Provenience of cell lines and cell culture conditions

A673 and HEK293T cells were purchased from American Type Culture Collection (ATCC). MHH-ES1, RDES, RH1, SK-ES1, and SK-N-MC cells were provided by the German Collection of Microorganisms and Cell Cultures (DSMZ). TC-32, TC-71, and CHLA-10 cells were kindly provided by the Children’s Oncology Group (COG) and EW1, EW3, EW7, EW16, EW17, EW18, EW22, EW23, EW24, LAP35, MIC, ORS, POE, STA-ET1, STA-ET8 cells were provided by O. Delattre (Institute Curie, Paris). A673/TR/shEF1 cells were kindly provided by J. Alonso (Madrid, Spain)35. The SK-N-MC cell line is listed in the database of commonly misidentified cell lines, ICLAC (http://iclac.org/databases/cross-contaminations), as it was initially described to be a neuroblastoma cell line. Indeed, it is a EwS cell line expressing the pathognomonic fusion oncogene EWSR1-FLI1. All cell lines were grown in humidified atmosphere at 37 °C and 5% CO 2 . Cells were cultured in RPMI 1640 medium supplemented with stable glutamine (Biochrom), 10% tetracycline-free FCS (Sigma-Aldrich), 100 Uml−1 penicillin (Biochrom), and 100 µg ml−1 streptomycin (Biochrom). Cells were routinely checked by nested PCR for mycoplasma infection, and their purity was confirmed by STR-profiling and, if applicable, by PCR-based detection of specific fusion oncogenes.

DNA/RNA extraction, reverse transcription, and qRT-PCR

DNA was extracted with the NucleoSpin Tissue kit (Macherey-Nagel); plasmid DNA was extracted from bacteria with the PureYield kit (Promega). RNA extraction was performed with the NucleoSpin RNA kit (Macherey-Nagel) and RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). qRT-PCRs were performed using SYBR Select Master Mix (Applied Biosystems) and reactions were run on a Bio-Rad CFX Connect instrument and analyzed using the Bio-Rad CFX Manager 3.1 software. All oligonucleotides were purchased from MWG Eurofins Genomics. For primer sequences see Supplementary Data 11.

Transient transfection

For siRNA transfection, cells were seeded in a six-well plate at a density of 1.5 × 105 per well in 1.6 ml of growth medium. The cells were transfected with either a negative control non-targeting siRNA (Sigma-Aldrich MISSION siRNA Universal Negative Control #1) or specific siRNAs (25–65 nM, depending on the cell line and the siRNA) and HiPerfect (Qiagen). Cells were retransfected 48 h after the first transfection and harvested 96 h after the first transfection. siRNA sequences are given in Supplementary Data 11. For plasmid transfection, cells were seeded in a six-well plate at a density of 2 × 105 per well in 1.8 ml of growth medium. Plasmids were transfected with Lipofectamine LTX and Plus Reagent (Invitrogen). The pGL3 vector used for reporter assays has been described before16.

Doxycycline (DOX)-inducible shRNA constructs

Either a non-targeting negative control shRNA (MWG Eurofins Genomics) or specific shRNAs targeting EWSR1-FLI1 or MYBL2 (both MWG Eurofins Genomics) were cloned in the pLKO-Tet-on-all-in-one system36. Oligonucleotide sequences are given in Supplementary Data 11. Lentiviruses were produced in HEK293T cells. A673 and SK-N-MC EwS cells were infected with respective lentiviruses and selected with 1.5 µg ml−1 puromycin (Invivogen). After single-cell cloning, knockdown efficacy of individual clones was assessed by qRT-PCR 48 h after addition of DOX (1 µg ml−1; Sigma-Aldrich).

