Abstract 4499: Genomic editing of EWS-FLI1 and its targets, and its therapeutic potential in treatment of Ewing sarcoma

Abstract

Abstract Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor. These tumors have a low mutational burden and are mainly driven by chimeric transcription factor, EWS-FLI1 or equivalent fusion gene. Tumor dependency on EWS-FLI1 makes it an ideal therapeutic target but targeting this disordered protein has proven to be a challenge. Here we show that CRISPR-Cas9-mediated genomic editing can a) be employed to effectively target EWS-FLI1 and its target genes, both coding and non-coding, and b) be safely delivered to tumors in mice, thus highlighting its potential as a therapeutic agent. All CRISPR-Cas9 reagents (ThermoFisher Scientific) including Cas9 protein, single guide RNA (sgRNA) directed against various gene targets and controls, and LentiArrayTM lentiviruses were obtained and optimized for in vitro and in vivo use. Cas9-sgRNA ribonucleoprotein (RNP) complexes were transfected using CRISPRMAXTM or delivered to ES cells by CD99-targeted nanoparticles. We were able to achieve about 70% genomic cleavage when we targeted the fusion gene, EWS-FLI1, and its coding target, NR0B1, using two different sgRNA delivered as RNP complexes. Loss of EWS-FLI1 slowed down cell growth, but increased cell adhesion, and cell invasion. For EWS-FLI1 target, long non-coding RNA, FEZF1-AS1, we used paired sgRNA targeting two different regions of the RNA to achieve effective genomic editing (70%) and loss of transcript expression. We further used a lentiviral-generated stable Cas9 expressing A673 cell line to effectively knockout FEZF1-AS1 using lentiviruses expressing paired sgRNA. Both the RNP and lentiviral mediated FEZF1-AS1 knockdown did not affect cell growth but significantly decreased cell invasion. Dual knockdown of EWS-FLI1 and FEZF1-AS1 decreased cell growth and cell invasion. Mice with ES tumors in their flanks were treated with intravenous injections of CD99-targeted nanoparticles carrying Cas9-sgRNA RNP. Mice injected with Cas9-scrambled gRNA RNP had regular tumor growth. Cas9-EWSR1 sgRNA RNP-treated mice had nearly completely suppressed tumor growth for the duration of treatment but failed to totally eliminate the tumors. In conclusion, we show that CRISPR-Cas9 mediated genomic editing can effectively target EWS-FLI1 and disrupt its function in ES pathogenesis. We further demonstrate that modified CRISPR strategy can effectively knockout non-coding RNA and that the combined targeting of EWS-FLI1 and its lncRNA target had an added therapeutic response in controlling tumor growth and invasion. The pilot animal studies (more detailed studies underway) emphasize the importance of developing CRISPR-Cas9 as a therapeutic tool provided we can safely deliver it in vivo using vehicles like targeted nanoparticles to help reduce toxicity caused by untoward targeting effects. Citation Format: Sheetal A. Mitra, Namritha Ravinder, Veronica Magnon, Jon Nagy, Timothy J. Triche. Genomic editing of EWS-FLI1 and its targets, and its therapeutic potential in treatment of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4499.