WEE1 inhibitor, AZD1775, overcomes trastuzumab resistance by targeting cancer stem-like properties in HER2-positive breast cancer

Journal Pre-proof WEE1 Inhibitor, AZD1775, Overcomes Trastuzumab Resistance by Targeting Cancer Stem-like Properties in HER2-positive Breast Cancer Andrea Sand, Mitchel Piacsek, Deborah L. Donohoe, Aspen T. Duffin, Geoffrey T. Riddell, Chaoyang Sun, Ming Tang, Richard A. Rovin, Judy A. Tjoe, Jun Yin PII:

S0304-3835(19)30637-8

DOI:

https://doi.org/10.1016/j.canlet.2019.12.023

Reference:

CAN 114619

To appear in:

Cancer Letters

Received Date: 5 February 2019 Revised Date:

27 November 2019

Accepted Date: 16 December 2019

Please cite this article as: A. Sand, M. Piacsek, D.L. Donohoe, A.T. Duffin, G.T. Riddell, C. Sun, M. Tang, R.A. Rovin, J.A. Tjoe, J. Yin, WEE1 Inhibitor, AZD1775, Overcomes Trastuzumab Resistance by Targeting Cancer Stem-like Properties in HER2-positive Breast Cancer, Cancer Letters, https:// doi.org/10.1016/j.canlet.2019.12.023. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 The Author(s). Published by Elsevier B.V.

Author Contributions J. Yin designed, conceived, performed the experiments and wrote the manuscript. A. Sand, M. Piacsek, D.L. Donohoe, A.T. Duffin, G.T Riddell and C.Y. Sun performed the experiments and edited the manuscript. M. Tang analyzed the whole exome sequencing data for mutation calling. R.A. Rovin provided expertise and feedback. J.A. Tjoe provided clinical expertise and coordinated the project and provided feedback on the manuscript.

Abstract Although trastuzumab has greatly improved the outcome of HER2-positive breast cancer, the emergence of resistance hampers its clinical benefits. Trastuzumab resistance is a multi-factorial consequence predominantly due to presence of cancer stem-like cells (CSCs). AZD1775, a potent anti-cancer agent targeting WEE1 kinase to drive tumor cells with DNA damage to premature mitosis, has previously shown high efficacies when targeting different cancers with a well-tolerated cytotoxic profile, but has not been evaluated in trastuzumab-resistant (TrR) breast cancer. We sought to investigate the effect of AZD1775 on cancer stem-like cell (CSC) properties, apoptosis, cell cycle regulation in TrR breast cancer. Our study for the first time demonstrated that AZD1775 induces apoptosis and arrests TrR cells at G2/M phase. More importantly, AZD1775 effectively targeted CSC properties by suppressing MUC1 expression levels. AZD1775 administration also induced apoptosis in our in-house patient-derived tumor cell line at passage 0, implying its significant clinical relevance. These findings highlight the potential clinical application of AZD1775 in overcoming trastuzumab resistance in breast cancer.

Title Page Title: WEE1 Inhibitor, AZD1775, Overcomes Trastuzumab Resistance by Targeting Cancer Stem-like Properties in HER2-positive Breast Cancer Authors: Andrea Sand, MS1, Mitchel Piacsek, BS1, Deborah L. Donohoe, BS1, Aspen T. Duffin, BS1, Geoffrey T. Riddell, BS1, Chaoyang Sun, PhD2, Ming Tang, PhD3, Richard A. Rovin, MD4, Judy A. Tjoe, MD1,5, *, Jun Yin, PhD1,* Affiliations: 1. Translational Oncology Research (TORQUE), Advocate Aurora Health Research Institute, Milwaukee, WI 53233, USA 2. Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 3. Harvard FAS informatics, Harvard University, Cambridge, MA 02138, USA 4. Aurora Neuroscience Innovation Institute, Aurora St. Luke’s Medical Center, Milwaukee, WI 50302, USA 5. Comprehensive Breast Care Center, Aurora Sinai Medical Center, Advocate Aurora Health, Milwaukee, WI 53233, USA *To whom correspondence should be addressed: Jun Yin, PhD (main corresponding author) Aurora Research Institute, 960 N 12th Street, Milwaukee, WI 53233 Tel: 414-219-4527 Email: [email protected] Or Judy A. Tjoe, MD Aurora Heath Care, 945 N 12th Street, Milwaukee, WI 53233 Tel: 414-219-6809 Email: [email protected] 1

Abstract Although trastuzumab has greatly improved the outcome of HER2-positive breast cancer, the emergence of resistance hampers its clinical benefits. Trastuzumab resistance is a multi-factorial consequence predominantly due to presence of cancer stem-like cells (CSCs). AZD1775, a potent anti-cancer agent targeting WEE1 kinase to drive tumor cells with DNA damage to premature mitosis, has previously shown high efficacies when targeting different cancers with a well-tolerated cytotoxic profile, but has not been evaluated in trastuzumab-resistant (TrR) breast cancer. We sought to investigate the effect of AZD1775 on cancer stem-like cell (CSC) properties, apoptosis, cell cycle regulation in TrR breast cancer. Our study for the first time demonstrated that AZD1775 induces apoptosis and arrests TrR cells at G2/M phase. More importantly, AZD1775 effectively targeted CSC properties by suppressing MUC1 expression levels. AZD1775 administration also induced apoptosis in our in-house patient-derived tumor cell line at passage 0, implying its significant clinical relevance. These findings highlight the potential clinical application of AZD1775 in overcoming trastuzumab resistance in breast cancer.

