NIH Research Festival
Chromosomal translocations in the EWSR1 locus and a second breakpoint, are known as the initiating mutation and catalyst for several cancer types. Ewing Sarcoma (EWS), a soft tissue and bone cancer, is a cancer that is associated with rearrangements between the EWSR1 gene and a gene from the ETS DNA binding proteins. Treatments for EWS include harmful and general methods like surgery, radiation, and chemotherapy and, unfortunately, investigations into more targeted treatments have not proven fruitful. Therefore, to target the EWSR1-fusion-driven cancers more efficiently, we conducted RNAi screens aimed at identifying regulators of the EWSR1 gene, with future aims to conduct a similar screen for the EWSR1-fusion oncoprotein. To accomplish this, our team utilized CRISPR-Cas9 gene editing to create a TC32 cell line containing the EWSR1 gene tagged with a HiBiT luminescence reporter. With the cell line, we validated our negative, positive, and biological siRNA controls, and then optimized three assay parameters: the TC32 cell number, lipid transfection reagent concentration, and incubation time point. Then, we conducted the genome-wide RNAi screen in a 384-well arrayed format. From our analysis of the luminescence readings, we found genes that when silenced could effectively downregulate or upregulate EWSR1 expression. Our results suggest that these gene hits could be utilized as effective regulators of EWSR1 and therapeutic targets of EWSR1-driven cancers.
Scientific Focus Area: RNA Biology
This page was last updated on Monday, September 25, 2023