Abstract 1467: A novel screening model to identify EWS::FLI1 regulators and their consequences on Ewing sarcoma plasticity

Abstract Ewing sarcoma (ES) is a rare, highly aggressive cancer of the bone and soft tissues that mainly affects children and young adults. The most common driver of ES is a chromosomal translocation between the EWSR1 gene and an ETS family member, most frequently FLI1. This leads to the expression of an aberrant transcription factor called EWS::FLI1 (EF1) that acts as an epigenetic reprogrammer and transcriptional modulator. Previous studies on ES cell lines showed that EF1 levels are dynamic where cells with high EF1 levels are more proliferative, while cells with low EF1 levels have a strong propensity to migrate and metastasize. However, there is no direct evidence of EF1 fluctuations on a functional protein level. Moreover, the upstream modulators or pathways regulating EF1 thresholds and their corresponding consequences on ES tumorigenesis are poorly understood. Thus, we aimed to develop an ES screening model to identify EF1 regulators by performing a high-throughput drug screen and a genome-wide CRISPR/Cas9 knockout screen. This model was established by endogenously tagging EF1 of ES cell lines (A673, TC71) with a fluorescent tag (mNeonGreen) together with a degradation tag (dTAG) using CRISPR/Cas9 knock-in. The fluorescent tag serves for the readout of altered EF1 expression on multiple levels (transcriptional, post-transcriptional, translational, post-translational, and epigenetic). At the same time, dTAG allows mimicking rapid and acute EF1 perturbation-specific cell states upon treatment with a VHL-recruiting ligand (dTAGv-1). The successful knock-in clones with edited EF1 were extensively characterized compared to the wildtype EF1 cells with respect to cellular morphology, subcellular EF1 localization, stability, and target gene expression to rule out a loss of EF1 function due to the addition of the tags. The results from the high throughput drug screen (2.5K custom compound library) resulted in a candidate list of EF1 up- and down-regulators involving genes of various cancer signaling pathways (PI3K/AKT/mTOR, IGF-1, ALK pathway), cell cycle regulators (CDKs, AURKA, PLK1) and epigenetic regulators (HDACs, BETs). In the next steps, the drug targets will be compared to the gene hits from the CRISPR screen in an unbiased manner to identify novel EF1-regulatory pathways. Moreover, the functional consequences of treatment with EF1-modulatory compounds on ES tumorigenesis will be studied. Together, our study will promote a better understanding of factors affecting EF1 thresholds and ES plasticity and help identify potential therapeutic targets for ES. Citation Format: Veveeyan Suresh, Valerie Fock, Beat Schäfer, Heinrich Kovar. A novel screening model to identify EWS::FLI1 regulators and their consequences on Ewing sarcoma plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1467.