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. Overall design: 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.