# Suppressing the Aberrant Transcriptional Functionality of EWS::FLI1 Oncoprotein by Designer polyQ Fusions with Its Homologous Peptides

## TL;DR

This paper explores a new method to fight Ewing sarcoma by trapping a harmful protein, EWS::FLI1, using specially designed polyQ fusion proteins.

## Contribution

The novel use of polyQ fusion proteins to sequester and reduce the activity of the EWS::FLI1 oncoprotein in Ewing sarcoma cells.

## Key findings

- PolyQ fusion proteins co-precipitate and co-localize with EWS::FLI1, forming insoluble aggregates.
- The polyQ fusions reduce the cellular availability of EWS::FLI1 and alter downstream gene expression.
- Atx793Q-N172-LCD increases P21 and decreases c-Myc, suggesting therapeutic potential.

## Abstract

Background/Objectives: The oncoprotein EWS::FLI1 is a chimeric transcription factor that aberrantly brings transcriptional deregulation relevant to Ewing sarcoma. It is also regarded as a therapeutic target for suppressing oncogenic progression, but the inhibition and clearance of the EWS::FLI1 oncoprotein remain a challenge. Methods: We apply a polyglutamine (polyQ) fusion strategy to directly target EWS::FLI1 in suppression of its transcriptional malfunction in A673 cells derived from Ewing sarcoma. Based on the template of the N-terminal fragment of polyQ-expanded ataxin-7 (Atx793Q-N172) and the homologous peptides of EWS::FLI1, we have designed and constructed three polyQ fusion proteins, namely Atx793Q-N172-SYGQ1, Atx793Q-N172-SYGQ2, and Atx793Q-N172-LCD. Results: Supernatant/pellet fractionation and immunofluorescence imaging reveal that the polyQ fusion proteins co-precipitate and co-localize with EWS::FLI1 in A673 cells, indicating that the polyQ fusions we have designed can sequester endogenous EWS::FLI1 into insoluble aggregates and reduce its cellular availability. Moreover, these polyQ fusions, especially Atx793Q-N172-LCD, alter the expression of EWS::FLI1 downstream genes, with an increase in P21 (CDKN1A) and a decrease in c-Myc. Conclusions: These results demonstrate that the engineered polyQ fusions entrap endogenous EWS::FLI1 protein into aggregates and reduce its soluble fraction in Ewing sarcoma cells. This study provides an alternative potential for treating Ewing sarcoma and other tumors by directly targeting the oncogenic proteins in the future.