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1. Introduction Breast carcinomas with human epidermal growth factor receptor 2 (HER2) receptor amplification and overexpression account for approximately 20% of all breast cancers have a more aggressive phenotype and are associated with worse prognosis [1-3]. The current strategy is to target the HER2 receptor with trastuzumab. Trastuzumab is a humanized monoclonal antibody that blocks HER2 activation by binding to its extracellular domain IV, leading to antibody-dependent cell-mediated cytotoxicity [3, 4]. Despite the success of trastuzumab in improving disease-free and overall survival in patients with HER2-positive breast cancer, nearly 70% of these patients experience primary or acquired resistance [5]. Therefore, understanding and overcoming this resistance would be of benefit to trastuzumab-resistant (TrR) patients. There are several proposed mechanisms contributing to trastuzumab resistance, including the over-activation of PI3K/AKT pathway via direct or alternative pathways, e.g., abnormal expressed EGFR family and their ligands, and loss of PTEN function [6]. Among the newly investigated mechanisms, MUC1 has been well recognized as a functional biomarker for trastuzumab resistance. The cleaved form of MUC1 acts as a growth factor receptor on cancer cells and embryonic stem cells. Fessler et al. [7] have demonstrated a dramatic increase in MUC1 expression in a cancer cell line which was induced to acquire trastuzumab resistance. Meanwhile, emerging evidence suggests that cancer stem-like cells (CSCs) are important contributors to trastuzumab resistance, driving tumor recurrence and metastatic spread [8, 9]. Interestingly, upregulation of the MUC1 gene has been reported to be associated with CSC growth [10-13], however, its function in regulating CSC proliferation has not been studied in the TrR breast cancer cohort. One drug, which is currently undergoing many clinical trials as a potential treatment option for several types of cancer, such as ovarian cancer, and uterine cancer, is the WEE1 inhibitor – AZD1775. Preliminary data from the existing clinical studies on AZD1775 has shown great response rate with well-tolerant toxicity profiles [14]. WEE1 is a tyrosine kinase regulator of the G2/M cell cycle checkpoint. It prevents cell cycle progression for tumor cells with DNA damage by inhibiting the phosphorylation of CDC2 3

(CDK1) to maintain low CDC2 activity, allowing sufficient time for DNA repair. Therefore, targeting WEE1 kinase by AZD1775 sensitizes tumor cells to their inherent genomic instability, driving cells to premature mitosis. Several studies have demonstrated that AZD1775 could overcome the CSC resistance to radiation therapy or chemotherapy [15, 16]. Therefore, we sought to assess the effect of AZD1775 on targeting TrR cells and to characterize the mechanism of action responsible for AZD1775-induced killing effect in targeting both CSC population via MUC1-related signaling pathways and the entire population by inducing apoptosis and cell cycle regulation. The ability of AZD1775 being able to target CSCs could possibly be extended to other drug-resistant cancer cohorts of which resistance is due to the presence of cancer stem-like populations.

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2. Materials and Methods Cell Culture Human breast cancer cell lines BT-474 (American Type Culture Collection (ATCC)) and BT474R (BT-474 Clone 5; ATCC) were cultured in DMEM (Corning) supplemented with 10% FBS (GE Healthcare-Hyclone). Additionally, the breast cancer cell lines T47D (ATCC), HCC1954 (ATCC) and ZR75-30 (ATCC) were cultured in RPMI (Gibco) media, supplemented with 10% FBS. MCF-10A cells (ATCC) were cultured in complete MEGM (Lonza) and SK-BR-3 cells (ATCC) were cultured in McCoy’s media (Gibco) supplemented with 10% FBS. The in-house patient-derived cell line, 8071, was derived from a metastatic breast to brain HER2-positive tumor in brain with the method per previously described [17]. It was further maintained in NeuroCult Proliferation Media (Stemcell). All cell lines were maintained at 37 °C in an atmosphere of 5% CO2. Cell preparation for STR analysis and whole exome sequencing 5x105 patient-derived cells were collected and washed with DPBS (Lonza). DNA of the collected cells was extracted using AllPrep DNA/RNA Mini Kit (Qiagen) and quantified using NanoDrop (Thermo Fisher Scientific (TFS)). The extracted DNA was further subjected to STR analysis and whole exome sequencing service, performed by Cell Line Genetics (Madison, WI) and MD Anderson Cancer Center (Houston, TX), respectively. Dose Response Curves Cell lines were plated at a density of 2000 cells per well (Nexcelom Bioscience Cellometer Auto 2000) in 96 well tissue culture plates using the appropriate culture medium, previously optimized from plate surface area and time of treatment incubation. AZD1775 (SelleckChem), Pertuzumab (SelleckChem), and Trastuzumab (SelleckChem) were serially diluted and the treated cells were incubated at 37 °C for 5 days, at which point the PrestoBlue cell viability assay (Invitrogen) was performed to determine relative cell population on a Synergy H1 microplate reader with Gen5 software (BioTek). The experiments were conducted in experimental triplicate, and the measurement was normalized to that of untreated cells. 5

