Examining oncofusion-driven transcript and protein isoforms that underpin fitness relationships essential for Ewing sarcoma tumor formation and metastasis

Ewing sarcoma (EwS) is the second most common childhood bone malignancy, primarily affecting children, adolescents and young adults. The mutational landscape of the EwS genome is relatively quiet and is primarily defined by chromosomal translocations that result in fusions between EWSR1 (EWS) and ETS family transcription factor genes (e.g. FLI1, ERG). EWS fusion oncoproteins act as aberrant transcription factors whose specificity is defined through binding to particular DNA sequence motifs. Although the collective efforts of multiple groups has resulted in a deep understanding of genomic loci that are targeted by EwS fusion oncoproteins, widespread definition of transcript and protein variants, called isoforms, that emerge from these regions and whether they support aggressive phenotypes including metastasis, remains underexplored. We hypothesize that the mechanism of EWS fusion driven oncogenesis extends beyond previously established classical transcriptional activation (or repression) events. Our latest data from temporal transcriptome and proteome profiling of EwS cells with modified EWS-FLI1 expression indicate that apart from widespread promotion and inhibition of selected genes, regulation of specific transcript isoforms is a further defining feature. In this proposal, we will investigate isoform-level and non-classical transcription events triggered by EWS-fusion oncoproteins and use CRISPR-Cas9 targeting to identify variant dependency relationships that can be targeted to disrupt EwS tumor formation and progression. To generate a high-resolution set of transcript isoforms and their translated products driven by EWS-fusion oncoproteins, we will utilize a combination of full-length and short-read RNA sequencing along with multi-protease mass spectrometry-based proteomics to profile EwS cells with modifiable EWS-FLI1 expression. We will use these data to define a candidate set of known and novel transcript and translation events that will be examined using in vivo CRISPR-Cas9 library screens of cell line and patient-derived xenograft EwS cells to assign fitness relationships that underpin the ability of these cells to form tumors. Together, these data will establish if isoform variant expression events are triggered by EWS fusions, whether these transcripts result in translated products, and if their expression is essential for EwS tumor formation and progression. Given the specific nature of gene regulation by EWS fusions, we anticipate this study will yield multiple EwS-specific markers that can be exploited to redefine treatment options for this aggressive disease. This 12 month project has two aims: Aim 1 – Mapping transcript and protein isoforms regulated by the EWS-FLI1 fusion. Aim 2 – In vivo CRISPR screening of selected isoform targets to identify fitness relationships linked to EwS tumor formation.