DNA constructs and reporter assays

MYBL2-associated GGAA-microsatellites (with ~440 bp 5′ and 3′ flanking regions) from three EwS cell lines were PCR-cloned upstream of the SV40 minimal promoter into the pGL3-Fluc vector (Promega)16. Primer sequences are given in Supplementary Data 11. The presence of additional variants devoid of the GGAA-microsatellite was ruled out by WGS of the parental cell lines and Sanger sequencing of the cloned fragments. A673/TR/shEF1 cells (2 × 105 per well) were transfected with the Firefly pGL3-Fluc vector containing respective microsatellites and the Renilla pGL3-Rluc vector (Promega) (ratio 100:1) in a six-well plate with 1.8 ml of growth medium. Four hours after transfection, transfection medium was replaced by medium with/without DOX (1 µg ml−1; Sigma-Aldrich). Cells were lysed and assayed with a dual luciferase assay system (Berthold) after 72 h. Firefly luciferase activity was normalized to that of Renilla.

CRISPR interference (CRISPRi) and analysis of cell growth

Due to the lack of functional DNAse, CRISPRi does not cause a knockout of the targeted DNA sequence, but blocks protein binding to it26,28. For the reported experiments, a DNAse-dead CAS9 (dCAS9) fused to the KRAB effector domain, which promotes an inhibiting chromatin state, is targeted to the genomic region of interest by specific gRNAs to silence the activity of a given enhancer26,28. To achieve this, we used a pHAGE TRE dCas9-KRAB vector (Addgene #50917) and a pLKO.1-puro U6 sgRNA BfuAI large stuffer vector (Addgene #52628), the latter containing either two gRNAs, targeting sequences adjacent to the MYBL2-associated GGAA-microsatellite, or a scrambled control (Supplementary Data 11). Lentivirus production was performed in HEK293T cells. RDES EwS cells were infected with the respective lentiviruses and selected with 1 µg ml−1 puromycin and 1.5 µg ml−1 G418 (both Invivogen). The cells were induced with DOX (1 μg ml−1; Sigma-Aldrich) for 5 days, after which MYBL2 and EWSR1-FLI1 levels were measured by qRT-PCR.

For measurement of cell growth, cells were grown in medium containing selection antibiotics and DOX (2 µg ml−1) for 14 days as described37. Thereafter, 8 × 104 cells/well were plated in quadruplicate wells of 24-well plates in the presence of DOX. After four additional days, cells were washed and fixed with trichloroacetic acid for 1 h at 4 °C. Then, plates were washed with phosphate-buffered saline (PBS), air dried, and cells were stained with crystal violet (Sigma-Aldrich) for 30 min. Surplus crystal violet was removed by rinsing the plates with PBS. Cell-bound crystal violet was dissolved in 10% acetic acid, and optical density was measured at 595 nm in a DS-11 spectrophotometer (DeNovix Inc.).

Western blot

Protein from A673/TR/shEF1 cells was extracted at d0, d7, d11, d14, and d17 with RIPA and anti-protease cocktail (Roche). Western blots were performed following routine protocols and specific band detection was achieved by the use of rabbit monoclonal anti-FLI1 antibody (1:1000, ab133485; Abcam)38, rabbit polyclonal anti-MYBL2 antibody (1:500, sc-725; Santa Cruz)39, and mouse monoclonal anti-ß-actin (1:10,000, A-5316; Sigma-Aldrich). Anti-rabbit IgG horseradish peroxidase-coupled antibody (1:3000, Amersham Bioscience) and anti-mouse IgG horseradish peroxidase coupled antibody (1:3,000; Amersham Bioscience) was used as secondary antibody. Proteins were visualized using chemiluminescence (Pierce ECL Western blot chemiluminescent substrate; Thermo Fisher Scientific).

Proliferation assays

Cells were seeded in a six-well plate at a density of 1.5 × 105 per well in 1.6 ml of growth medium. The cells were transfected with either a negative control non-targeting siRNA in duplicate wells (Sigma-Aldrich MISSION siRNA Universal Negative Control #1) or up to four specific siRNAs (MWG Eurofins Genomics) (25–65 nM, depending on the cell line and siRNA) using HiPerfect (Qiagen). siRNA sequences are given in Supplementary Data 11. Retransfection was performed 48 h after the first transfection. Ninety-six hours after the first transfection, cells were harvested (including supernatant), stained with Trypan blue (Sigma-Aldrich), and counted in a standardized hemocytometer (C-Chip, NanoEnTek).

