Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

Abstract

The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNAseq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron-exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.T he alternative splicing of mRNA expands the diversity of the human proteome through evolution and ontogeny (1, 2). Spliceosomal network interactions, including proteins that recognize splice enhancer and silencer regions, are critical for the regulation of alternative splicing leading to protein isoforms with disparate functionality (3). Alternative splicing provides both a method by which to categorize subsets of cancers and an avenue for more effective targeted treatments (4). However, the spliceosomal protein interaction networks that are specific to cancer have not been systematically defined; alternative splicing can also change protein-protein interactions within networks (5). A systems biology approach can be used to study the relationship between splicing and oncogenesis by using tumor models with chromosomal translocations whose expressed fusion proteins have putative roles in splicing (6, 7).Fusion proteins produced by chromosomal translocations in sarcomas often contain the amino-terminal portion of EWS and are classified as transcription factors due to the presence of a canonical carboxyl-terminal DNA-binding domain (6). EWS-FLI1 is a well-established ES oncoprotein and is re...

Pediatric Malignant Bone Tumors: A Review and Update on Current Challenges, and Emerging Drug Targets

“…84 This molecule binds directly to EWS-FLI1 and inhibits RNA splicing, a critical oncogenic function of the EWS-ETS fusion protein. 85 This will be the first direct target therapy against the EWS-ETS fusion protein evaluated in clinical trials for the ESFT.…”

Molecular genetics of Ewing sarcoma, model systems and finding novel (immuno-) therapeutic targets

“…Fusion of the N-terminal region of EWSR1, which contains a strong transcriptional activation domain [21,22] , and these DNA binding domains causes aberrant transcription of a multitude of genes. Additionally, EWSR1-ETS is known to affect epigenetic programs [23,24] , splicing [25,26] , and metabolic activity [27] of EWS. Oncogenic fusions between ETS genes and transcriptional activators are not unique to EWS: in 50%-70% of prostate cancers, similar chromosomal rearrangements are found [28] .…”

“…Targeting the fusion protein directly has proven to be challenging due to its flexibility, but progress has been made and a phase I clinical trial (NCT02657005) is ongoing with a small molecule inhibitor [108] . Aside from targeting the EWSR1-ETS fusion protein directly, inhibiting its dominant direct targets has been investigated as therapeutic alternative, as EWSR1-ETS is a key driver in the epigenetics [23,24] , transcription [109] , splicing [25,26] , and metabolic [27] reprogramming of EWSR1-ETS.…”

Fusion Genes and their Detection through Next Generation Sequencing in Malignant Hematological Disease and Solid Tumors

“…In the study carried out by Saravana et al [10], genes such as CLK1, CASP3, PPFIBP1 and TERT, which potentially participate in oncogenesis, are alternatively spliced by EWS-FLI1. Thus EWS-FLI1 can be used as a diagnostic biomarker for Ewing's sarcoma.…”