**6. Combining targeted drugs with protein degradation**

To identify potential targeted therapeutic compounds that can promote fusion protein degradation, high-throughput chemical (HTS) screening can be used in a model system that reports on the stability of the target protein [96]. Thus, cell-based systems expressing a fluorescent dye-labeled protein of interest and a different color fluorescent control can be used for image-based screening that can identify compounds that measure the stability of the fluorescently labeled protein. The identified compounds can be further investigated and the mechanism affecting protein stability can be identified [96].

An example of the successful use of such a system was recently published for Ewing sarcomas: Using a high-throughput drug screen (HTS) enriched with FDA-approved drugs coupled with global protein stability (GPS) approach revealed that the dual HDAC and phosphatidylinositol 3-kinase (PI3K) inhibitor Fimepinostat (CUDC-907) is an excellent candidate to modulate EWSR1-FLI1 stability. Fimepinostat greatly reduced the amount of fusion protein, decreased the viability of several Ewing sarcoma cell lines and PDX primary cells, and delayed tumor growth in a xenograft mouse model, while not significantly affecting healthy cells. They demonstrated that EWSR1- FLI1 protein levels were mainly regulated by the HDAC activity of Fimepinostat [97].

A second approach to degradation of fusion oncoproteins is their targeted protein degradation mediated by degradation molecules or proteolysis targeting chimeras (PROTACs). While there are several strategies for targeted protein degradation [98–100], PROTACs are small molecule-based and thus a drug-like method to degrade a target protein of interest. The methodology combines small molecules that can bind directly to E3 ligases such as CRBN and VHL [101–103] with molecules that bind to the desired target protein-coupled through a chemical linker such as polyethylene glycol. Thus, these compounds bring the target protein and an E3 ligase complex into close proximity, resulting in polyubiquitination of the target protein, followed by proteasome-mediated degradation [100].

This strategy requires a small molecule that can bind to the desired fusion protein but does not necessarily need to enter the enzyme pocket or specifically inhibit the activity of the target protein, which has historically been an obstacle to the development of drugs targeting transcription factors. Small molecule inhibitors of proteins with bromodomains and extra terminal domains (BET) such as JQ1 and OTX015 have been successfully converted into degraders [104, 105].

On the other hand, there are ways to directly tag fusion proteins for proteasomal degradation. For example, it has been shown that EWSR1-FLI1 degradation involves polyubiquitination at lysine-380, which marks the fusion protein for proteasomal degradation [106]. Lysine-380 is located within the DNA-binding domain and is also present in wild-type FLI1 and conserved in several other members of the ETS family like ETS1. However, this may limit specificity [107]. Although, given the short half-life of EWSR-FLI1, a PROTAC targeting a lysine-380-containing motif could create a therapeutic window [106]. On the other hand, EWSR1-FLI1-specific PROTACs have not yet been developed. However, PROTACs targeting fusion protein interacting with BET or CK proteins have been successfully tested in Ewing sarcoma cells [108, 109].

In the search for small molecules that can bind to a protein of interest, the HTS method is now being used very successfully. A wide variety of target-specific HTS methods and assay formats can be used (see review in Coussens et al. [110]). With improvements in stability and delivery of PROTACS targeting fusion proteins, they may represent a viable approach to identify new drugs for targeted therapy of FP sarcomas.
