Cancer cell cultures. Cell lines were obtained from the American Type Culture Collection (ATCC), grown under recommended conditions and maintained at 37°C in a humidified atmosphere of 5% CO2/95% air. The cells lines were authenticated by genotyping for TP53 and other known mutations. All cell lines used in this study were mycoplasma-free. Genomic DNA and total RNA were extracted by standard protocols. One primary lung cancer cell line PCD11 was derived from malignant pleural effusions37. Two primary cancer cell lines cultures were derived from orthoxenografts/PDOXs generated in nude mice from two primary tumours of two SCCOHT patients (OVA250L and OVA259L) that were obtained from Bellvitge Hospital and the Catalan Institute of Oncology (ICO) with the approval of the Ethical Committee (CEIC Bellvitge). Ethical and legal protection guidelines of human subjects, including informed consent, were followed. Fresh orthoxenografts/PDOXs grown in the mouse ovaries were collected when mice were sacrificed at passage #1, then minced with sterile scalpels. Single cells and clumps were transferred to cell culture plates and maintained in DMEM supplemented with 10% FBS plus 50 U/mL penicillin and 50 mg/mL streptomycin under standard culture conditions. When cell colonies with epithelial cell morphology were observed, cells were trypsinized and expanded. Both primary cell lines were considered established after > 6 passages in vitro. Specific informed consent was obtained from all patients for tumour implantation into mice, and the study was approved by the IDIBELL Ethics Committee (No. AAALAC-1155).
Antibodies and western blots. The following primary antibodies were used for western blots: anti-TUBULIN, T6199 mouse (1/10000, Sigma Aldrich, St. Louis, MO, USA); anti-Beta-ACTIN, 13854 (1/20000 Sigma Aldrich); anti-SMARCA4 49360S (1:1000, Cell Signaling Technology); anti-EZH2 5246S (1:1000, Cell Signaling); anti-H3K27ac D5E4 (1:1000, Cell Signaling) anti-H3K27me3 07-449 (1:1000, Cell Signaling); anti-UTX (KDM6A) D3Q1I (1:1000, Cell Signaling); anti-KDM6B (JMJD3) AB154126 (1:1000 Abcam) (see also Supplementary Tables). For western blots, whole-cell lysates were collected in a buffer containing 2% SDS 50 mM Tris-HCl (pH 7.4), 10% glycerol and protease inhibitor cocktail (Roche Applied Science). Protein concentrations were determined using a Bio-Rad DC Protein Assay kit (Life Science Research). Equal amounts of lysates (20 µg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane that was blocked with 5% nonfat dry milk. Membranes were incubated with the primary antibody overnight at 4°C, then washed before incubation with species-appropriate IRDye 680CW (925-68022) or IRDye 800CW (925-32213) fluorescent secondary antibodies (1:10.000 LI-COR, NE, USA) for 1 h at room temperature.
Treatments and shRNAs. Chemicals were obtained from the following sources: SAHA, suberoyl anilide hydroxamic acid (Cayman Chemical Company, Ann Arbour, MI, USA); GSK-J4 (Shelleckchem); GSK-126 (Cayman Chemical Company). shRNAs against SMARCA4, KDM6a and KDM6B were purchased from SIGMA-MISSION (LentiExpressTM Technology, Sigma-Aldrich) as a glycerol stock of five pLKO plasmids carrying specific shRNA sequences. A non-target shRNA (shNT) (Sigma MISSION shRNA non-mammalian control SHC002) was used as a control. The lentiviruses were generated within the 293T packaging cells.
Cell growth analysis and calculation of EC50 and combination index (CI). For cell viability assays, cell lines were incubated in 96-well plates. Before harvesting, cells were treated for 5-7 days with the indicated concentrations of each compound (SAHA, GSK-J4, GSK-126) or combinations. For the assays, 10 µl of a solution of 5 mg/mL MTT [3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma Chemical Co.) was added. After incubation for 3 h at 37°C, the medium was discarded, the formazan crystals that had formed were dissolved in 100 µl of lysis buffer (50% N-N-dimethylformamide in H2O, 20% SDS, 2.5% glacial acetic acid, NaOH 5 mol/L, pH 4.7), and absorbance was measured at 596 nm. Results are presented as the median of at least two independent experiments performed in triplicate for each cell line and for each condition. For EC50 calculations, cells were treated with each drug and their various combinations for 5 days. Estimates of EC50 were derived from the dose response curves. To assess the drug concentration effect and to calculate the combination index (CI), cells were plated in 96-well plates and incubated with a concentration of SAHA ranging from 0.07 μM to 10 μM (0.07, 0.15, 0.3, 0.6, 1.25, 2.5, 5.0 and 10 μM), and the same for GSK-J4 for 5 days. MTT assays were performed and the EC50 was determined by fitting the dose-response curve utilizing the CompuSyn software. The CI values for each dose and the corresponding effect level were calculated. The combination index offers a quantitative definition for drug combinations in which CI < 1, CI = 1 and CI > 1 indicate synergism, an additive effect, and antagonism, respectively.
