A breakthrough approach targeting the EWS-FLI1 protein offers new hope for childhood cancer treatment
Imagine a cancer driver so fundamental that without it, the tumor cells cannot survive. This is the reality for Ewing sarcoma, an aggressive bone and soft tissue cancer that primarily affects children and adolescents. For patients with metastatic or relapsed disease, survival rates remain a dismal 20-30% despite intensive chemotherapy, radiation, and surgery 2 3 4 . The field desperately needs targeted therapies that can attack this cancer more precisely while sparing young patients the devastating side effects of conventional treatment.
At the heart of this cancer lies a singular genetic aberration—a chromosomal translocation that creates a Frankenstein-like protein called EWS-FLI1. This oncoprotein functions as a master control switch, reprogramming cells to become cancerous 1 . For decades, scientists have struggled to target EWS-FLI1 directly because, like most transcription factors, it lacks the traditional "pockets" that drugs can easily bind to. However, recent innovative research has uncovered a potential weakness: by preventing this protein from being recycled within cancer cells, we might finally have a way to defeat it 1 .
In approximately 85% of Ewing sarcoma cases, a specific genetic accident occurs—pieces of chromosomes 11 and 22 break off and swap places. This translocation fuses the N-terminal domain of the EWSR1 protein with the DNA-binding domain of the FLI1 protein, creating the aberrant EWS-FLI1 oncoprotein 4 .
This chimeric protein acts as a malignant transcription factor, hijacking the cellular machinery to activate cancer-promoting genes while shutting down protective tumor suppressor genes. What makes EWS-FLI1 particularly dangerous is that it's completely absent from normal cells, making it an ideal therapeutic target—if only we could find a way to attack it 3 4 .
| Protein Domain | Origin | Function in the Fusion Protein |
|---|---|---|
| N-terminal transactivation domain | EWSR1 | Recruits epigenetic modifiers and transcription machinery; contains intrinsically disordered region |
| DNA-binding domain | FLI1 | Binds to GGAA microsatellite regions in DNA to control target genes |
| Prion-like domain | EWSR1 | Enables phase separation and forms protein complexes |
Cancer cells are utterly dependent on EWS-FLI1—a concept known as oncogene addiction. When researchers experimentally reduce EWS-FLI1 levels, Ewing sarcoma cells stop proliferating and eventually die 1 . This dependency creates a therapeutic opportunity.
To understand the groundbreaking research on EWS-FLI1 destabilization, we first need to explore how cells normally manage their protein levels. Cells have a sophisticated quality control system called the ubiquitin-proteasome system (UPS) that identifies and destroys damaged or unnecessary proteins 1 .
A series of enzymes attaches small ubiquitin proteins to target proteins, marking them for destruction.
The proteasome—a cellular "shredder"—recognizes these tagged proteins and breaks them down into amino acids.
Specialized enzymes called deubiquitinases (DUBs) can remove ubiquitin tags, potentially saving proteins from destruction and effectively extending their lifespan 1 .
The constant battle between ubiquitinating enzymes and deubiquitinases determines the stability and half-life of proteins within cells—including cancer-driving proteins like EWS-FLI1.
In 2019, a research team made a crucial discovery: they identified a specific deubiquitinating enzyme called USP19 that stabilizes EWS-FLI1 in Ewing sarcoma cells 1 . Their investigation began with a systematic search for DUBs that might be protecting EWS-FLI1 from degradation.
The researchers employed an innovative siRNA screening approach to identify which deubiquitinases affect EWS-FLI1 stability:
They selected 21 deubiquitinating enzymes from the ubiquitin-specific protease family based on their high expression in Ewing sarcoma tumors and cell lines 1 .
Using two different Ewing sarcoma cell lines (A673 and RDES), they engineered cells to express flag-tagged EWS-FLI1 at levels similar to the endogenous protein.
They then systematically silenced each of the 21 candidate DUBs using three different siRNA molecules per target and measured the resulting EWS-FLI1 protein levels.