Western Blot 1x106 cells were plated in 6-well cell culture dish (Corning), collected and lysed in RIPA buffer (Abcam) with protease inhibitors (Cell Signaling Technologies (CST)). Lysates were run in 4-12% Criterion Tris-HCL Protein gels (BioRad) in a Criterion Cell electrophoresis system (BioRad), followed by being transferred to Nitrocellulose, 0.2 µm membranes. Primary antibodies were applied at various dilutions: Actin (1: 1000, Sigma-Aldrich, St. Louis, MO), Beta-tubulin (1:500, CST), phospho-Histone H2A.X (Ser139) (1:1000, γH2AX; Millipore, Burlington, MA), phosho- Histone H3 (Ser10) (1: 500, CST), HER2/ErbB2 (1: 500, CST) , MUC1(1:500, CST), Cleaved Caspase-3 (Asp175) (1: 1000, CST), and phospho-CDC2 (Y15) (1: 500, CST). The blots were then incubated with HRP-conjugated mouse (Santa Cruz Biotechnology (SCB)) or rabbit IgG antibodies at 1:1000 for one hour and the signals were detected on a LiCor3000 instrument (Li-Cor) using Western Clarity ECL (BioRad). Signals were measured and normalized, and experiments were repeated in triplicates. Immunofluorescence Cells were grown in 4-well chamber slides (TFS) and treated with and without 1µM AZD1775 for 48 hours. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.1-0.25% Triton X-100 in PBS and blocked with 5% BSA and incubated with primary antibodies: γH2AX, p-Histone H3 overnight at 4 °C at 1:100 dilution. The slides were washed and incubated with Alexa Fluor 488 (TFS) and Alexa Fluor 594 (TFS) at 1:400 dilution. Cells were mounted with mounting media containing DAPI (Invitrogen). Images were acquired using an Olympus IX83 microscope at 20x magnification (Olympus, Waltham, MA). Flow Cytometry For CD44/CD24 staining, cells were incubated with Cy7-conjugated anti-CD44 and CF594-conjugated anti-CD24 (BD Biosciences) at a dilution of 1:500 per the previously described protocol [18]. Cy7- and CF594-conjugated anti-mouse IgG (BD Biosciences) were used as negative controls. For cell cycle analysis, cells were harvested and fixed with 70% cold ethanol for 24 H, followed by incubation with propidium iodide (PI, 50 6

µg/ml) and RNase (50 µg/ml) for 30 min, and analyzed by flow cytometry using Flowjo software. For Annexin V/PI assay, FITC-conjugated Annexin V apoptosis detection kit (BD Biosciences) was used, as previously described [19]. For HER2 expression profile, cells were incubated with FITC-conjugated anti-HER2 at a dilution of 1:500 and further subjected to flow cytometry. In Vitro Gene Silencing 1x106 cells were transfected using the Neon electroporatiofonen system (TFS) with 5 ng siMUC1 (Dharmacon), 5 ng siWee1 (Dharmacon) or 5 ng control siRNA (Dharmacon) vectors. The cells were electroporated for 30 ms at 1100 volts with two pulses. The cells were plated in 6-well plates with the appropriate media, incubated at 37⁰C for 48 hours and processed for further experiments. Mammosphere Formation Assay 20,000 cells of each cell line were plated in ultralow attachment dishes (Corning) and cultured in Mammocult Media (Stemcell) with necessary supplements [20] for two weeks. Bright field images were acquired using EVOS XL CORE microscopy system at 10x magnification. Quantitative PCR 1x106 cells were plated and RNA was extracted using RNeasy mini kit (Qiagen). Extracted RNA was further subjected to NanoDrop for RNA quantification. An iScript cDNA synthesis kit (BioRad) was used for cDNA synthesis from the extracted RNA. Quantitative PCR was prepared in biological triplicate using ITAQ™ Universal SYBR® Green Supermix (BioRad) and was performed by Roche Light Cycler 480 II instrument (Roche, NJ). Specific primers for MUC1 (Integrated DNA Technologies (IDT), Assay ID: Hs.PT.58.38790646.g) and B2M (IDT, Assay ID: Hs.PT.58v.18759587) were used in the assay. MUC1 expression level was normalized to the level of B2M expression, which is the internal standard and mRNA expression level was calculated with the 2−∆∆Ct method. Soft Agar assay