Analysis of cell cycle and apoptosis

Analysis of cell cycle phases was performed by propidium iodide (PI) (Sigma-Aldrich) staining. Cells were transfected with siRNAs equivalently to the proliferation assays (see above), harvested after 96 h (including supernatant), fixed in ethanol (70%) at 4 °C, and stained with PI solution (50 µg ml−1, with 20 µg ml−1 RNAse A (Invitrogen)). Analysis of apoptosis has been performed by combined Annexin V-FITC/PI staining (BD Pharmingen FITC Annexin V Apoptosis Detection Kit II; BD Biosciences). Cells were transfected with siRNAs equivalently to the proliferation assays (see above) and harvested after 96 h (including supernatant). The samples were assayed on an Accuri C6 flow cytometer and analyzed with the Accuri C6 CFlow Plus software. An example of the gating strategy is given in Supplementary Fig. 6.

Colony-forming assays

A673 and SK-N-MC cells containing either a DOX-inducible non-targeting control shRNA or MYBL2-targeting specific shRNAs were seeded in triplicate wells of a 12-well plate at a density of 500 cells (A673) or 1000 cells (SK-N-MC) per well in 2 ml of growth medium. Cells were grown with/without DOX (1 µg ml−1; Sigma-Aldrich) for 10–14 days depending on the cell line and afterwards stained with crystal violet (Sigma-Aldrich). Colony number was determined on scanned plates using Fiji (ImageJ)40,41.

cDNA library and RNA sequencing (RNA-seq)

A673, SK-N-MC, and RDES EwS cell lines were transfected in triplicates with either a negative control non-targeting siRNA or a specific siRNA targeting MYBL2 (siMYBL2_1). Total RNA was extracted using the NucleoSpin II kit (Macherey-Nagel). Complementary DNA libraries were sequenced with an Illumina HiSeq2500 instrument using 150 bp paired-end sequencing. Obtained reads were aligned on the human genome (hg19) using TopHat (version 2.0.6)42. Counting of reads on annotated genes from the GRCh37 gene build was done using htseq-count (v. HTSeq-0.5.3p9)43 with the following parameters: htseq-count -a 10 -q -s no -m union. Sample-to-sample normalization and differential expression analyses were performed using the R package DESeq2 (v.1.18.0)44. RNA-seq data were deposited at the Gene Expression Omnibus (GEO; accession code GSE119972).

Chromatin immunoprecipitation and sequencing (ChIP-seq)

DNA–protein cross-linking was done in the presence of 1% of paraformaldehyde on 12 × 106 A673 or 4 × 106 RDES cells, respectively, for each condition for 10 min. Cell lysis, chromatin shearing, immunoprecipitation, and DNA purification were performed with reagents from iDeal ChIP-seq kit for Transcription Factors (Diagenode, ref: C01010054). Chromatin shearing was carried out in a Bioruptor (Diagenode) using 20 cycles of sonication (30 s high, 30 s off) in TPX tubes (Diagenode, ref: 50001). For immunoprecipitation of activated MYBL2, 2 µg of a monoclonal ChIP-grade rabbit anti-p-MYBL2 antibody (Abcam, ab76009, lot GR113270–6)45 were used. MYBL2 ChIP and input were sequenced on an Illumina HiSeq2500 instrument (100 bp single-end). For immunoprecipitation of EWSR1-FLI1, 2 µg of a polyclonal ChIP-grade rabbit anti-FLI1 antibody (Abcam, ab15289, lot GR293950-1)18 was used. EWSR1-FLI1 ChIP and input were sequenced on an Illumina HiSeq2500 instrument (150 bp single-end). ChIP-seq reads were aligned to the human genome (hg19 version) with Bowtie2 (ref. 46). Peaks were called with MACS2 with option narrow47. To normalize, we took the input dataset from the same cell line. PAVIS was used for peak annotation and visualization48. For analysis of the number of EWSR1-FLI1 ChIP-seq reads spanning the different haplotypes of the MYBL2-associated GGAA-microsatellite in RDES cells, exclusively the spanning reads were extracted from the BAM file with SAMtools, and mapped to the corresponding haplotype according to CIGAR scores49. Significance levels were calculated using a binomial test (p = 0.5). ChIP-seq data concerning MYBL2 were deposited at the GEO (accession code GSE119972).

