Soumya Sundara Rajan, Katelyn R. Ludwig, Javed Khan, Raj Chari, Natasha J. Caplen.

• Submitter: Soumya Rajan (Postdoc or fellow)
• email: soumya.sundararajan@nih.gov

A sub-set of chromosomal rearrangements result in the expression of chimeric proteins that facilitate malignant transformation. Ewing sarcoma (EWS) is one such cancer, where a chromosomal translocation results in the expression of a chimeric protein. This fusion oncoprotein consists of the N-terminus of the RNA-binding protein EWSR1 and the C-terminus of a member of the ETS transcription family, in most cases, FLI1. The C-terminal DNA binding domain of EWS-ETS fusion oncoproteins is responsible for the transcriptional reprogramming required for tumorigenesis. However, in addition to the EWSR1 regulatory regions responsible for promoting EWS-ETS protein expression, there is increasing evidence that the EWSR1 N-terminal domain of EWS-ETS fusion proteins also contributes to the disease phenotype by facilitating the oncoprotein’s multimerization at GGAA microsatellite sequences. This multimeric binding activates a subset of EWS-ETS target genes, at least some of which contribute to transformation. There is also evidence that the wild-type EWSR1 protein expressed from the unaffected allele contributes to EWS biology, including the response to DNA damage. To assess the contributions of EWSR1 and EWS-ETS to the phenotype of EWS, we will need complementary model systems that report on their expression or function. Here, we discuss the results of generating a panel of EWS cell lines using CRISPR knock-in strategies to tag the 5’ end of the EWSR1 gene.

Using sgRNAs targeting the first exon of EWSR1 and repair templates that allowed insertion of either a fluorescent or antibiotic selection marker, we report here the successful generation of multiple endogenously EWSR1-tagged clones of two EWS cell lines, A673 and TC-32. Optimization of each step in this process, including methods that assess the allelic-specific insertion of each reporter or tag, has resulted in the successful identification of multiple clones of interest. To date, we have confirmed the modification of at least two clones per EWS cell line that express EWSR1 N-terminal in-frame fusion proteins tagged with either the fluorescent reporter mNeonGreen, a small HiBit peptide that can be excited to luminesce, or the degradation tag FPKB12F36V. Preliminary analysis of the EWS cells expressing the degradation tag FPKB12F36V has shown that an appropriate degrader molecule can induce a substantial decrease in EWSR1 protein levels in as little as 20 minutes. The analysis of the phenotype of these tagged clones upon manipulation of EWSR1 expression is ongoing. Our current work provides a much-needed community resource to enable the functional, genomic and transcriptional assessment of EWSR1’s hitherto unexplored role in Ewing sarcoma while providing a platform for the modification of any other candidates of interest, including complementary efforts targeting the EWS-ETS fusion protein.