For clonogenic assays, the plates were seeded with 5,000 cells from each cell line, then treated with SAHA (1 µM), GSK-J4 (1 µM) or GSK-126 (1 µM) for 5 days. Cells were stained with crystal violet solution (0.5% Crystal Violet in 25% of methanol).
Quantitative RT-PCR. To assess mRNA levels of the KDMs in different cells qPCR analysis was performed. 1 μg of total RNA was reverse-transcribed using SuperScript™ II reverse transcriptase (Invitrogen) and Random primers (Promega), according to the manufacturers’ instructions. qRT-PCR was performed in a Quantstudio 5 Real-Time PCR Instrument using SYBR Green PCR Master Mix (Applied Biosystems). Three biological replicates were carried out. Primer sequences are provided in the Supplementary Tables.
ChIP-sequencing. For ChIP, cells were grown in P-150 cm cell dishes and fixed with 1% methanol-free formaldehyde (Thermo Scientific) for 10 min at room temperature, then quenched by 125 mmol/L glycine for 15 min at room temperature, washed with ice-cold PBS twice and centrifuged at 200g, 4°C for 5 min. The pellet was resuspended in 1 mL of cell lysis buffer (10 mmol/L Tris-HCl, 10 mmol/L NaCl, 0.5% NP-40, protease inhibitor) and kept at 4°C, rotating for 30 min. After centrifugation, the pellet was resuspended in 1 mL of nuclear lysis buffer (1% SDS, 10 mmol/L EDTA, 50 mmol/L Tris-HCl pH 8.0, protease inhibitor) and kept at 4°C for 60 min. After another centrifugation, the lysate was sonicated with a Covaris M220 instrument to yield chromatin fragments of an average size of 0.25–1.00 kb, and then frozen at -20ºC for 30 min. The chromatin was thawed on ice and centrifuged at 2,500 g. For each ChIP reaction, 60 μL of Magna ChIP™ Protein A+G Magnetic Beads (Merck Millipore) was used in accordance with the manufacturer’s protocol. Before addition of the sheared chromatin to the beads, Triton X-100 and Na-deoxycholate was added to a final concentration of 10% each. 1% of the chromatin volume was used for input. At least two independent ChIP experiments were performed.
Immunoprecipitated chromatin was deep-sequenced in the Genomics Unit of the Centre for Genomic Regulation (CRG, Barcelona, Spain) using the Illumina HiSeq 2500 system (Illumina). Briefly, library preparation included end-repair, generation of dA overhangs, adapter ligation, size selection and removal of non-ligated adapters by agarose gene electrophoresis and amplification (18 cycles) before loading the samples into the sequencer.
For ChIP-sequencing data analysis, reads were aligned to the human reference genome hg38, using Bowtie v1.2.2, with default parameters and disallowing multi-mapping (–m 1)38. PCR duplicates were removed using PICARD (http://broadinstitute.github.io/picard/). Ambiguous and multi-mapped reads were discarded. Peaks were called using MACS2 v2.1.139. To avoid false positives, peaks were discarded if they were present in the ChIP-seq of SMARCA4 in the SMARCA4-deficient cells. Genomic peak annotation was performed with the R package ChIPpeakAnno v3.15, considering the region ± 2 kb around the TSS as the promoter40. All analyses considered peaks overlapping with promoter regions, unless otherwise specified. Peak lists were then transformed to gene target lists.
For heatmap and intensity plot representation of ChIP-seq signal, bedgraph files were generated using the makeUCSCfile function in HOMER with default parameters normalizing for differences in sample library size, and BigWig files were generated using the function bedGraphToBigWig from UCSC. Heatmaps were derived using the functions computeMatrix, in a window of ± 2 kb around the centre in the TSS, and plotHeatmap from deepTools41.
