The Ewing sarcoma cell line atlas (ESCLA) - The relevance of the transcription factors GATA2 and E2F2 in a Ewing sarcoma cell line model

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 identified GATA2 and E2F2 as EWSR1-ETS-driven putative co-regulatory transcription factors. To evaluate the relevance of these transcrition factors, RNA interference in a Ewing sarcoma cell line was employed with subsequent Affymetrix Exon array profiling. The expression data were analysed in synopsis with the effects of EWSR1-FLI1 in the same cell line. The Ewing sarcoma cell line A673 was transfected with 30 nM siPools targeting E2F2 or GATA2, or with 30nM of a non-targeting control siRNA pool (all siTOOLs Biotech, Planegg, Germany) using Opti-MEM (Thermo Fisher) and lipofectamine RNAiMax reagent according to the manufacturer’s recommendations (Thermo Fisher). 48 h after the first transfection, cells were re-transfected. 96 h after the first transfection, total RNA was isolated from three replicates, each. The transcriptome was profiled on Affymetrix Clariom D Human microarrays. Raw data were processed using the Transcriptome Analysis Console (version 4.0) by ThermoFisher, the SST-RMA analysis algorithm and the manufacturer's array description file (version 2).