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Ewing sarcoma (EwS), a highly aggressive malignancy affecting children and young adults, is primarily driven by a distinctive oncogenic fusion, the EWSR1-ETS, whose activity is a key source of epigenetic and clinical heterogeneity. CD99 is constantly present in EwS cells, known to modulate the EwS genetic profile and tumor malignancy. However, the relevance of CD99 alone, or in association with EWSR1-ETS chimeras, is poorly understood. We explored the dynamic relationship between CD99 and EWS::FLI1, the main fusion observed in EwS, by means of model systems with inducible expression of either molecule. The transcriptomic dynamics of cells with or without expression of EWS::FLI1 or CD99 were analyzed and correlated with tumor cell growth. The CD99-associated EwS gene profile was found to have commonalities with the profile induced by EWS::FLI1, but also peculiar differences. Both EWS::FLI1 and CD99 are regulated targets of the DREAM complex, but the CD99 expression specifically impacted genes that are the targets of FOXM1 and are involved in the setting of the G2/M phase of the cell cycle. Most CD99-regulated FOXM1-targeted genes were found to correlate with bad prognosis in two public clinical datasets (R2 platform), further supporting the clinical relevance of CD99-mediated regulation of EwS gene expression., Competing Interests: No potential conflicts of interest were disclosed by the other authors., (© 2024 The Author(s). Journal of Cell Communication and Signaling published by John Wiley & Sons Ltd.)

Ewing sarcoma (EWS) is the second most common pediatric bone tumor. The EWS tumor microenvironment is largely recognized as immune-cold, with macrophages being the most abundant immune cells and their presence associated with worse patient prognosis. Expression of CD99 is a hallmark of EWS cells, and its targeting induces inhibition of EWS tumor growth through a poorly understood mechanism. In this study, we analyzed CD99 expression and functions on macrophages and investigated whether the concomitant targeting of CD99 on both tumor and macrophages could explain the inhibitory effect of this approach against EWS. Targeting CD99 on EWS cells downregulated expression of the "don't eat-me" CD47 molecule but increased levels of the "eat-me" phosphatidyl serine and calreticulin molecules on the outer leaflet of the tumor cell membrane, triggering phagocytosis and digestion of EWS cells by macrophages. In addition, CD99 ligation induced reprogramming of undifferentiated M0 macrophages and M2-like macrophages toward the inflammatory M1-like phenotype. These events resulted in the inhibition of EWS tumor growth. Thus, this study reveals what we believe to be a previously unrecognized function of CD99, which engenders a virtuous circle that delivers intrinsic cell death signals to EWS cells, favors tumor cell phagocytosis by macrophages, and promotes the expression of various molecules and cytokines, which are pro-inflammatory and usually associated with tumor regression. This raises the possibility that CD99 may be involved in boosting the antitumor activity of macrophages., (©2023 The Authors; Published by the American Association for Cancer Research.)