Analysis of published ChIP-seq and DNAse-seq data

Publicly available ENCODE SK-N-MC DNAse-seq data (GSM736570) and pre-processed A673 and SK-N-MC ChIP-seq data (GSE61944) were retrieved from the GEO and displayed in the UCSC genome browser. Samples used: GSM1517544 SK-N-MC_shGFP_48h_FLI1; GSM1517553 SK-N-MC_shFLI1_48h_FLI1; GSM1517569 A673_shGFP_48h_FLI1; GSM1517572 A673_shFLI1_48h_FLI1; GSM1517548 SK-N-MC_shGFP_96h_H3K4me1; GSM1517557 SK-N-MC_shFLI1_96h_H3K4me1; GSM1517545 SK-N-MC_shGFP_48h_H3K27ac; GSM1517554 SK-N-MC_shFLI1_48h_H3K27ac; GSM1517568 A673 whole-cell extract (WCE).

CDK2 inhibitor assays in vitro

Cells were seeded in a 96-well plate at a density of 5 × 103 per well. In case of cells containing DOX-inducible constructs, cells were treated with/without DOX (1 µg ml−1; Sigma-Aldrich). Twenty-four hours after seeding or pre-incubation with DOX, respectively, CDK2 inhibitors (CVT-313 or NU6140; Merck and Tocris) were added in serially diluted concentrations ranging from 0.001 to 100 µM. Each well contained an equal concentration of 0.5% DMSO (Sigma-Aldrich). Cells only treated with 0.5% of DMSO served as a control. After 72 h of inhibitor treatment, the plates were assayed on a Thermo Fisher Varioskan plate reader after incubation with Resazurin (20 µg ml−1; Sigma-Aldrich) for 6 h.

Xenotransplantation and CDK2 inhibitor treatment in vivo

All mouse experiments were approved by the local authorities in compliance with all relevant ethical regulations (including, but not limited to, tumor size). Sample size was predetermined using power calculations with β = 0.8 and α = 0.05 based on preliminary data and in compliance with the 3 R system (replacement, reduction, refinement). 3 × 106 A673 and SK-N-MC cells, containing either a DOX-inducible negative control shRNA or specific shRNAs against EWSR1-FLI1 or MYBL2, were injected subcutaneously with a 1:1 mix of PBS (Biochrom) and Geltrex (LDEV-Free Reduced Growth Factor Basement Membrane Matrix, Thermo Fisher Scientific; max volume 100 µl) in the right flanks of 3–9 months old female or male NSG mice (Charles River Laboratories). For shRNA sequences see Supplementary Data 11. When tumors were palpable, mice were randomized to the control group (17.5 mg ml−1 sucrose (Sigma-Aldrich) in drinking water) or the treatment group (2 mg ml−1 DOX (Beladox, bela-pharm) and 50 mg ml−1 sucrose (Sigma-Aldrich) in drinking water). Tumor size was measured with a caliper every 2 days and tumor volume was calculated as V = a × b2/2 with a being the largest diameter and b being the smallest diameter. Once the tumors reached a volume of 1500 mm3 respective mice were sacrificed by cervical dislocation. Other humane endpoints were determined as follows: Ulcerated tumors, loss of 20% body weight, constant curved or crouched body posture, bloody diarrhea or rectal prolapse, abnormal breathing, severe dehydration, visible abdominal distention, obese Body Condition Scores (BCS), apathy, and self-isolation. For CDK2 inhibitor treatment in vivo, cells were injected as described above. When tumors were palpable, mice were assigned to either the vehicle (DSMO) or a treatment group (20 or 40 mg kg−1), each with or without addition of DOX to the drinking water (2 mg ml−1 DOX; Beladox, bela-pharm). In case of CVT-313 (Tocris) treatment, DOX was not applied. The CDK2 inhibitors NU6140 or CVT-313 (both Tocris) were administered i.p. for 12 days, with a break of 1 day every 4 days of treatment. The experimental endpoint was predetermined as 14 days after first injection of either inhibitor, or if humane endpoints as described above were reached before. To check histomorphological changes of inner organs upon CDK2 inhibitor treatment, we examined hematoxylin and eosin (HE) stained slides of heart, lungs, liver, stomach, pancreas, intestines, kidneys, adrenal glands, bone marrow, and spleen from treated and non-treated mice. Tumor tissues were subjected to HE staining, as well as immunohistochemical staining for p-MYBL2 and cleaved caspase 3 (as described below). Animal experiments were approved by the government of Upper Bavaria and conducted in accordance with ARRIVE guidelines, recommendations of the European Community (86/609/EEC), and UKCCCR (guidelines for the welfare and use of animals in cancer research).

