions, respectively. (F) THP1 and MOLM-13 cells were treated with VE-822, AZD1775, or a combination for 24 h. Whole cell lysates were subjected to western blotting and probed with anti-PARP-1, anti-cf-caspase-3, anti-γH2AX, and anti-GAPDH. The fold changes for the densitometry measurements, which were normalized to GAPDH and compared to the untreated control, are indicated below the corresponding blot.

Figure 4. AZD1775 improves VE-822-induced cell apoptosis in AML cell lines through downregulation of Wee1 and RNR in a time-dependent manner. (A and C) THP-1 (Panel A) and MOLM-13 (Panel C) cells were treated with VE-822, AZD1775, or the combination for up to 24 h and then subjected to Annexin VFITC/PI staining and flow cytometry analysis. THP-1 and MOLM-13 cells representative dot plots for up to 24 h are shown. (B and D) The mean percentage of Annexin V-positive cells ± SEM is shown. ns indicates not significant, *indicates p<0.05, and ***indicates p<0.001. (E) THP-1 and MOLM-13 cells were treated with VE-822 (VE), AZD1775 (AZD), or VE+AZD for up to 12 h. Whole cell lysates were subjected to western blotting and probed with the indicated antibodies. The fold changes for densitometry measurements, normalized to GAPDH and compared to the 25

untreated control, are indicated below the corresponding blot.

Figure 5. AZD1775 significantly enhances VE-822-induced DNA replication stress and DNA damage in AML cells. (A and B) THP-1 (Panel A) and MOLM-13 (Panel B) cells were treated with VE-822 and AZD1775, alone or in combination for 24 h. Chromatin-bound RPA32 and γH2AX were analyzed by western blotting and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to histone H4 and then compared to untreated control, are indicated below the corresponding blot. (C and D) THP-1 (Panel C) and MOLM-13 (Panel D) cells were treated with VE-822 and AZD1775, alone or in combination for 16 h

and then subjected to alkaline comet assay analysis. Representative images are

shown. (E and F) Comet assay results are graphed as the median percentage of DNA in the tail from three replicate gels ± SEM. *indicates p<0.05, **indicates p<0.01, and ***indicates p<0.001.

Figure 6. Proposed mechanisms for the anti-leukemic interaction of VE-822 and AZD1775 in AML cells. (A) THP-1 and MOLM-13 cells were treated with VE-822, AZD1775, or VE+AZD for 24 h. Whole cell lysates were subjected to western blotting

and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to GAPDH and compared to the untreated control, are shown below the corresponding blot. (B) Proposed mechanism for the anti-leukemic interaction between VE-822 and AZD1775 in AML cells. Inhibition of Wee1 led to 26

reduced phosphorylation of CDK1/CDK2, allowing CDK1/CDK2 to maintain activity and ultimately resulting in DSBs. DNA damage triggered activation of ATR/CHK1, which repaired DNA damage to maintain cell survival. When we added the ATR inhibitor VE-822 to inhibit DNA damage repair, more cells died without repair. VE822 and AZD1775 decreased levels of RRM1 and RRM2, key enzymes in dNTP synthesis, thereby reducing dNTP synthesis consequentially. The outcome of dNTP consumption must result in more DNA damage and cell death.

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