ASPSCR1-TFE3 orchestrates the angiogenic program of alveolar soft part sarcoma [ChIP-seq]

Alveolar soft part sarcoma (ASPS) is a rare soft part malignancy affecting adolescents and young adult. ASPS is characterized by its alveolar structure consisting of tumor cells and highly integrated vascular network, and its high metastatic potential indicates the importance of the prominent angiogenic activity of ASPS. Here we find that the expression of ASPSCR1-TFE3, the fusion transcription factor causatively associated with ASPS, is dispensable for in vitro tumor maintenance but required for in vivo tumor development via angiogenesis. ASPSCR1-TFE3 frequently associates with active enhancers including super-enhancers (SE) upon its DNA binding, and the loss of its expression induces dynamic modification of SE distribution related to genes belonging to the angiogenesis pathway. Using epigenomic CRISPR/dCas9 screening, we identify Pdgfb, Rab27a, Sytl2, and Vwf as critical target genes associated with reduced enhancer activities due to the ASPSCR1-TFE3 loss. ASPSCR1-TFE3 thus orchestrates higher ordered angiogenesis via enhanced intracellular trafficking of angiogenic factors. Overall design: Mouse ASPS was induced by expressing ASPSCR1-TFE3 in embryonic mesenchymal cells followed by subcutaneous transplantation into nude mice. Subcutaneous tumors were subjected to cell culture. Human ASPS cell lines were used for gene knockdown experiments.肺泡软组织肉瘤（Alveolar soft part sarcoma, ASPS）是一类罕见的软组织恶性肿瘤，好发于青少年与年轻成年人。该肿瘤以由肿瘤细胞与高度整合的血管网络构成的肺泡样结构为典型特征，其高转移性提示ASPS显著的血管生成活性具有关键生物学意义。 本研究发现，与ASPS发病存在因果关联的融合转录因子ASPSCR1-TFE3，其表达对体外肿瘤维持并非必需，但可通过调控血管生成途径参与体内肿瘤的发生发展。 ASPSCR1-TFE3结合DNA后，常与包括超级增强子（super-enhancers, SE）在内的活性增强子区域结合；当其表达缺失时，会引发血管生成通路相关基因的超级增强子分布发生动态改变。 本研究通过表观基因组学CRISPR/dCas9筛选，鉴定出Pdgfb、Rab27a、Sytl2与Vwf为ASPSCR1-TFE3缺失后增强子活性降低的关键靶基因。 由此可见，ASPSCR1-TFE3可通过增强血管生成因子的胞内运输，调控层级化的血管生成过程。 实验整体设计：通过在胚胎间充质细胞中过表达ASPSCR1-TFE3构建小鼠ASPS模型，随后将细胞接种至裸鼠皮下成瘤；提取皮下瘤组织进行细胞培养，并使用人源ASPS细胞系开展基因敲低实验。