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Soft Agar assay were performed using CytoSelect 96-well Cell Transformation Assays (Cell Biolabs, Inc), as per manufacturer’s instructions or protocol as described previous [21]. Briefly, 2x104 cells mixed with cell media and commercial agar were plated on top of the agar base provided by the Assays. Cell culture media containing vehicle and 1µM AZD1775 were applied each sample well weekly for two weeks. Once colony formation was observed, images were taken using EVOS XL core imaging system. Animal Studies All animal procedures were conducted in compliance with National Institute of Health guidelines for animal research and approved by Institutional Animal Care and Use Committee (IACUC) at Aurora Research Institute. Five-week-old female Crl:Nu(NCr)Foxn1nu nude mice were purchased from Charles River Laboratories and housed for another week in a specific pathogen-free environment, followed by subcutaneously injection into mammary fat pads with 1x106 cells suspended in 100µl ice-cold Matrigel (Corning) at a 1:1 ratio for each mouse (n=10/each group). After tumors reached approximately 50mm3, vehicle (0.5% methyl cellulose) or AZD1775 (120mg/kg) was administered through oral gavage for 4 weeks (5 days on, 2 days off/week). Tumor volumes, calculated by V = (length x Width2)/2, and body weight were measured twice per week during treatment. Immunohistochemistry Tissues were fixed in 10% formalin over 24 hours and embedded in paraffin. Paraffin embedded sections were dehydrated with Xylene and stained using EnVision Detection SystemsPeroxidase/DAB, Rabbit/Mouse kit (Agilent) kit per protocol previously described [22]. Briefly, tissue sections with primary antibodies: Ki67(1:20, Abcam), MUC1 (1: 500, CST), pCDC2 (1:20, SCB), rH2AX (1: Millipore) were incubated overnight at 4°C, followed by 1-hour incubation with Labelled Polymer-HRP at room temperature. Negative controls were treated identically, but without primary antibody. Subsequently,

slides

were

incubated

with

DAB+

Chromogen,

followed

by

counterstaining with hematoxylin. Pictures of slides after mounting with Permount (TFS) were captured under microscope (Olympus). Scores of IHC for all markers are calculated by the percentage of tumor cells multiplied by the intensity of markers. 8

Statistical analysis Data were analyzed by Student’s t-test and one- or two-way ANOVA per experiment design. A two-way ANOVA was used to assess the effects and interactions of two variables and multiple comparisons were achieved using Bonferroni’s post hoc test. All statistical analysis was completed using GraphPad Prism 6 and results are presented as mean ± SD of three independent experiments. For whole exome sequencing analysis, raw FASTQ files were quality controlled using FASTQC and then aligned to hg38 using BWA-MEM without trimming. The aligned BAM files were processed using GATK4 following best practice. Mutations were called using mutect2 without normal control.

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3. Results AZD1775 specifically and effectively reduces cell viability of TrR cells. To identify potential targetable signaling pathways that are more vulnerable in TrR cells, we used the drug screening database from J.W. Gray [23]. We first classified commercially available HER2-positive breast cancer cell lines as trastuzumab-resistant (TrR) or sensitive (TrS) (Gray Ref) (Fig. 1A). We next used the Reverse Phase Protein Array (RPPA) database [24] to find differentially expressed proteins between pairs of TrS and TrR cell lines. Interestingly, we observed marked changes, with at least two-fold change, in cell cycle regulators: P16, CYCLIN B1 and P53 [25-27]. We also found up-regulation of multiple components involved in the G2/M DNA damage checkpoint in TrR cells, such as CDK1 (CDC2) and CHK1 with at least two-fold change [28-30] (Fig. 1B). In addition, reactome analysis confirmed the predicted vulnerability in cell cycle progression and G2/M DNA damage checkpoint pathways in TrR cells (Fig. 1C). Together, the data raise the possibility that targeting checkpoint defects in TrR cells could be a novel treatment. Therefore, we probed the endogenous levels of a marker indicative of DNA damage checkpoint blockage, pCDC2 [31] and found that all TrR cells are positive for pCDC2 in compared to TrS cells, which is consistent with observations from previous studies [32, 33], albeit for very different levels of γH2AX (Supplementary Fig. S1A). We carefully tested the effect of AZD1775 on three TrR cell lines, two TrS cell lines and a normal-like breast cell line, MCF10A, to avoid any colony effect. We found that AZZD1775 treatment significantly reduced viability of TrR cells compared to MCF10A and all matched TrS cell lines (Fig. 1D, left panel). Further, the susceptibility of TrR cell lines is independent of ER status. Mechanistically, we observed significantly reduced phosphorylation of CDC2 upon WEE1 Kinase activity by AZD1775 treatment, at 48 H and 72 H post-treatment without changing HER2 expression levels (Fig. 1D, right panel). Consistently, inhibition WEE1 levels by siRNA, which decreased pCDC2 levels, also significantly reduced growth rate of TrR cells without changing HER2 expression levels (Fig. 1E), which possibly confers the absence of synergistic effect between WEE1 inhibition and trastuzumab treatment (Supplementary Fig. S2A).

