Systematic multi-omics profiling of Ewing sarcoma cell lines

Orth, Martin Franz (2021): Systematic multi-omics profiling of Ewing sarcoma cell lines. Dissertation, LMU München: Medizinische Fakultät

Abstract

100 years after its first description, Ewing sarcoma (EwS), the second most common bone-associated cancer in children and young adults, is still poorly understood. Neither the cell of origin is known, nor the detailed mechanism of expression regulation by the pathognomic fusion oncogene. Similarly, factors causing overt clinical heterogeneity and advanced/targeted therapeutic strategies for patients with non-localized disease remain to be identified. An apparent paradox of EwS is its clinical heterogeneity compared to its silent landscape of genomic mutations. The only highly recurrent mutation in EwS is the characteristic fusion oncogene composed of EWSR1 and an ETS-transcription factor. Interactions of this single driver with the genome have been described and associated with gene expression regulation several times, but always in a small number of cell line models. This thesis aimed at creating a multidimensional dataset on a large number of EwS cell line models with and without fusion oncogene knockdown, the Ewing Sarcoma Cell Line Atlas (ESCLA), to both enable further investigations of expression regulation in EwS and model heterogeneity. In 18 well-characterized EwS cell lines, with three distinct fusion types, an inducible shRNA construct targeting the fusion oncogene was stably integrated. The whole genomes of the cell lines were sequenced with relatively long reads (150 bp) and >30 coverage. For the respective fusion and the histone marks H3K27ac, H3K27me3 and H3K4me3 chromatin immunoprecipitation with subsequent next-generation sequencing (ChIP-Seq) was performed. The transcriptome of the cells with and without fusion knockdown was assessed by ClariomD DNA microarrays, as was the protein expression by mass spectrometry and the CpG island methylation by MethylationEPIC BeadChip arrays. Whole genome sequencing enabled genotyping of several polymorphic potentially fusion binding microsatellites with GGAA motif. ChIP-Seq data were in line with previous publications and identified 50 additional consensus fusion binding sites. Transcriptome and proteome data strongly correlated with each other and displayed expression rearrangement upon fusion knockdown. Only for CpG methylation not any uniform effect of fusion oncogene knockdown was observed. Cell lines with distinct fusion types, EWSR1-FLI1 type 1, 2 and EWSR1-ERG, were for the first time systematically compared to each other. Neither expression regulation, nor methylation profile were dependent on the respective fusion. However, the fusion types differed in their rate of chromoplexy as developmental process. All EWSR1-ERG fusions and 55% of EWSR1-FLI1 type 1 fusions developed from chromoplexy, whereas all EWSR1-FLI1 type 2 fusions were the result of reciprocal translocation. Binding of the fusion to GGAA motifs appeared as multifactorial and still poorly understood process. Among others, high numbers of consecutive GGAA motifs, additional nearby motifs and microsatellites as well as and copy number gains correlated with fusion binding probability. Genes differentially expressed upon fusion knockdown differed from not affected genes in their distance to the next fusion bound GGAA mSat, the number of nearby GGAA mSats, and in the presence of transcription factor bindings sites for NFAT5, NFYC, and E2F2 in their promoters. All these transcription factors were also regulated by the fusion oncogene. A set of 22 genes were identified to be regulated to different extends in the 18 cell line models upon fusion knockdown. This heterogeneity in regulation was in line with heterogeneous expression in patients, which correlated with overall survival. These genes were mainly associated with cell-cycle progression and cell division, transcription factors and targets of those. Yet, evaluated and identified parameters of EWSR1-ETS mediated gene expression regulation were not sufficient to fully explain inter-cell line differences in gene regulation. Several studies demonstrated previously an interaction between the fusion oncogene and GGAA microsatellites, but were limited to few loci. Previous whole exome sequencing projects missed out on these relevant regulatory regions. Reporter assays in vitro revealed enhancer activity of GGAA microsatellites, but in an artificial only mono-allelic approach. Studies and experiments on gene regulation in EwS with only two to three cell lines could hardly model heterogeneity. The here generated ESCLA overcame these obstacles, and supported, refined and expanded previously elaborated models of fusion oncogene mediated gene regulation genome wide. In conclusion, a multidimensional and comprehensive dataset was generated on a collection of EwS cell line models clearly outnumbering previous studies. Moreover, the dataset has already enabled first novel insights on the mechanisms and dependencies of fusion mediated gene regulation and modelled heterogeneity. The generated cell lines and the ESCLA likely constitute a rich resource for the Ewing sarcoma research community. Additionally, the capability of the dataset to model heterogeneity might enforce research on personalized medicine and the development of new treatment strategies for so far incurable advanced disease patients.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Ewing sarcoma, omics, GGAA-mSat
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 22. März 2021
• 1. Bericht­erstatter:in: Grünewald, Thomas