Integration with TCGA breast cancer patient data
Breast invasive carcinoma patient data from The Cancer Genome Atlas Breast Cancer (TCGA BRCA) were retrieved using the UCSC Cancer Browser (https://genome-cancer.ucsc.edu/). For graphical view of genomic data, whole-exome sequencing (n = 526) data were analyzed using the Xena browser at the cBioPortal (http://www.cbioportal.org). A detailed description of data generation and instructions can be viewed on https://xenabrowser.net .
Gene expression was analyzed in KB1P and KP mouse mammary tumors. Illumina TruSeq mRNA libraries were generated and sequenced with 50–65 base single reads on a HiSeq 2500 using v4 chemistry (Illumina Inc., San Diego). The resulting reads were trimmed using Cutadapt (version 1.15) to remove any remaining adapter sequences and to filter reads shorter than 20 bp after trimming to ensure good mappability. The trimmed reads were aligned to the GRCm38 reference genome using STAR (version 2.6.1a ). Gene expression counts were generated by feautureCounts (version 1.5.0-p1 ) using genome definitions from Ensembl GRCm38 version 76. Normalized expression values were obtained by correcting for differences in sequencing depth between samples using DESeq median-of-ratios approach  and the log-transforming normalized counts. To statistically test the differences between the groups, ANOVA and pairwise t-test with multiple testing correction was used. The RNA sequencing data reported in this study are available in the NCBI GEO database (GSE182448).
Cell lines and culturing
Murine tumor cell lines were generated from individual tumors arising in female KB1P or KP mice as described previously . Established cell lines were cultured at 37 °C with 5% carbon dioxide under low oxygen conditions (3%) in DMEM/F12 medium (Thermo Fisher, Cat#31331028) supplemented with 10% FCS, 1% Penicillin–Streptomycin (5000 U/mL, Thermo Fisher, Cat#12140122), 5 mg/mL insulin (Sigma, Cat#53003-018), 5 ng/mL epidermal growth factor (Thermo Fisher, Cat#I6634), and 5 ng/mL cholera toxin (Sigma-Aldrich Israel (Cat#C8052).
KB1P-G3 and KP-3.33 cells stably expressing shEzh2 were cultured in DMEM/F12 medium supplemented with 10% FCS, 1% Penicillin–Streptomycin, 5 µg/mL insulin, 5 ng/mL epidermal growth factor, and 5 ng/mL cholera toxin. shRNA expression was induced with 100 ng/ml doxycycline (Dox) (Sigma-Aldrich, Cat#D9891) in culture medium. Medium of uninduced cells was supplemented with the respective volume of PBS.
Human SUM149 cells were cultured in DMEM/F12 medium supplemented with 10% FCS, 1% Penicillin–Streptomycin, 5 µg/mL insulin, and 1 µg/mL hydrocortisone (Sigma, Cat#0315). Human CAL120 cells were cultured in RPMI1640 (Thermo Fisher, Cat#12633012) supplemented with 10% FCS, 1% Penicillin–Streptomycin.
All cell lines were tested negative for Mycoplasma contamination upon thawing using a PCR Mycoplasma Test Kit (AppliChem, Cat#A3744).
The following compounds were purchased from Selleckchem: AZD1390 (ATM inhibitor, Cat#S8680), AZD7762 (CHK1/2 inhibitor, Cat#S1532), BI2536 (PLK1 inhibitor, Cat#S1109), BKM120 (PI3K inhibitor, Cat#S2247), crizotinib (ROS1 inhibitor, Cat#S1068), dinaciclib (CDK inhibitor, Cat#S2768), gefitinib (EGFR inhibitor, Cat#S1025), GSK126 (EZH2 inhibitor, Cat#S7061), JQ1 (BET inhibitor, Cat#S7110), KU60019 (ATM inhibitor, Cat#S1570), KU60648 (DNA-PK inhibitor, Cat#S8045), LDC67 (CDK9 inhibitor, Cat#S7461), MK1775 (Wee1 inhibitor, Cat#S1525), olaparib (PARP inhibitor, Cat#S1060), palbociclib (CDK4/6 inhibitor, Cat#S1579), panobinostat (HDAC inhibitor, Cat#S1030), PF477736 (CHK1 inhibitor, Cat#S2904), purvalanol A (CDK1/2 inhibitor, Cat#S7793), RO3306 (CDK1 inhibitor, Cat#S7747), selisitat (SIRT1 inhibitor, Cat#S1541), selumetinib (MEK inhibitor, Cat#S1008), senexin A (CDK8/19 inhibitor, Cat#S8520), TH287 (MTH1/NUDT1 inhibitor, Cat#S7631), THZ1 (CDK7 inhibitor, Cat#S7549), VE822 (ATR inhibitor, Cat#S7102), venetoclax (BCL2 inhibitor, Cat#S8048). NSC663284 (Cdc25 inhibitor, Cat#383907-43-5) was purchased from Cayman Chemical, PF3644022 (MK2 inhibitor, Cat#B5549) from ApexBio, ZLD1039 (EZH2 inhibitor, Cat#AOB9716) from AOBIOUS. All compounds were dissolved in DMSO (Carl Roth, Cat#A994.2) at a concentration of 10 mM. Equal amounts of DMSO added to the cell culture medium served as vehicle control.
