RNA-seq profiling of Ewing sarcoma cells after TRIM8 knockout and EWS/FLI over-expression

Fusion-transcription factors (fusion-TFs) represent a class of driver oncoproteins that are difficult to therapeutically target. Recently, protein degradation has emerged as a strategy to target these challenging oncoproteins. The mechanisms that regulate fusion-TF stability, however, are generally unknown. Using CRISPR-Cas9 screening, we discovered tripartite motif- containing 8 (TRIM8) as an E3 ubiquitin ligase that ubiquitinates and degrades EWS/FLI, a driver fusion-TF in Ewing sarcoma. Moreover, we identified TRIM8 as a selective dependency in Ewing sarcoma compared to >700 other cancer cell lines. Mechanistically, TRIM8 knockout led to an increase in EWS/FLI protein levels that was not tolerated. EWS/FLI acts as a neomorphic substrate for TRIM8, defining the selective nature of the dependency. Our results demonstrate that fusion-TF protein stability is tightly regulated and highlight fusion-oncoprotein specific regulators as selective therapeutic targets. This study provides a tractable strategy to therapeutically exploit oncogene overdose in Ewing sarcoma and potentially other fusion-TF driven cancers. Overall design: TC71 Ewing sarcoma cells engineered to express FKBP12F36V-HA-TRIM8 was treated with either DMSO control or dTAGV-1 for 24 hours to induce TRIM8 degradation. In addition, TC71 Ewing sarcoma cells engineered to express doxycycline-inducible EWS/FLI-HA was treated with either water control or doxycycline (500ng/mL) for 8 hours. Triplicates for all groups were used for paired-end RNA-sequencing.