Abstract 1669: Transient IGF1 exposure stably reprograms EWS-FLI1 immortalized embryonal limb-derived mesenchymal stem cell-like cells to full transformation
Authors: Noorizadeh Rahil , Halbritter Florian , Sax Barbara , Mikulits Wolfgang , Moriggl Richard , Kovar Heinrich 
Published: June 2022

Ewing sarcoma (EwS), an aggressive bone and soft tissue cancer predominantly occurring during adolescence, is driven by the chimeric EWS-FLI1 oncogene. It is assumed that a permissive cellular context or potentially additional complementing mutations are necessary for the EWS-FLI1 fusion to exert its full oncogenic effect. Prior efforts to generate genetic EwS mouse models failed because of high EWS-FLI1 induced apoptosis when expressed in various candidate progenitor cell types. In our study, we restricted EWS-FLI1 expression to mesenchymal stem cells during endochondral bone formation by crossing a mouse containing EWS-FLI1 knocked into the Rosa26 locus with a mouse expressing Prx1-driven Cre recombinase. However, the resulting EFprx1 progeny died after birth as functional consequence of severe skeletal malformations resulting from differentiation arrest of early chondrocytes. In vitro, these embryonic EWS-FLI1 transgenic chondrocyte-like mesenchymal stem cells (EFprx1 MSCLC) are immortal, but not fully transformed. However, we observed elevated levels of cleaved PARP-1, a surrogate marker of apoptosis. This apparent increased cellular turnover was paralleled by down-regulation of anti-apoptotic Bcl2a family genes as identified by RNA-seq analysis of EFprx1 MSCLC. The peak incidence of human EwS occurs during puberty, a developmental period in which several growth promoting hormones are highly enriched in the bone microenvironment. Among them is IGF1, which is known for its anti-apoptotic activity through upregulation of Bcl2 family genes. As previous studies implied IGF1 signaling essential in EwS pathogenesis, we speculated that IGF1 treatment may aid in the transformation process of EFprx1 MSCLC. In fact, a significant increase in soft-agar colony formation was observed upon IGF1 treatment for 4 weeks. To test whether IGF1 had irreversibly reprogrammed EFPrx1 MSCLC, we picked colonies grown in presence of IGF1, expanded and re-assayed them after 5 weeks in absence of the hormone. Strikingly, EFprx1 MSCLC retained their transformed phenotype not only in the soft-agar assay but also upon xenotransplantation in mice. Gene set enrichment analysis of upregulated genes from these stably hormone-reprogramed fully transformed EFprx1 MSCLC showed a significant enrichment for the EWS-FLI1 specific transcriptional signature and cell cycle-related pathways, while apoptotic gene sets were downregulated. Our data suggest that transient IGF1 exposure in the bone microenvironment may be required for EWS-FLI1 driven transformation. We propose that EwS formation could be influenced by hormonal changes during puberty. Based on these findings, we postulate that correct developmental timing of EWS-FLI1 expression may play a pivotal role for the generation of an EwS mouse model.

Citation Format: Rahil Noorizadeh, Florian Halbritter, Barbara Sax, Wolfgang Mikulits, Richard Moriggl, Heinrich Kovar. Transient IGF1 exposure stably reprograms EWS-FLI1 immortalized embryonal limb-derived mesenchymal stem cell-like cells to full transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1669.