Cell viability measurement
Optimal seeding density per cell line was derived from growth curves performed prior to screening experiments. Cells from subconfluent cell culture dishes were filtered through a cell strainer and viable cells were counted with Countess™ II FL Automated Cell Counter (Invitrogen) to allow optimal exponential growth (log phase) during the whole experiment. Cells were plated into 384-well plates (KB1P and KP cells: 500 cells/well, SUM149 and CAL120 cells: 1000 cells/well) in 30 µl complete culture medium. The compounds were added after 24 h by using the TECAN D300e digital dispenser (HP). After 72 h of treatment, cell viability was assessed by measuring ATP content in each well using CellTiter-Glo Reagent (Promega, Cat#G7573) 1:1. Luminescence intensity was measured using a plate reader (Tecan Infinite M1000 Pro) and normalized to intensities of control wells.
Compound synergy screen
Before screening for drug synergy, single agent effects of all compounds were profiled on the KB1P and KP mouse mammary tumor cell lines for 10 different concentrations in two-fold serial dilutions, ranging from 20 nM to 20 µM and assessed by cell viability measurement using CellTiter-Glo. The concentration-effect relationship (IC50 values) was determined by logistic interpolation using GraphPad Prism. See Additional file 8: Table S1 for IC50 values.
Next, in a pre-screen for drug synergy, GSK126 was tested against each compound in two-fold serial dilutions to determine the optimal drug concentrations for synergy analysis using a 6 × 6 matrix for drug combinations (5 concentrations for each compound and DMSO control). For the main synergistic combination screen, 5 representative concentrations titrated around the previous determined IC50 values were used for each compound. The compounds were then profiled in combination with GSK126 using the 6 × 6 matrix layout, and cells were treated as described above See Additional file 8: Table S1 for compound concentrations. Cell viability was detected after 72 h using CellTiter-Glo Reagent. See Additional file 9: Table S2 for normalized cell viability data used for calculation of synergy scores.
Analysis of synergy
Analysis of synergy scores was determined using the Bliss independence model calculating the difference between observed and expected compound effects  as described before . Briefly, single agent effects of compounds A and B (αA, αB) at concentrations CxA and CyB were used to calculate the expected effect for additive compound interactions (αexp = αA + αB − αA * αB), and subsequently compared to the effect observed under combination of both compounds (αobs). The delta score (Δα = αobs − αexp) calculated as the difference between observed and expected effects over the full dose–response matrix characterizes the synergistic effect. A score of > 15 was used as threshold indicating a synergistic compound interaction . Synergistic effects were visualized using the web-application tool SynergyFinder version 2.0 .
Clonogenic survival assay and crystal violet staining
Cells were seeded at a density of 25,000 cells (KB1P and KP cells) or 50,000 cells (SUM149 and CAL120) per well into 6-well cell culture plates. After overnight incubation, cells were continuously treated with GSK126 (7.5 µM) or with AZD1390 (2 µM) alone or in combination for 7 days. DMSO was used as vehicle control.
KB1P-G3 and KP-3.33 cells stably expressing shEzh2 or shRandom were seeded into 10-cm cell culture dishes in the appropriate cell culture media 24 h before treatment. To induce Ezh2 knockdown, cells were continuously treated with Dox (100 ng/µl) for 7 days. For the colony formation assay, cells treated with Dox for 7 days were replated at 10,000 cells per well into 12-well cell culture plates and cultivated in Dox for the duration of the entire experiment. Twenty-four hours after replating, cells were exposed to AZD1390 (2 µM) and incubated for 7 days in Dox-supplemented culture medium. Uninduced (PBS) and DMSO-treated cells were used as negative control.
