The EWS-FLI1 fusion oncoprotein deregulates transcription to initiate the paediatric cancer Ewing sarcoma. Here we used a domain-focused CRISPR screen to implicate the transcriptional repressor ETV6 as a unique dependency in this tumour. Using biochemical assays and epigenomics, we show that ETV6 competes with EWS-FLI1 for binding to select DNA elements enriched for short GGAA repeat sequences. Upon inactivating ETV6, EWS-FLI1 overtakes and hyper-activates these cis-elements to promote mesenchymal differentiation, with SOX11 being a key downstream target. We show that squelching of ETV6 with a dominant-interfering peptide phenocopies these effects and suppresses Ewing sarcoma growth in vivo. These findings reveal targeting of ETV6 as a strategy for neutralizing the EWS-FLI1 oncoprotein by reprogramming of genomic occupancy.

EWS-FLI1 融合癌蛋白会解除转录调控，引发儿童癌症尤文肉瘤。在这里，我们使用以域为中心的 CRISPR 筛选来暗示转录抑制子 ETV6 作为该肿瘤的独特依赖性。通过生化分析和表观基因组学，我们发现 ETV6 与 EWS-FLI1 竞争结合，以选择富含短 GGAA 重复序列的 DNA 元件。在使 ETV6 失活后，EWS-FLI1 取代并过度激活这些顺式元件以促进间充质分化，其中SOX11是关键的下游靶点。我们证明，用显性干扰肽抑制 ETV6 可复制这些效应并抑制体内尤文肉瘤的生长。这些发现揭示了 ETV6 的靶向作为通过基因组占用重编程中和 EWS-FLI1 癌蛋白的策略。