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AZD1775 reduces cell viability by inducing G2/M phase arrest and apoptosis in TrR HER2-positive breast cancer. To explore the possible mechanism of AZD1775mediated inhibition of TrR cell growth, we firstly assessed the endogenous levels of cell cycle related proteins: p-Histone H3 (Ser10) (p-His H3) and ß-tubulin and found that TrR cells are relatively positive for both p-His H3 and ß-tubulin (Supplementary Fig. S1A). Previous studies have revealed the association of high levels of p-His H3 and ßtubulin with worse prognosis in cancer [34]. Consistently, we observed significantly shorter doubling time in TrR cells in comparison to TrS cells (Supplementary Fig. S1B). We further accessed the cell cycle distribution of the three TrR cell lines before and after AZD1775 treatment. p-His H3 and γH2AX expression levels were significantly elevated in each cell line after treatment; however, β-tubulin was significantly decreased after treatment in HCC1954 and T47D cell lines, while no significance was detected in BT474R cells. (Fig. 2A-B). p-His H3, a mitosis-specific marker, was preferentially expressed in TrR cells in the presence of AZD1775 (Fig. 2A-B). The analysis of cell cycle distribution showed that AZD1775 significantly increased the proportion of cells in G2/M phase (P < 0.001) (Fig. 2C). Additionally, we observed AZD1775-induced apoptosis in TrR cells at 1µM starting at 48 H and 72 H post-treatment. The annexin V/PI apoptotic assay, an assay for characterizing both early and late apoptosis with high sensitivity, showed that the early apoptotic population was significantly increased at 48 H post treatment in BT474R and HCC1954 cells, and 72 H post treatment in T47D cells (Fig. 3A-B). In addition, a significant elevation of late apoptotic population in BT474R cells was also detected, but not in others. The apoptosis induced by treatment was further demonstrated by activation of caspase 3 signaling, which can be detected at both early and late stages (Fig. 3B, bottom panel). AZD1775 targets CSC properties in HER2-positive breast cancer cells through inhibition of MUC1. Emerging evidence suggests a significant correlation between drug resistance and increased breast CSC populations [35]. These cells give rise to tumorigenesis, e.g., tumor growth, propagation, and are also associated with tumor recurrence and resistance to treatment. CSCs constitute a small subset of tumor cells with tumor-initiating potential, characterized by the CD44high/CD24low phenotype [36, 37]. 11

To preliminarily check whether TrR cells harbor more of the CSC-like properties, we performed both a mammosphere formation assay and assessed the CD44high/CD24low phenotype of the isogenic pair – BT474 and BT474R, as shown in Fig. 4A. Our results show that BT474R, the TrR cell line, forms more mammospheres and has a greater inherent CD44high/CD24low CSC population than BT474, the TrS cell line. Furthermore, we checked whether inhibition of WEE1 kinase activity by either AZD1775 or siRNA would inhibit the growth of CSC population. Intriguingly, we found a significant reduction of CSC population in each of the TrR cell lines upon 1uM AZD1775 treatment (Fig. 4BC) and siRNA (Supplementary Fig. S2B-C). To further investigate the mechanism of AZD1775-induced killing effect on CSCs, we probed the endogenous expression levels of MUC1, which is a well-recognized biomarker for trastuzumab resistance in HER2-positive breast cancer cells [7] in three pairs of TrS and TrR cell lines. Overexpression of MUC1 at both RNA levels and protein levels was observed, shown in Fig. 5A and Fig. S3A. Interestingly, AZD1775 significantly inhibited MUC1 levels as early as 48 H amongst the three TrR cell lines. The treatment does not alter HER2 expression levels across all TrR cell lines, which suggests the inhibition of AZD1775 on the CSC population might be due to downregulation of MUC1, but not the HER2 related pathways, as shown in Fig. 5B. To validate this hypothesis, we knocked down MUC1 expression levels by siRNA in all three TrR cell lines and characterized the CSC population by both mammosphere formation assay and CD44high/CD24low phenotype. Consistent with our hypothesis, we found significant inhibition of the CSC population after silencing MUC1 expression and that this regulation is independent of cell cycle arrest, HER2 expression or the activation of CDC2, as shown in Fig. 5C-E. AZD1775 suppresses trastuzumab-resistant HER2-positive patient-derived and xenograft-derived tumor cell growth and anchorage-independent growth in TrR cells. In addition to the observation from commercial patient-derived tumor cell lines, we used a metastatic HER2-positive breast cancer cell line derived in house (Pt) from tumor dissected from brain site at passage 0 and further validated its heterogeneity and HER2 positivity by flow cytometry (Fig. 6A). Although this tumor hasn’t been previously 12