Survival analysis

Microarray data of 166 primary EwS tumors (GSE63157, GSE34620, GSE12102, GSE17618) for which clinical annotations were available were downloaded from the GEO. Data were either generated on Affymetrix HG-U133Plus2.0 or on Affymetrix HuEx-1.0-st microarray chips and separately normalized by RMA using custom brainarray chip description files (CDF, v20). ComBat was used to remove batch effects50,51. Patients were stratified by their quintile or median intra-tumoral gene expression levels. Mantel–Haenszel test was performed to calculate significance levels, using either a custom code (GenEx) for batch queries or GraphPad PRISM version 5 for individual genes (GraphPad Software Inc., CA, USA). P values < 0.05 were considered as statistically significant. Survival data were crossed with gene expression microarray data (Affymetrix HG-U133A2.0) generated in A673/TR/shEF1 cells (GSE27524; 53 h DOX-treatment), which were normalized as described above (RMA with brainarray CDF, v19).

Gene-set enrichment analysis

Using the Affymetrix gene expression dataset comprising 166 primary EwS patients, enrichment of gene-sets that are among MYBL2 co-regulated genes were identified by ranking of Pearson´s correlation coefficient of the expression of every gene with MYBL2 expression and performance of a pre-ranked GSEA with 1000 permutations52. Using the RNA-seq dataset containing DEGs after siRNA-mediated MYBL2 knockdown compared to a non-targeting siControl in A673, SK-N-MC, and RDES EwS cell lines, all genes were ranked by their mean log2 FC and a pre-ranked GSEA was performed with 1000 permutations52.

GGAA-microsatellite analysis using HipSTR

EwS tumors and/or matched blood samples were collected with informed consent from EwS patients treated in the Hospital for Sick Children (SickKids) in Toronto, Canada, in accordance with Research Ethical Board (REB) guidelines (approval no. 1000053452). In addition, publicly available EwS reference samples from the International Cancer Genome Consortium (ICGC) with matched tumor/germline WGS data were used for analysis6. WGS was performed in all tumors and available matched germline samples using established protocols on Illumina instruments (paired-end 150/150 bp for the Toronto cohort, and paired-end 100/100 bp for the ICGC cohort). Paired-end FASTQ files were aligned to the human genome (hg19/GRCh37) using BWA-MEM (v.0.7.8). Indel realignment and base quality scores were recalibrated using the Genome Analysis Toolkit (v.2.8.1). For the Toronto cohort, published gene expression data were available from RNA-seq which was deposited at the European Genome-phenome Archive (EGA) under accession number EGAS00001003062; and for the ICGC cohort from matched Affymetrix HG-U133A or HG-U133Plus2.0 gene expression arrays (GSE37371; GSE7007; GSE34620). Affymetrix gene expression data were normalized separately for each chip type by RMA53 using custom brainarray CDF (v20, ENTREZ)54. Batch effects were removed using ComBat50,51. For eQTL analyses, only tumor samples with a minimum tumor purity of >60%, corresponding to TCGA standard tissue requirements (http://cancergenome.nih.gov/cancersselected/biospeccriteria), were used. Tumor purity estimates were made using the AscatNGS (Toronto cohort)55 or the ESTIMATE algorithm (ICGC cohort)56. To call the genotypes of the MYBL2-associated GGAA-microsatellite, we applied HipSTR (v.0.6.2)23 on the WGS data using a minimum threshold of ten reads. All genotypes passed the following HipSTR default filters: --min-call-qual 0.9; --max-call-flank-indel 0.15; --max-call-stutter 0.15; --min-call-allele-bias -2; --min-call-strand-bias -2.

