BRCA1, R-loops and Recombination defects in Ewing's sarcoma (DRIP-seq)

Ewing’s sarcoma family of tumors (ESFT) is an aggressive pediatric bone and soft tissue cancer. It is the prototypical example of mesenchymal tumors driven by a fusion oncogene involving the ewing sarcoma break point region 1 (EWSR1) gene, most frequently– EWS-FLI1. We have discovered that loss of EWSR1 leads to accumulation of R-loops, replication stress and impaired homologous recombination, recapitulating breast cancer 1, early onset (BRCA1) deficiency. EWS-FLI1 acts dominant negatively in ESFT to impart the same phenotypes. Further we demonstrate that in ESFT, BRCA1 predominantly associates with the elongating transcription machinery and is unavailable for DNA strand break repair. Gene expression profiling identified upregulated compensatory mechanisms in ESFT cells to process increased R-loops (RNASEH2 and FEN1) and replication stress (Fanconi Anemia). Taken together, our data identifies BRCA1 sequestration due to transcription stress as the mechanistic basis for ESFT chemosensitivity and suggests potential targets for the much lacking second-line therapy. Examination of R-loops was conducted using DRIP-Seq. Three different Ewing's sarcoma cell lines, IMR90 control cell line and U2OS cells transfected with either siRNA against EWSR1 or EWS-FLI1 vector along with appropriate controls were used.Samples were further treated with either vehicle or LD65 dose of etoposide for 6 hours. Genomic DNA was extracted, digested with a restriction enzyme cocktail and used for pulldown of DNA:RNA hybrids.

尤因肉瘤家族肿瘤（Ewing’s sarcoma family of tumors, ESFT）是一类侵袭性儿童骨与软组织恶性肿瘤，其作为由涉及尤因肉瘤断点区域1（ewing sarcoma break point region 1, EWSR1）基因的融合致癌基因驱动的间叶源性肿瘤的典型代表，最常见的融合类型为EWS-FLI1。本研究发现，EWSR1缺失会导致R环（R-loops）积累、复制应激以及同源重组功能受损，重现了乳腺癌易感基因1早期发病型（breast cancer 1, early onset, BRCA1）缺陷的表型。EWS-FLI1在ESFT中以显性负性方式发挥作用，赋予细胞相同的异常表型。

本研究进一步证实，在ESFT中，BRCA1主要与延伸中的转录机器结合，无法参与DNA链断裂修复。基因表达谱分析显示，ESFT细胞中存在上调的代偿机制，以应对增多的R环（由RNASEH2与FEN1介导）及复制应激（由范可尼贫血通路介导）。

综上，本研究数据表明，转录应激介导的BRCA1螯合是ESFT化疗敏感性的分子机制，并为目前极度匮乏的二线治疗手段提供了潜在靶点。

本研究采用DRIP-Seq技术开展R环检测：使用3株不同的尤因肉瘤细胞系、IMR90对照细胞系，以及分别转染针对EWSR1的小干扰RNA（siRNA）或EWS-FLI1过表达载体的U2OS细胞，并设置相应对照。随后将样本分别用溶剂对照或依托泊苷LD65剂量处理6小时，提取基因组DNA，经限制性内切酶混合物酶切后，用于DNA:RNA杂交复合物的下拉（pulldown）实验。