treated with HER2 blockage agents, we validated its resistance and the resistance of its xenograft-derived cell line (PDX1), which possess a more homogenous self-propagated tumor cell population, to both trastuzumab and pertuzumab treatment, as shown in Fig. 6B. To assess whether this intrinsic resistance is associated with any HER2 mutations in this tumor, we performed whole exosome sequencing on both cell lines and detected two nonsynonymous single-nucleotide polymorphisms (SNVs, with allele frequencies of 0.922 and 0.955) in HER2. Although both SNVs (AA change: Ile655Val and Pro1170Ala) were reported to be associated with trastuzumab-induced cardiotoxicity [38, 39] but their association with trastuzumab resistance remains inconclusive due to extremely limited reports on Pro1170Ala variant and the contradictory conclusions on Il655Val variant [4042]. Intriguingly, both Pt and PDX1 lines responded to AZD1775 treatment (Fig. 6C), with the IC50 of approximately 50 µM for mixed population in Pt line and 500µM for PDX1 line, respectively. Prior to test our hypothesis that AZD1775 could effectively suppress tumor growth in vivo, we also performed soft agar assay, a well-established anchorage-independent assay characterizing the capability tumorigenesis and carcinogenesis [43], to investigate whether inhibition of WEE1 kinase activity could inhibit this capability. Consistent with our observation for cell growth inhibition, the inhibition of WEE1 kinase by either AZD1775 or siRNA significantly reduced the numbers of colonies formed (Fig. 6D-E) in TrR cells. However, no colony formation was detected in 8071, possibly due to its high heterogeneity at early passage. AZD1775 suppresses tumor growth in trastuzumab-resistant TrR cell-derived Xenografts. To confirm the clinical relevance of our in vitro observations, we further explored the efficacy of AZD1775 on tumor growth and the relevant mechanisms in vivo. BT474R, HCC1954 and T47D cells (1 x 106), which have demonstrated their capabilities to form colonies in soft agar assay, were orthotopically injected into the mammary fat pads of 6-week-old female nude mice (n=10 each group). Mice bearing tumors were then treated with AZD1775 (120mg/kg, 5 days on, 2 days off) or vehicle control. Strikingly, AZD1775 administration effectively reduced tumor growth and tumor burden in BT474R (Fig. 7A, left panel), HCC1954 (Fig. 7A, right panel) and T47D 13

(Supplementary Fig. S4). No significant differences in body weight were observed between AZD1775 and control groups (Fig. 7B). The antitumor effect occurred concomitantly with a significant reduction in Ki-67-positive cells and the mechanism observed in vitro was further validated by significantly decreased levels MUC1 and pCDC2 in treated tumors (Fig. 7C-D).

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4. Discussion In this study, we investigated the efficacy of a potent and highly selective inhibitor of the WEE1 kinase, AZD1775, on TrR HER2-positive breast cancer cells. Targeting DNA damage repair (DDR)–related pathways has been a promising therapeutic strategy to sensitize tumor cells to their inherent genomic instability and further improve response rates for the treatment of various types of cancers [44]. Currently, there exist 49 clinical studies listed in ClinicalTrials.gov (accessed January 2019) for AZD1775 (MK1775), covering a range of cancer types such as solid tumors, carcinomas, leukemia as either monotherapy or in combination with other DNA-damage agents such as carboplatin or cisplatin. Preliminary data from the existing clinical studies on AZD1775 show promising results with an acceptable toxicity profile [45-47]. Nonetheless, no trial has yet been registered to assess its efficacy for treating TrR HER2-positive breast cancer. Following this thought, by carefully choosing cell line models that well represent both acquired resistance (BT474R) and initial resistance (HCC1954 and T47D) in patients, we for the first time demonstrate that AZD1775 can overcome trastuzumab resistance in HER2positive breast cancer cells. One of the major mechanisms of resistance to trastuzumab involves HER2 interactions with the epitope masking of HER2 by other cell surface proteins, particularly MUC1 [48, 49]. MUC1 was also reported as a determinant for trastuzumab resistance in breast cancer cells and functions as a growth factor receptor on cancer cells and embryonic stem cells [7]. Another well-recognized mechanism of this resistance found by previous studies is that the relatively quiescent CSC subpopulation in TrR breast cancers, which forms a reservoir for new tumor relapses, is a major hindrance to drug treatment. Though MUC1 has also been reported to play an important role in promoting CSC formation across other different cancers [35, 36], its role in CSC has not yet been validated in this cohort. Our results have demonstrated the significant inhibition of CSC formation upon AZD1775 treatment via suppression of MUC1 levels, evident by a marked inhibition of the CD44high/ CD24low subpopulation and the impairment of mammosphere formation in vitro upon both AZD1775 treatment or through silencing MUC1 expression levels. This finding provides a new insight into a sustainable 15

therapeutic approach to HER2-positive breast cancer treatment by targeting CSC-like properties. As the TrR breast tumor consists of CSCs and their more differentiated counterparts, the evaluation of AZD1775-induced cell death in the entire tumor cell population is also necessary. We found that exposure of TrR cells to 1uM AZD1775 remarkably induces both early and late apoptosis, which occurs with significantly increased Annexin V-positive population and the activation of caspase-3 (17kDa). In addition, AZD1775 can induce double strand breaks (DSBs), indicated by an observed increased population of γH2AX-positive cells upon AZD1775 treatment. In the presence of DSBs, ATM is preferentially activated, leading to phosphorylation and activation of CHK2 to maintain CDK in an inactive form, thus preventing cells with DSBs to enter into mitosis [50]. AZD1775, at the same time, effectively inhibits the phosphorylation of CDK1, observed in TrR cells in our study, to force cells with DSBs to enter premature mitosis, not allowing sufficient time for DNA repair. However, we also found AZD1775 could effectively arrest TrR cells at G2/M phase, preventing them from division, which is contrary to its function as a driver for premature mitosis. Interestingly, similar findings have also been reported by other groups: Lewis et al. [51] and Webster et al. [52] have demonstrated that AZD1775 could enhance 5-FU cytotoxicity through increased DSBs but not premature mitosis, and it also induces mitotic arrest resulting in cell death regardless of the cell cycle phase, suggesting that AZD1775 is also an anti-mitotic agent. In our immunochemistry staining, we found no overlap between γH2AX-positive cells and p-His H3-positive cells, indicating the mechanisms of AZD1775-induced cell death might be different within TrR cancer cells – for the subpopulation with DSBs induced by AZD1775, it functions as a driver to premature mitosis while for other subpopulations, it selectively arrests cells at G2/M phase to inhibit proliferation. However, further investigation and evidence is needed to clarify this mechanism for better development of treatment strategies in the clinic. In summary, our study highlights a new potential strategy to target TrR HER2-positive breast cancer by AZD1775 via inhibiting tumor cell viability and proliferation as well as inducing apoptosis. More importantly, AZD1775 targets CSC formation via suppression 16