Human samples and ethics approval

Archived human tissue samples were retrieved from the Institute of Pathology of the LMU Munich (Germany) and the Gerhard-Domagk Institute of Pathology of the University Hospital of Münster (Germany). All patients provided written informed consent. Retrospective and blinded analysis of anonymized samples was carried out upon ethical approval of LMU Munich’s ethics committee (approval no. 550-16 UE).

Tissue microarrays and immunohistochemistry

Formalin-fixed paraffin-embedded samples were collected at the Institute of Pathology of the LMU Munich57. We harvested at least two cores per sample with a core-diameter of 1 mm from all blocks to construct tissue microarrays. All EwS samples showed cytogenetic evidence for a translocation of the EWSR1 gene either as determined by fluorescence in situ hybridization and/or qRT-PCR. The samples were reviewed by a reference pathologist. Four-micrometer sections were cut for immunohistochemistry and antigen retrieval was performed with microwave treatment using the antigen retrieval ProTaqs I Antigen-Enhancer (Quartett) for p-MYBL2 or the Target Retrieval Solution (Agilent Technologies) for cleaved caspase 3. In total, 7.5% aqueous H 2 O 2 solution (room temperature) and blocking serum from the corresponding kits were used for 20 min for blockage of endogenous peroxidase. Then slides were incubated for 60 min with the primary antibodies anti-p-MYBL2 (1:100 dilution; Abcam, ab76009) and anti-cleaved caspase 3 (1:100 dilution, Cell Signaling, #9661). Afterwards slides were incubated with a secondary anti-rabbit IgG antibody (MP-7401, ImmPress Reagent Kit, Peroxidase-conjugated) followed by subsequent target detection using DAB+chromogen (Agilent Technologies). Slides were counterstained with hematoxylin Gill’s Formula (H-3401; Vector).

Evaluation of immunoreactivity and quantification of mitoses

Evaluation of p-MYBL2 immunostaining was carried out semi-quantitatively by a blinded observer in analogy to the Immune Reactive Score (IRS), which is used routinely by pathologists for quantification of hormone receptor expression in mammary carcinoma, ranging from 0 to 12 as described58. The intensity of p-MYBL2 immunoreactivity (score 0 = none, score 1 = low, score 2 = intermediate, and score 3 = strong) and the percentage of cells stained with each intensity (score 0 = 0%, score 1 = 0–9%, score 2 = 10–50%, score 3 = 51–80%, and score 4 = 81–100%) was determined per high-power field (×40). The product of the predominant intensity score and its percentage score defined the final IRS. For cleaved caspase 3 immunostaining, automated quantification of the percentage of positive high-power field area was performed using Fiji (ImageJ)40,41. Mitoses were quantified in HE-stained slides by a blinded observer per high-power field. Final scores/quantifications were determined by examination of 4–16 high-power fields of at least one section for each sample.

Statistical analysis and software

Statistical data analysis was performed using GraphPad PRISM 5 (GraphPad Software Inc., CA, USA) on the raw data. If not otherwise specified in the figure legends comparison of two groups in functional in vitro experiments was carried out using a two-tailed Mann–Whitney test; ***P < 0.001, **P < 0.01, *P < 0.05. Comparison of three groups with data in ordinal scale was performed using Kruskal–Wallis test. If not otherwise specified in the figure legends, data are presented as dot plots with horizontal bars representing means, and whiskers representing the standard error of the mean (SEM). Sample size for all in vitro experiments was chosen empirically. In Kaplan–Meier survival analyses, curves were calculated from all individual survival times of patients or mice, respectively. Curves were compared by Mantel–Haenszel test to detect significant differences between the groups. For in vivo experiments, sample size was predetermined using power calculations with β = 0.8 and α = 0.05 based on preliminary data and in compliance with the 3R system (replacement, reduction, refinement).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.