CD99 contributes to the EWS:FLI transcriptome by specifically affecting FOXM1-targets involved in the G2/M cell cycle phase

Ewing sarcoma (EwS), a highly aggressive malignancies affecting children and young adults, is primarily driven by a distinctive oncogenic fusion (EWSR1-ETS), whose activity is a principal source of epigenetic and clinical heterogeneity. However, another molecule (CD99) is constantly present in EWS cells. Despite the fact that this molecule is widely reported to modulate EWS genetic profile and tumor malignancy, the relevance of CD99 alone or in association with the chimera has been largely ignored. In this study, we explored the dynamic relationship between EWS::FLI1, the main fusion observed in EWS, and CD99 through experimental inducible models for the expression of one or the other molecule. The transcriptome of cells with or without expression of EWS::FLI or CD99 were analyzed in dynamics and associated with in tumor cell growth. CD99-associated EWS gene profile was found to have commonalities with that induced by EWS::FLI but also some peculiar diversities. In particular, both EWS::FLI and CD99 regulates targets of the DREAM complex but CD99 expression specifically impacts on those genes that are targets of FOXM1 and involved in the setting of G2/M phase of cell cycle. Most of these CD99-regulated genes were found to be associated to worst clinical prognosis in two different clinical datasets on the freely available R2 platform , further supporting the clinical relevance of CD99-mediated regulation of EWS gene expression. We developed two inducible cell models using a tetracycline-inducible shRNA system targeting EWS::FLI1 or CD99 transcripts (named as A673pTERshEWS::FLI1 or A673pTERshCD99 respectively)to determinate whether these two molecules work in a dependent or synergistic manner.We performed the gene expression profiles associated with their silencing and recovery by SurePrint G3 Human Gene Expression v3 8x60K Microarray Kit, cat# G4851C, Agilent Technologies in two biological replicate fro both models.