of MUC1 expression, which has not been previously reported. Consistent with our findings, Mir et al. also reported that the WEE1 inhibitor overcomes CSC resistance to radiation [53] and Zhou et al. demonstrated that the combination therapy of WEE1- or CHK1- targeting agents with histone deacetylase (HDAC) inhibitors appears to be effective against some leukemia stem cell-like cells [15]. We postulate that AZD1775 may also be effective on targeting other cancers which have previously demonstrated resistance to first–line therapy due to the CSC subpopulation, but further evidence is needed. Additionally, further research is warranted to find biomarkers for the selection of patients who will benefit from this therapy.

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5. Acknowledgement This study is supported by Vince Lombardi Cancer Foundation and Aurora Health Care Seed Grant, Wisconsin, USA. We thank Department of Biorepository at Aurora Research Institute for patient sample collection and we thank the cell line processing core, Kate Dennert and Logan Friedrich at Aurora Research Institute for processing and maintaining patient derived cell lines. We thank ACL laboratories for their generous help with processing tissue sections for tumors harvested from animal Xenografts.

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6. Conflict of Interest The authors declare no conflict of interest.

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Figure 1 B.

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Highlights Trastuzumab-resistant (TrR) cells are sensitive to AZD1775 AZD1775 induces apoptosis and arrests cell cycle progression in TrR cells AZD1775 effectively targets cancer stems-like properties via suppressing MUC1 AZD1775 suppresses in-house patient-derived TrR tumor cells at early passage

Conflict of Interest The authors declare no conflict of interest.

Figure Legends Figure 1. AZD1775 significantly and specifically reduces viability of TrR HER2-positive breast cancer cells. (A) Drug response curves of eight HER2-positive breast cancer cell lines to trastuzumab were obtained from the drug-screening database of JW Gray (21) and analyzed by non-linear regression. They were further classified as TrS cell lines (blue curves) and TrR cell lines (red curves) by two-way ANOVA (**P < 0.01). (B-C) RPPA data of two pairs of TrS (BT474, SKBR3) and TrR (T47D, HCC1954) cell lines were processed to assess differentially regulated proteins (**P < 0.01) and signaling pathways by reactome analysis (48-50) between TrS and TrR cell lines . (D) Left panel: Six different cell lines: normal-like breast cell (MCF10A), two TrS cell lines (BT474, ZR75-30) and three TrR cells (BT474R, T47D, HCC1954), were treated with AZD1775 at various concentrations for 5 days and cell viability was determined by Presto Blue assay. Right panel: Western blot showed effect of AZD1775 (1µM) on pCDC2 and HER2 at 48- and 72-hours post-treatment in all three TrR cells. Actin was used as an internal control.

(E) Left Panel: Three TrR cell lines transfected with control siRNA, labeled as

Scrambles (blue curves) and Wee1 siRNA, labeled as siWee1 (red curves) were monitored for their growth rate for 9 days. Data points were fitted in exponential growth curve. Differences in growth rate between Scrambles and siWee1 group were compared (BT474R, ***P<0.001, T47D, **p<0.01, HCC1954, ***P<0.001). Right panel: Western blot showed WEE1, pCDC2 and HER2 levels 48 hours post siRNA perturbation. Actin was used as an internal control. Figure 2. AZD1775 inhibits proliferation of TrR cells by arresting cells at G2/M phase and inducing apoptosis. (A) TrR cells were treated with and without 1µM AZD1775 for 48 hours to assess expression levels of p-Histone H3, β-tubulin and γH2AX by western blot. Actin was used as an internal control. Experiments were conducted in triplicates and quantified with error bars indicating SD (right panel). (B) TrR cells were immunostained for γH2AX (green), p-Histone H3 (red), and DAPI (nuclei, blue) with and without AZD1775 treatment (1µM, 48 hours). Images were assessed (shown in the left panel for HCC1954, scale bar = 100µm) and foci-positive cells were quantified (shown on the right panel for all three lines), where the groups of treatment were labeled with “Trt” with errors bars representing SD. (C) TrR cells were treated with AZD1775 for 24, 48 and 72 hours and were further subjected to flow cytometry for cell cycle analysis. Statistical significance was assessed by two-way ANOVA. Figure 3. AZD1775 induces apoptosis in TrR cells by caspase activation. (A-B) TrR cells were treated with AZD1775 (1µM) for 48 and 72 hours and were subjected to flow cytometry to assess the percentage of early apoptotic cells (annexin V-positive only cells) and late apoptotic

