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[Preprint]. 2024 Oct 29:2024.10.27.620498.
doi: 10.1101/2024.10.27.620498.

Endogenous EWSR1-FLI1 degron alleles enable control of fusion oncoprotein expression in tumor cell lines and xenografts

James H McGinnis  1   2 , Alberto Bremauntz Enriquez  1   2 , Florentina Vandiver  1 , Xin Bai  1 , Jiwoong Kim  3 , Jessica Kilgore  2 , Purbita Saha  4   5   6 , Ryan O'Hara  4   5   6 , Yang Xie  3 , Laura A Banaszynski  7   4   5   6 , Noelle Williams  2 , David G McFadden  1   8   2   7
Affiliations

Endogenous EWSR1-FLI1 degron alleles enable control of fusion oncoprotein expression in tumor cell lines and xenografts

James H McGinnis et al. bioRxiv. .

Abstract

Pediatric malignancies frequently harbor chromosomal translocations that induce expression of fusion oncoproteins. The EWSR1-FLI1 fusion oncoprotein acts as a neomorphic transcription factor and is the dominant genetic driver of Ewing's sarcoma. Interrogation of the mechanisms by which EWSR1-FLI1 drives tumorigenesis has been limited by a lack of model systems to precisely and selectively control its expression in patient-derived cell lines and xenografts. Here, we report the generation of a panel of patient-derived EWS cell lines in which inducible protein degrons were engineered into the endogenous EWSR1-FLI1 locus. These alleles enabled rapid and efficient depletion of EWSR1-FLI1. Complete suppression of EWSR1-FLI1 induced a reversible cell cycle arrest at the G1-S checkpoint, and we identified a core set of transcripts downstream of EWSR1-FLI1 across multiple cell lines and degron systems. Additionally, depletion of EWSR1-FLI1 potently suppressed tumor growth in xenograft models validating efforts to directly target EWSR1-FLI1 in Ewing's sarcoma.

Keywords: EWSR1-FLI1; Ewing’s sarcoma; auxin-inducible degron; fusion oncoprotein; inducible degron; small molecule assisted shutoff; xenograft.

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Conflict of interest statement

DECLARATION OF INTERESTS: The authors declare no competing interests

Figures

Figure 1:
Figure 1:. Degron tags enable depletion of endogenous EWSR1-FLI1.
A) Schematic depicting SMASh and AID based degron approaches for depletion of endogenous EWSR1-FLI1. B) Immunoblot for EWSR1-FLI1 (FLI1) in indicated cell lines. Cell lines were exposed to either DMSO or IAA (100 μM) for 24 hours prior to collection. C) Immunoblot for EWSR1-FLI1 (FLI1) in indicated cell lines. Cell lines were exposed to either DMSO or IAA (100 μM) for the indicated time prior to collection. D) Immunoblot for EWSR1-FLI1 (FLI1) in indicated cell lines. Cell lines were exposed to either DMSO or danoprevir (1 μM) for 24 hours prior to collection. E) Immunoblot for EWSR1-FLI1 (FLI1) in indicated cell lines. Cell lines were exposed to either DMSO or danoprevir (1 μM) for the indicated time prior to collection.
Figure 2:
Figure 2:. C-terminal AID tag on EWSR1-FLI1 does not disrupt DNA binding.
A) Venn diagram representing overlap of FLI enriched regions identified from A673 and A673 EFAID;TIR1 F74A cells B) Average profiles (top) and heatmaps (bottom) of FLI CUT&RUN enrichment at FLI enriched regions in A673 cells (n=33,743) in A673 cells and A673 EFAID;TIR1 F74A cells treated with DMSO or 5-Ph-IAA (300 nM) for 24 hrs. 0.5 kb around the peak center are displayed for each analysis C) Genome browser representations of FLI CUT&RUN in A673 cells and A673 EFAID;TIR1 F74A cells treated with either DMSO or 5-Ph-IAA (300 nM) for 24 hrs. The y-axis represents read density in reads per million mapped reads (rpm).
Figure 3:
Figure 3:. EWSR1-FLI1 depletion induces G1/S arrest
A) Population doublings after 6 days of treatment. Indicated cell lines were exposed to vehicle or 1 μM danoprevir (DSV). B) Population doublings after 6 days of treatment. Indicated cell lines were exposed to DMSO or IAA (100 μM). C) Immunoblot for EWSR1-FLI1 (FLI1) in TC32 EFSMASh cells with indicated pLVX constructs. Cell lines were exposed to either Vehicle or danoprevir (1 μM) for 24 hours prior to collection. D) Population doublings after 6 days of treatment. TC32 EFSMASh cells with indicated pLVX constructs (C) were exposed to vehicle or 1μM danoprevir. E-F) Cell cycle analysis using propidium iodide. Flow cytometry plots (left two panels) for indicated cell lines treated with either vehicle or 1 μM danoprevir for 72 hours before cells were collected. Plot (left panel) of cells (percentage) in each phase of the cell cycle based on flow cytometry data.
Figure 4:
Figure 4:. Core set of EWSR1-FLI1 response genes shared across EWS cell lines.
A) Volcano plots of RNA sequencing data obtained from indicated cell lines following treatment with 1 μM danoprevir. Red dots represents genes that are significantly differentially induced. Blue dots represents genes that are significantly differentially repressed. .B) Venn diagram comparing up-regulated genes (left) or down-regulated genes (right) across the three cell lines tested.
Figure 5:
Figure 5:. EWSR1-FLI1 is required for tumor maintenance in vivo
A) Tumor volume (left) and tumor mass (right) for A673 EFAID;TIR1 xenografts treated with vehicle (n = 8) or 200 mg/kg IAA (n = 8). Vehicle or IAA was administered twice daily via oral gavage for a total of 11 days. B) Tumor volume (left) and tumor mass (right) for A673 EFAID: xenografts treated with vehicle (n = 6) or 200 mg/kg IAA (n = 6). Vehicle or IAA was administered twice daily via oral gavage for a total of 11 days.
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