Rewiring the fusion oncoprotein EWSR1::FLI1 in Ewing scarcoma with bivalent small molecules [RNA-Seq]. Rewiring the fusion oncoprotein EWSR1::FLI1 in Ewing scarcoma with bivalent small molecules [RNA-Seq]

Dysregulated transcription is a defining hallmark of cancer. Recently, novel chemically induced proximity approaches have enabled the rewiring of transcriptional machinery to drive expression of pro-apoptotic genes using bivalent small molecules. In this work, we demonstrate that this strategy is amenable to relocalizing DNA bound transcriptional machinery, such as fusion transcription factors that commonly drive pediatric malignancies. Targeting fusion transcription factors, such as EWSR1::FLI1 in Ewing sarcoma, with these bivalent compounds may open new therapeutic avenues. Here, we develop a small molecule, EB-TCIP, that recruits FKBP12F36V-tagged EWSR1::FLI1 to DNA sites bound by the transcriptional regulator BCL6, leading to rapid chromatin remodeling and expression of BCL6 target genes. This proof-of-concept study demonstrates that DNA binding proteins with pioneering transcription factor activity, such as EWSR1::FLI1, can be relocalized on chromatin to induce expression of repressed genes. Insights herein will guide the development of future bivalent molecules that rewire DNA binding transcriptional machinery. Overall design: Investigate alterations in gene expression in parental EWS502 Ewing sarcoma cells and EWS502 cells expressing FKBP treated for 4 hours with DMSO, BI3812, EB-TCIP, NEG-1, or NEG-2. Three replicates were performed for each conditon. NEG-1 samples were harvested from a latter passage of cells with a separate DMSO control labelled DMSO-2. Under GEO GSE290895 (RNA-Seq), 8 hours and 24 hours samples, EB-TCIP is labelled as BAK212, BI3812 is labelled as BI, and NEG-1 is labelled as O89.

转录失调是癌症的核心标志性特征。近年来，新兴的化学诱导邻近技术（chemically induced proximity）已可借助双价小分子化合物，实现转录调控机器的重编程，从而驱动促凋亡基因的表达。本研究证实，该策略可用于重新定位结合DNA的转录调控机器，例如在儿童恶性肿瘤中常见的致癌融合转录因子。利用此类双价小分子化合物靶向融合转录因子——例如尤因肉瘤（Ewing sarcoma）中的EWSR1::FLI1——或将开辟全新的治疗途径。

本研究开发了一款名为EB-TCIP的小分子化合物，其可将携带FKBP12F36V标签的EWSR1::FLI1募集至转录调控因子BCL6（BCL6）结合的DNA位点，进而快速诱导染色质重塑并激活BCL6靶基因的表达。本概念验证研究证实，具有先锋转录因子（pioneering transcription factor）活性的DNA结合蛋白——例如EWSR1::FLI1——可在染色质上被重新定位，从而诱导沉默基因的表达。本研究所得见解将为后续开发可重编程DNA结合型转录调控机器的双价小分子化合物提供指导。

实验整体设计：探究亲本EWS502尤因肉瘤细胞，以及经二甲基亚砜（DMSO）、BI3812、EB-TCIP、NEG-1或NEG-2处理4小时的FKBP表达型EWS502细胞的基因表达变化。每个实验组均设置3次生物学重复。NEG-1样本取自后续传代的细胞，其对应的二甲基亚砜对照组标记为DMSO-2。在基因表达综合数据库（Gene Expression Omnibus, GEO）的GSE290895（RNA测序（RNA-Seq））数据集中，8小时和24小时样本的EB-TCIP标记为BAK212，BI3812标记为BI，NEG-1标记为O89。