cells (annexin V-positive and PI-positive cells). Experiments were conducted in triplicate and the results are presented as mean ± SD and analyzed by one-way ANOVA followed by Bonferroni’s post hoc test. (C) TrR cells were treated with and without 1µM AZD1775 for 24, 48 and 72 hour and cell lysate was collected to evaluate the expression level of cleaved caspase-3 by western blot. Figure 4. AZD1775 disrupts CSC-like properties in TrR cells. (A) BT474 and BT474R were used for mammosphere formation assay and images of mammosphere formation were captured (top left panel, scale bar = 200µm). Numbers and size (threshold = 50 µm) of mammospheres were quantified by optical microscopy and results are represented as mean ± SD from five different fields. Identification and quantification of CD44high/CD24low population by flow cytometry (isotype controls were used for negative control) are also shown. (B) TrR cells were treated with and without AZD1775 (1µM, two weeks) for mammosphere formation assay. (C) TrR cells were treated with and without AZD1775 (1µM, 48 H) and were further subjected to flow cytometry to characterize the CD44high/CD24low population. Experiments were conducted in triplicates and the results are presented as mean ± SD. Figure 5. AZD1775 targets CSC-like properties through downregulation of MUC1. (A) Comparison of both RNA (left panel) and protein (right panel) expression levels of MUC1 between TrR cells and TrS cells. RNA expression levels of MUC1 in TrR cells were normalized to those in TrS cells (isogenic pair: BT474 vs. BT474R; ER+/HER2+ pair: SKBR3 vs. HCC1954; ER-/HER2+ pair: ZR75-30 vs. T47D, **P<0.05)). Error bars represent SD from three independent experiments. (B) TrR cells treated with and without 1µM AZD1775 for 48 H and 72 H were collected and probed for protein expression levels of MUC1, HER2 and pCDC2 by western blot (L: long exposure, S: short exposure for HER2 blot). Actin was used as an internal control. (C) TrR cells were also transfected with control siRNA (scramble) and Muc1 siRNA (siMuc1) for 48 H. Transfected cells were collected and used for western blot to evaluate protein expression levels of MUC1, p-His H3, HER2 and pCDC2. Actin was used as an internal control. (D) Transfected TrR cells were also subjected to mammosphere formation assay and quantified. (E) The CD44high/CD24low population in transfected TrR cells was quantified by flow cytometry. Each experiment was conducted in triplicates and the results are presented as mean ± SD. Figure 6. AZD1775 inhibits the growth of both early passage patient-derived and xenograft-derived tumor cells and reduces colony formation of TrR cells in soft agar assay in vitro. (A) HER2 expression levels were evaluated for a HER2-positive tumor-derived cell line (8071) at passage 0. Efficacies of trastuzumab (B), pertuzumab (B) and AZD1775 (C)

were evaluated for patient tumor-derived cell line (labeled as pt, blue curves) and xenograftderived cell line (labeled as PDX1, red curves) by Presto Blue assay after 5 days of treatment. Experiments were conducted in triplicate and the results are presented as mean ± SD. (D and E) TrR cell lines were subjected to soft agar assays to assess their capability of anchorageindependent growth with and without inhibition of WEE1 kinase activity by either 1µM AZD1775 (D) and siWee1 (E). Images of formed colonies (threshold = 20µm) were acquired from five different fields (scale bar = 200µm). Experiments were performed independently in triplicates and the results are presented as mean ± SD. Figure 7. AZD1775 inhibits tumor growth in TrR cell-derived xenografts in vivo. (A-B) Effect of AZD1775 on tumor growth in vivo. BT474R (1x106) and HCC1954 (1X106) were injected into the mammary fat pads of Foxn1nu nude mice (n = 10/group). Mice bearing tumors were administered with vehicle (0.5% methyl cellulose) and AZD1775 (120mg/kg, body weight, 5 days on, 2 days off) through oral gavage for 28 days when tumors reached approximately 50mm3. Tumor volume measured with a caliper and animal weight were documented twice a week. AZD1775-treated xenografts exhibited statistically significant reduction in tumor growth (A, **P < 0.01 for BT474R and ***P<0.001 for HCC1954) but no significant changes of body weight (B). Individual tumor growth and weight tracking per animal with and without treatment are illustrated (A-B, bottom panel). Tumors of mice were harvested upon end of treatment cycles or sacrificing when tumor size reached 2cm3 in diameter and were further subjected to (C) immunohistochemical analysis (IHC) and (D) western blot to probe protein expression levels with indicated antibodies. Representative images of immunohistochemistry (IHC) are shown with and without treatment (scale bar = 100µm). Both IHC and western blot indicated protein changes of pCDC2, MUC1 and Ki67 levels in tumor tissues after treatment with AZD1775.