Dynamic modelling of EWS::FLI1 fluctuations reveals molecular determinants of phenotypic tumor plasticity and prognosis in Ewing sarcoma [RNA-seq]

The mechanisms underlying tumor cell plasticity driving drug resistance and disease progression remain poorly understood. In Ewing sarcoma (EwS), variations in EWS::FLI1 (EF) activity have been associated with epithelial-mesenchymal plasticity (EMP). Using degron technology, we titrated endogenous EF in an EwS cell line and linked phenotypic states to distinct EF thresholds. Strikingly, modest EF depletion promoted a pro-metastatic phenotype, which was diminished upon near-complete EF loss. Transcriptomic analysis revealed distinct gene clusters with heterogenous response patterns to varying EF dosage. Target genes most sensitive to subtle EF depletion contained GGAA microsatellites in EF-bound enhancers. Furthermore, we identified Krüppel-like zinc-finger transcription factors to be associated with EF-repressed EMP genes. EF rescue after partial depletion for a transient period identified persistently dysregulated genes associated with poor prognosis. This study underscores the therapeutic challenge of insufficient EF inhibition and provides a foundation for exploiting oncoprotein dynamics to uncover therapeutic vulnerabilities in fusion-driven cancers. Overall design: In one part of the experiment, A673 cells in bulk, untagged A673 single cell clones, EF-dTAG clones A2.2 and B3.1 were sequenced without treatment (ssc_experiment). In the other parts, the two EF-dTAG clones were treated with various dTAGV-1 concentrations for various durations with either no ligand washout after treatment, or 7 day washout applied.

驱动肿瘤细胞可塑性、介导耐药与疾病进展的分子机制迄今仍未被充分阐明。在尤因肉瘤（Ewing sarcoma, EwS）中，EWS::FLI1融合蛋白（EF）的活性变化与上皮-间质可塑性（epithelial-mesenchymal plasticity, EMP）相关联。本研究利用降解子（degron）技术，在尤因肉瘤细胞系中对内源性EF进行剂量梯度调控，并将细胞表型状态与不同的EF表达阈值相关联。令人意外的是，轻度EF耗竭可促进促转移表型的形成，而当EF近乎完全缺失时该表型则会被逆转。转录组分析揭示了不同基因簇对EF剂量变化呈现异质性应答模式。对轻度EF耗竭最为敏感的靶基因，其EF结合的增强子区域中含有GGAA微卫星（GGAA microsatellites）序列。此外，本研究发现Krüppel样锌指转录因子（Krüppel-like zinc-finger transcription factors）与EF抑制的EMP相关基因存在关联。对EF进行短暂部分耗竭后的恢复实验，鉴定出了与不良预后相关的持续失调基因。本研究凸显了EF抑制不足所带来的治疗挑战，并为利用癌蛋白动态调控特征、挖掘融合基因驱动型癌症的治疗靶点提供了理论基础。实验整体设计：本实验分为两部分，其一为未经过处理的批量培养A673细胞、未标记A673单细胞克隆以及EF-dTAG克隆A2.2与B3.1的测序实验（ssc_experiment）；其二为将两种EF-dTAG克隆分别用不同浓度的dTAGV-1处理不同时长，处理后或不进行配体洗脱，或施加7天的洗脱处理。