After the treatment period, colonies were fixed with methanol on ice, stained with 0.5% crystal violet (Sigma-Aldrich, Cat#HT90132) and imaged using Carl Zeiss Stemi 2000-C Stereo microscope equipped with a CCD camera (Zeiss) at 0.65× magnification. Colonies were counted on whole surface area using the Clono-counter software and values were normalized to control wells .
Real-time cell proliferation assay
Cells were plated into 384-well plates (500 cells/well) in 30 µl complete culture media. After overnight incubation, cells were treated with GSK126 (7.5 µM), AZD1390 (2 µM) or the combination of both. DMSO served as vehicle control. Cells were allowed to grow for 120 h. Phase-contrast images were automatically acquired by IncuCyte FLR (Essen Bioscience) from the incubator at 4-h intervals. Proliferation was monitored by analyzing the occupied area (% confluence) of cell images over time by IncuCyte software (Essen Bioscience).
Cell lysis and immunoblotting
Whole-cell lysates were prepared in RIPA lysis buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl, 0.1% SDS, 0.5% Sodium deoxycholate) supplemented with 1 mM PMSF, 1% SDS, and phosphatase inhibitors (1x PhosphoSTOP Phosphatase Inhibitor Cocktail, Roche Diagnostics, Cat#4906845001). Samples were lysed on ice for 20 min, sheared by passing cells through a G25 needle (B. Braun, Hypodermic Needle-Pro®, Cat#4658304), cleared by centrifugation (14,000 × g, 15 min), and quantified using Pierce™ Bradford protein assay kit (Thermo Scientific, Cat#23200). Lysates were boiled 5 min at 95 °C with 6x reducing Laemmli buffer (0.12 M Tris pH 6.8, 47% glycerol, 12% SDS, 0.6 M DTT, 0.06% bromophenol blue). Samples were separated on a polyacrylamide gel and transferred to PVDF membranes using the mini wet/tank blotting system (Bio-Rad). After blocking with 5% BSA in Tween-20/PBS, membranes were probed with primary antibodies prepared in blocking solution overnight at 4 °C on a roller, followed by incubation with horseradish peroxidase-conjugated secondary antibody in blocking solution for 1 h at room temperature and ECL detection (Thermo Fisher, Cat#34096) by the ChemiDoc XRS + system (Bio-Rad). Primary and secondary antibodies used for immunoblotting are listed in Additional file 8: Table S1. Quantitative analysis of protein expression relative to GAPDH was done using Image Lab software (Bio-Rad).
Analysis of apoptosis by flow cytometry
Cell lines (150,000 cells/well) were seeded into 6-well plates at 40–60% confluency. After overnight incubation, medium was replaced with growth medium containing single inhibitors GSK126 (7.5 µM) or AZD1390 (2 µM) or in combination for 48 h. Culture medium was collected, cells were trypsinized, washed with ice-cold PBS, and incubated with Annexin-V (BD Bioscience, Cat#556420) and propidium iodide (PI, Carl Roth, Cat#CN74, 5 µg/ml) in antibody binding buffer (2.5 mM CaCl2, 10 mM HEPES (pH 7.4), 140 mM NaCl, 20% accutase solution (Sigma-Aldrich, Cat#A6964), 70% PBS). Annexin-V/propidium iodide staining was detected by flow cytometry (Beckman Coulter, Gallios Flow Cytometer). The fraction of apoptotic cells was quantified as the Annexin-V, PI, and double positive stained populations using the Kaluza analysis software (Beckman Coulter).
Immunofluorescence staining and high-throughput microscopy
Cells (5000 cells/well) were seeded into 96-well imaging plates (Greiner Bio-One, µclear, Cat#655090). After overnight incubation, cells were treated with single or combined agents of GSK126 (7.5 µM) and AZD1390 (2 µM). Treatment with 1 µM cisplatin was used as positive control. For pre-extraction of nonchromatin-bound proteins, after 48 h of treatment cells were incubated with sucrose buffer (25 mM HEPES pH 7.5, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 300 mM sucrose, 0.5% TritonX-100) for 2 min, followed by fixation in 4% paraformaldehyde for 10 min at RT. After 1 h of blocking (5% BSA, 2% normal goat serum, 0.1% TritonX-100, 0.05% Tween-20) at RT, cells were incubated with primary and secondary antibodies as listed in Additional file 8: Table S1. After three final washes, cells were covered with PBS and stored at 4 °C until scanning. Quantitative high-throughput microscopy was performed similarly as described before . We used a Thermo Fisher Cellomics CellInsight CX7 LZR High Content Analysis (HCA) Platform with laser light source to scan stained cell models in 96-well imaging plates. 2 × 2 binned images (1104 × 1104 pixels) were acquired with a 20× (0.4 NA) Achroplan objective using the laser-based autofocus and analyzed using the Spotdetector V4.1 Bioapplication of the Cellomics software package (Version 6.6.2, Built 8533). Cell nuclei were identified by Hoechst 34580 staining in background corrected images (3D surface fitting) according to the object identification parameters size: 100–1500 μm2, ratio of perimeter squared to 4π area: 1–5, length-to-width ratio: 1–5, average intensity: 500–8000, total intensity: 2x105–5x107. Foci were identified within the nuclear region using the Box method with a value of 3. Object selection parameters for foci were 1–30 μm2, ratio of perimeter squared to 4π area: 1–5, length-to-width ratio: 1–5, average intensity: 500–16,000, total intensity: 3x102–1x106.
