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 with >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.

融合转录因子（fusion-TFs）代表了一类难以治疗靶向的驱动癌蛋白。最近，蛋白质降解已成为针对这些具有挑战性的癌蛋白的策略。然而，调节融合-TF 稳定性的机制通常是未知的。使用 CRISPR-Cas9 筛选，我们发现含有三方基序的 8 (TRIM8) 作为 E3 泛素连接酶，可泛素化和降解 EWS/FLI，这是尤文肉瘤中的驱动融合-TF。此外，与超过 700 种其他癌细胞系相比，我们将 TRIM8 鉴定为尤文肉瘤的选择性依赖性。机械上，TRIM8敲除导致无法耐受的 EWS/FLI 蛋白水平增加。EWS/FLI 充当 TRIM8 的新形态基板，定义了依赖性的选择性。我们的结果表明融合-TF 蛋白的稳定性受到严格调控，并突出融合癌蛋白特异性调节剂作为选择性治疗靶点。这项研究提供了一种易于处理的策略，以在尤文肉瘤和其他潜在的融合 TF 驱动的癌症中治疗利用致癌基因过量。