The Ewing Sarcoma Cell Line Atlas (ESCLA) - methylation profiling of 18 Ewing sarcoma cell lines in EWSR1-ETS-high versus -low condition

Cell lines have been essential for major discoveries in cancer including Ewing sarcoma (EwS). EwS is a highly aggressive pediatric bone or soft-tissue cancer characterized by oncogenic EWSR1-ETS fusion transcription factors converting polymorphic GGAA-microsatellites (mSats) into neo-enhancers. However, further detailed mechanistic evaluation of gene regulation in EwS have been hindered by the limited number of well-characterized cell line models. Here, we present the Ewing Sarcoma Cell Line Atlas (ESCLA) comprising 18 EwS cell lines with inducible EWSR1-ETS knockdown that were profiled by whole-genome-sequencing, DNA methylation arrays, gene expression and splicing arrays, mass spectrometry, and ChIP-seq for EWSR1-ETS and histone marks. Systematic analysis of these multi-dimensional data illuminated hundreds of new potential EWSR1-ETS target genes, the nature of EWSR1-ETS-preferred GGAA-mSats, and potential indirect modes of EWSR1-ETS-mediated gene regulation. Moreover, we identified putative co-regulatory transcription factors and heterogeneously regulated EWSR1-ETS target genes that may have implications for the clinical heterogeneity of EwS. Collectively, our freely available ESCLA constitutes an extremely rich resource for EwS research and highlights the power of leveraging multidimensional and comprehensive datasets to unravel principles of heterogeneous gene regulation by dominant fusion oncogenes. 18 established Ewing sarcoma cell lines were stably transduced with inducible shRNA expressing constructs targeting the respective EWSR1-ETS fusion oncogene. EWSR1-ETS knockdown was induced by 1 µg/ml doxycycline in culture medium for 96 h. Genomic DNA was isolated with a membrane based approach from three replicates after EWSR1-ETS-knockdown induction and three control replicates per cell line. DNA was processed and hybridized according to Illumina protocols to Illumina Infinium MethylationEpic BeadChip arrays. Data were processed using Illumina Genome Studio Software.