USP19 emerged as the primary regulator, with at least two different siRNAs against USP19 decreasing EWS-FLI1 protein levels by more than 25% in multiple screening rounds 1 .
| Experimental Approach | Key Result | Significance |
|---|---|---|
| siRNA knockdown of USP19 | 40% reduction in EWS-FLI1 protein levels | Proof that USP19 normally stabilizes the oncoprotein |
| USP19 overexpression | 2-fold increase in EWS-FLI1 levels | Demonstration that extra USP19 further stabilizes the oncoprotein |
| Catalytically inactive USP19 | Minimal stabilization of EWS-FLI1 | Evidence that deubiquitinating activity is required for the effect |
| Specificity testing | No stabilization of wild-type EWSR1 or FLI1 | Reveals unique vulnerability of the fusion protein |
Further experiments revealed the precise molecular mechanism:
When USP19 was silenced, EWS-FLI1 showed increased ubiquitination marks—the kiss of death that sends proteins to the proteasome 1 .
Stable knockdown of USP19 resulted in decreased cell growth, diminished colony-forming capacity, and significantly delayed tumor growth in animal models 1 .
The most striking finding was that USP19 specifically stabilizes the fusion oncoprotein but not the normal EWSR1 or FLI1 proteins from which it derives. This specificity suggests that targeting USP19 could affect cancer cells while sparing healthy tissues 1 .
The study that identified USP19's role employed sophisticated research tools that represent the cutting edge of cancer biology. The table below summarizes several key reagents and their applications in Ewing sarcoma research.
| Research Tool | Application in Ewing Sarcoma Research | Example Use Case |
|---|---|---|
| siRNA/shRNA libraries | Gene silencing screens | Identification of USP19 and other DUBs regulating EWS-FLI1 stability 1 |
| GPS (Global Protein Stability) system | Monitoring protein turnover in live cells | High-throughput drug screening for EWS-FLI1 destabilizers 2 |
| CRISPR/Cas9 knockout screening | Genome-wide identification of essential genes | Identification of C1GALT1 as regulator of EWS-FLI1 expression 3 |
| Co-immunoprecipitation | Detecting protein-protein interactions | Confirmation of binding between USP19 and EWS-FLI1 1 |
| Ubiquitination assays | Measuring protein ubiquitination levels | Detection of increased EWS-FLI1 ubiquitination after USP19 knockdown 1 |
| Xenograft mouse models | Preclinical testing of therapeutic strategies | Evaluation of tumor growth delay after USP19 inhibition 1 |
The discovery of USP19's role in stabilizing EWS-FLI1 has opened up new avenues for therapeutic intervention. Meanwhile, other research groups have been exploring complementary strategies to target the oncoprotein:
The dual HDAC/PI3K inhibitor fimepinostat (CUDC-907) was identified in a high-throughput screen as another compound that destabilizes EWS-FLI1 by enhancing its degradation through the ubiquitin-proteasome system 2 .
A 2025 study identified C1GALT1, a galactosyltransferase enzyme, as a promoter of EWS-FLI1 expression through activation of the Hedgehog signaling pathway. The FDA-approved antifungal drug itraconazole, which inhibits C1GALT1, reduced EWS-FLI1 levels and suppressed tumor growth in mouse models 3 .
The histone demethylase LSD1 forms part of the NuRD complex that EWS-FLI1 recruits to repress tumor suppressor genes. LSD1 inhibitors are currently under investigation as a way to disrupt this repressive function 4 .
While the prospect of targeting DUBs like USP19 is exciting, researchers remain cautious. DUBs typically regulate multiple protein substrates, so inhibiting them might cause unintended side effects. However, the unique dependence of EWS-FLI1 on USP19 for stability, combined with the specificity of the interaction, suggests that therapeutic windows might exist 1 .
The investigation into deubiquitinase inhibition for Ewing sarcoma represents more than just a potential treatment for one rare cancer—it pioneers a novel therapeutic strategy that could be applied to other fusion-driven cancers. The approach of targeting protein stability rather than protein function offers a promising path against transcription factors and other "undruggable" targets 1 2 .
As research advances, the dream of providing targeted, effective therapy for Ewing sarcoma patients moves closer to reality. The story of USP19 and EWS-FLI1 destabilization exemplifies how basic scientific investigation into cellular mechanisms can reveal unexpected therapeutic opportunities—offering hope for children facing this devastating disease.
The journey from laboratory discovery to clinical application is long and challenging, but each new understanding of cancer biology brings us one step closer to transforming patient outcomes.