Generation of orthotopic tumour models and treatments. Male and female athymic nu/nu mice (ENVIGO) aged 4-5 weeks were maintained in a sterile environment before use in the lung cancer orthotopic experiments. Female athymic nu/nu mice (ENVIGO), 4-6 weeks old, were used for the ovarian cancer orthotopic studies. The animals were housed in individually ventilated cages on a 12-hour light-dark cycle at 21-23ºC and 40-60% humidity. Mice were allowed free access to an irradiated diet and sterilized water. All animal experiments were approved by the IDIBELL Ethical Committee under protocol 9111 approved by the Government of Catalonian, AAALAC accredited Unit 1155, and performed in accordance with guidelines stated in the International Guiding Principles for Biomedical Research Involving Animals, developed by the Council for International Organizations of Medical Sciences (CIOMS). To generate orthotopic lung tumour xenografts the cell lines were injected subcutaneously into the back of the mice (n = 3 mice/cell line). Once the solid tumour had entered the exponential growth phase, mice were sacrificed, the tumour was isolated under sterile conditions, and the non-necrotic areas were selected and minced in small fragments of 2-3 mm3. These were then orthotopically implanted in the lung parenchyma, as previously described20,27. On day 15, the mice were randomized and intraperitoneally treated with GSK-J4 (50 mg/kg/day for each mouse) or corresponding vehicle only. For the lung orthotopic models, in most cases the animals were sacrificed when they displayed serious respiratory difficulty, which was subsequently confirmed to be associated with lung tumour growth
Orthoxenografts or patient-derived orthotopic xenografts of small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) were generated. The primary tumour specimens for the two primary SCCOHT samples were freshly obtained at Hospital Universitari de Bellvitge (Hospitalet de Llobregat, Barcelona, Spain). The study was approved by the Institutional Review Board, and written informed consent was obtained from both patients. The orthotopic ovarian tumours were engrafted in mice, following an established protocol27. Briefly, non-necrotic tissue pieces (2–3 mm3) from resected carcinoma were selected and placed in DMEM (BioWhittaker) supplemented with 10% FBS and penicillin/streptomycin at room temperature. Under isoﬂuorane-induced anaesthesia, animals were subjected to a lateral laparotomy, their ovaries exposed and tumour pieces anchored to the ovary surface with prolene 7.0 sutures. Tumour growth was monitored 2 or 3 times per week and when the tumour had reached a sufficient size, it was harvested, cut into small fragments, and transplanted into between two and four new animals. Engrafted tumours (named OVA250X) at early mouse passages were cut into 6-8 mm3 pieces and stored in liquid nitrogen in a cryopreservation solution of 90% FBS and 10% dimethyl sulfoxide, awaiting subsequent implantation.
To generate the cisplatin-resistant ovarian xenograft mouse model, orthotopically engrafted OVA250X tumours at passage#1 were allowed to grow (n=3 mice) until palpable intra-abdominal masses were noted. Cisplatin was i.v.-administered to the animals (cycle #1, 3, 5 mg/kg dose) for 3 consecutive weeks (days 0, 7 and 15; cycle#1 of treatment) (Supplementary Fig. 8b). Post-cisplatin tumours at relapse were harvested and engrafted in new animals. This process of cisplatin treatment was repeated up to four times by treating tumour-bearing mice with stepwise-incremental doses of cisplatin: cycle #2, 4 mg/kg; cycle #3, 5 mg/kg and cycle #4, 5 mg/kg (see Supplementary Fig. 8b). Cisplatin-resistant tumours were obtained (OVA250XR). At doses higher than 3.5 mg/kg, signs of cisplatin induced some toxicity that were ameliorated by 2 days administration of saline containing 5% glucose. Mice were transplanted with fragments of OVA250X and OVA250XR tumours, and when tumours reached a homogeneous palpable size were randomly allocated into the treatment groups (n = 3-7 mice/group): i) Placebo; ii) GSK-J4 (50 mg/kg) and iii) cisplatin (3.5 mg/kg); drugs were administered once a day, 5 days per week, for 4 consecutive weeks. Animals were sacrificed on day 21 of treatment, and their ovaries dissected out and weighed. Representative fragments were either frozen in nitrogen or ﬁxed and processed for parafﬁn embedding.
Histopathology and immunostaining. For histological analysis, tumours were fixed and embedded in paraffin. Necrosis/fibrosis were morphological assessed after staining with haematoxylin and eosin (H&E), using standard protocols, and then examined by light microscopy in a blinded fashion. For immunostainings, 4-μm-thick paraffin-embedded sections of lung and ovarian tumour samples were deparaffinized overnight at 62ºC and then immersed in xylene. Samples were rehydrated and, after microwaving with Tris/EDTA pH 9.0 for antigen retrieval, endogenous peroxidase was inhibited with a 3% hydrogen peroxide solution, blocked in 10% goat serum and incubated with primary antibodies overnight at 4ºC (Supplementary Tables). HRP-conjugated polyclonal goat (anti-mouse and anti-rabbit) secondary antibodies (NeoStain ABC Kit, NeoBiotech) were used in 1-h incubations at room temperature. Labelling detection was done using an ImmPACT DAB Peroxidase (HRP) Substrate kit (Vector Laboratories, Burlingame, CA, USA), and tissue sections were counterstained with haematoxylin. Once dehydrated in an ethanol battery and cleared in xylene for 1 h, samples were mounted with coverslips with DPX mounting medium (Merck Millipore, Darmstadt, Germany). Sections were evaluated under a Leica DM1000 microscope by two independent observers in a blind fashion. Areas of necrosis/fibrosis were quantified using Photoshop. The scoring criteria for determining H3K27me3 staining were based on the staining intensity (four categories, 1-4). The mean of values from three independent evaluators was determined.
Statistical analysis. Student’s t-tests, EC50 calculations, Kaplan-Meier estimates and log-rank (Mantel-Cox) test were performed using Prism software (GraphPad). Values of P less than 5% were considered statistically significant. The statistical methods used for each analysis are specified in the figure legends.
Data availability. The ChIP-seq data obtained in this study has been uploaded to the Gene Expression Omnibus-GEO (NCBI), under accession number GSE155129.
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