Abstract
Abstract Gene editing tools like CRISPR, using Cas9 protein and selected guide RNAs, are now the preferred method to study many coding gene functions, but historically have been hampered by low efficiency (typically <20% of cells), which limits their utility in suppressing highly expressed oncogenic drivers of cancer like fusion genes. Ewing sarcoma (ES) is driven by the EWS-FLI1 or equivalent fusion gene and expresses CD99 on its cell surface. CD99 itself is a direct target of the fusion gene, making it a universal target potentially exploited by CD99 targeted methods. We have utilized CD99 targeted nanoparticles containing plasmid constructs (Cas9-EGFP-guide RNA) targeting the EWS, FLI1, or ETS domains of the fusion gene to achieve up to ~70% knockdown after one transfection in TC32 ES cells. We show that targeting the 5' EWSR1 region of the fusion gene results in dramatically lower expression of the fusion gene (30% of control), with lesser effect when the 3' ETS region is targeted (40% of control). We then compared the genes targeted by the CRISPR EWS-FLI1 KO in TC32 cells with those whose expression was altered by knock down of the fusion gene using shRNA directed against the EWS-FLI1 3'UTR in CHLA9 ES cells and found >2,000 targets in common based on total RNA Seq data with a 2-fold or greater delta in expression. Most targets were down regulated after fusion gene suppression (~1,300 vs ~1,000), indicating they are up regulated by the oncoprotein. Importantly, over half of the affected transcripts were non-coding lncRNAs (Up regulated targets: 421 coding, 539 ncRNA; Down regulated targets: 299 coding, 448 ncRNA) Nearly 600 pseudogenes were also dysregulated. The most statistically significant targets were sense and anti-sense intragenic (regulatory) ncRNAs, documenting the likely importance of ncRNAs in the genesis of Ewing sarcoma. Further, while both coding and non-coding targets were enriched for multimeric GGAA repeats within 1 kb of their TSS when compared to a randomly selected control population, the vast majority of both classes of transcripts lacked multimeric GGAA repeats in their promoters or even GGAA repeats in enhancers within 50kb of the targeted transcripts, indicating that regulation of Ewing sarcoma target gene expression is mediated proximally by factors other than direct or indirect binding of the oncoprotein in cis with the target gene. Our results demonstrate that use of Ewing-specific, CD99 targeted nanoparticles allows high-efficiency (~70%) permanent editing and knockdown of the fusion gene. This enables analysis of fusion gene targets in a native, unperturbed tumor setting, free of artifacts introduced by transfection methods or induced fusion gene expression. It also allows functional studies of the fusion gene RNA targets themselves, using the same gene editing methods, which could enable pre-clinical therapeutic proof-of-concept studies directed against the fusion gene, its validated coding and non-coding targets, or both. Citation Format: Timothy J. Triche, Sheetal A. Mitra, Hyung G. Kang, Jonathan D. Buckley. Gene editing for functional analysis of Ewing sarcoma target genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 402.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have