Orthotopic tumor transplantation and in vivo drug intervention study
This study is compliant with all relevant ethical regulations regarding animal research. All animal experiments were approved by the Animal Ethics Committee of The Netherlands Cancer Institute (Amsterdam, the Netherlands) and performed in accordance with the Dutch Act on Animal Experimentation. Generation of conditional K14cre; Brca1F/F; Trp53F/F (KB1P) and K14cre; Trp53F/F (KP) breast cancer mouse models were described previously [68, 69]. Orthotopic transplantations, tumor monitoring, and tissue sampling were performed as described before . Briefly, donor KB1P tumor fragments were transplanted into the fourth mammary fat pad of 8-weeks old FVB females and treatments began upon reaching tumor outgrowth of approximately 100 mm3 (100%). Maximal tolerable dose (MTD) for the combined therapy with GSK126 and AZD1390 was determined prior to the intervention study using FVB females. GSK126 (Syncom) was reconstituted in 20% Captisol (CyDex Pharmaceuticals) and brought to a pH of 4.5 with 10 M potassium hydroxide, to create a working stock of 15 mg/mL. AZD1390 (Selleckchem) was dissolved at 40 mg/ml in ethanol and then dropwise added to 0.5% (w/v) HPMC, 0.1% (w/v) Tween-80 to achieve a final concentration of 2 mg/ml. Tumor-bearing mice were blindly randomized into four treatment groups and treated with vehicle (daily intraperitoneal injection), GSK126 (150 mg/kg, daily intraperitoneal injection), AZD1390 (bi-daily 20 mg/kg, by oral gavage for 5 days on, 2 days off) or a combination of GSK126 and AZD1390. Animals were treated for 28 consecutive days and mammary tumor volume (mm) was quantified by caliper measurements using the following formula: 0.5 × length × width2. For a better comparability, the tumor volume at day x was normalized to the initial tumor volume at treatment start (day 0) and defined as relative tumor volume (RTV). The endpoint of this study was reached when tumor size was 10 times the RTV, defined as progression-free survival (PFS). Animals were euthanized by CO2 when tumors extended a volume of 1500 mm3 or when severe side effects were observed. All procedures were carried out by animal technicians in a blinded fashion.
In vivo target inhibition by GSK126 and AZD1390
A small cohort of mice (n = 12) was sacrificed after 7 days of single agent or combined treatment with GSK126 and AZD1390, and KB1P tumors were harvested for immunohistochemistry analysis (IHC). IHC staining of EZH2, H3K27me3, and phosphorylated ATM was performed using formalin-fixed paraffin-embedded tumor tissue. The following monoclonal antibodies were used for immunohistochemistry: EZH2 (Rb, Cell signaling, Cat#5246, 1:200), H3K27me3 (Rb, Abcam, Cat#ab6002, 1:100) and phosphorylated ATM (phospho S1981, Rb, Abcam, Cat#ab81292, 1:400) overnight at 4 °C. These antibodies were extensively tested for target specificity . All slides were digitally processed using the Aperio ScanScope (Aperio, Vista, CA, USA) and captured using ImageScope software version 12.0.0 (Aperio).
Statistical analysis was performed using GraphPad Prism Version 8 and 9. Mann–Whitney U test, one-way ANOVA with Tukey multiple comparison testing or Kaplan–Meier survival testing with log-rank comparison were used as indicated in the figure legends. p values below 0.05 were considered statistically significant. Significance levels are indicated as: *—p ≤ 0.05; **—p ≤ 0.01; ***p ≤ 0.001; nonsignificant levels are not labeled. Number of experimental replicates are indicated in the figure legends.