Abstract B079: Development of model systems to investigate the roles of canonical and reciprocal EWSR1 fusion proteins in sarcomagenesis

Abstract Sarcomas constitute a large group of rare, heterogeneous cancers of the bone and soft tissue affecting children and young adults. There are over 80 clinically distinct subtypes of sarcoma, and their heterogeneity is a contributing factor to the moderate and poor survival outcomes observed in primary and metastatic disease, respectively. While they are heterogeneous, many subtypes harbor a balanced chromosomal translocation fusing the Ewing Sarcoma RNA binding protein 1 (EWSR1) gene with a gene encoding a transcription factor (TF). This translocation generates two distinct fusions: a canonical fusion consisting of the N-terminal transactivation domain of EWSR1 and the C-terminal DNA-binding domain of a TF, and a reciprocal fusion fusing the N-terminus of the TF and C-terminal RNA-binding domain of EWSR1. Canonical fusion protein products exhibit aberrant TF and pioneer factor activity, leading to alterations in transcriptional programs and the epigenetic landscape within a cell that contributes to oncogenesis. The functions of the reciprocal fusion proteins, however, have remained elusive. Ewing sarcoma (ES) and clear cell sarcoma (CCS), a bone and soft tissue sarcoma, respectively, are two subtypes that have been shown to express reciprocal fusions. The ES fusion implicates EWSR1 and Friend Leukemia Integration Factor 1 (FLI1), while the CCS fusion involves Activating Transcription Factor 1 (ATF1). A challenge in studying the roles of these and other fusion proteins in sarcoma development is the lack of effective model systems, primarily due to their toxicity upon exogenous introduction into fusion-naïve cells. Here, we outline the development of a cumate-inducible lentiviral vector to study the canonical and reciprocal EWSR1 fusion proteins present in ES and CCS. To track cellular localization of the fusion proteins and distinguish fusions from their respective wildtype proteins, we designed fusion sequences with N-terminal fluorescent tags. Tightly controlled expression of canonical and reciprocal fusion proteins reduces their toxicity when expressed in cells. We have successfully established 293T and HT-1080 cells that stably express these vectors. We have also validated cumate-inducible expression of fluorescently tagged canonical and reciprocal FLI1 and ATF1 fusions at the RNA and protein levels in both cell lines. The canonical fusion proteins produced by our model have the expected effects on expression levels of known downstream canonical fusion target genes. Initial cellular localization studies using our model and fluorescence microscopy indicate that reciprocal fusion proteins may localize in the nucleus, which was previously unknown. Overall, our cumate-inducible model represents a viable system for fusion protein expression. Using this model, we aim to gain insight into the roles of reciprocal fusions in ES and CCS development with hopes of uncovering potentially targetable vulnerabilities, opening the door for the development of new treatments in a patient population in need of novel and efficacious therapies. Citation Format: Sarah Gawlak, Joyce Ohm. Development of model systems to investigate the roles of canonical and reciprocal EWSR1 fusion proteins in sarcomagenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B079.