Among the various epigenetic mechanisms involved

Among the various epigenetic mechanisms involved in EwS pathogenesis, we focused on the PRC enzyme EZH2, since it is a direct target of EWSR1-FLI1 and a critical mediator of malignant cell growth.24, 30 Our observation that the inhibition of EZH2 in GD2neg EwS RGFP966 receptor induces GD2 expression leads us to propose that EZH2-mediated repression of genes involved in GD2 biosynthesis modulate GD2 expression in this cancer, resulting in the observed intratumor and interpatient heterogeneity of expression. This hypothesis is supported by our following findings: first, GD2 upregulation by EZH2 inhibition is associated with the removal of methylation marks at histone H3K27, and it is reversible by withdrawal of the agent; second, EZH2 directly binds to the promoters of genes encoding for the two critical enzymes in GD2 biosynthesis, GD3S and GD2S; and, finally, induction of GD2 by EZH2 inhibition is associated with upregulated gene expression of GD3S and, to a lesser extent, GD2S. Epigenetic regulation of GD2 expression in tumor cells likely involves more than a single genetic target. Synthesis of GD2 and other complex gangliosides and their precursors relies on various enzymatic steps, each of which alone or, more likely, in combination could be a target for modulation by EZH2 and other epigenetic regulators. Our finding of a key significance of GD3S for GD2 expression is well in line with data obtained in breast cancer. In addition, PRC2-independent effects of EZH2 and even activation of genes involved in the degradation of GD2 could contribute to the molecular mechanism. While we are the first to show upregulation of GD2 by EZH2 inhibition in EwS, another example supports epigenetic regulation of GD2 expression in cancer: in GD2-positive neuroblastoma cells, the histone deacetylase (HDAC) inhibitor vorinostat was reported to further enhance expression of the antigen. GD2 upregulation by vorinostat was associated with increased levels of GD2S protein, but not transcripts, and GD3S was not investigated. Since gangliosides have important biological functions in both normal and malignant cells, we were concerned that the manipulation of GD2 expression in EwS cells could affect their malignant phenotype. Intriguingly, GD2-expressing cells in breast cancer define a malignant population with a higher capacity to self-renew and reinitiate tumor growth36, 48 and a molecular profile associated with stem cell function and epithelial-mesenchymal transition (EMT). Inhibition of GD2 biosynthesis in breast cancer hampers self-renewal, mammosphere formation, tumor initiation, and cell motility, suggesting a functional contribution of GD2 to stem cell features.36, 48 In comparative experiments with EwS subpopulations selected for high and low or absent GD2 expression and by disruption of GD2 expression using targeted gene editing, we provide clear evidence that GD2 expression in EwS is functionally irrelevant for proliferation, stem cell-associated functions, and chemosensitivity. These findings support the safety of upregulating this antigen on EwS cells. By enhancing GD2 surface expression, EZH2 inhibitors emerge as promising new candidates for effective combination regimens with GD2-targeted therapies in EwS. Since H3K27 methylation by EZH2 regulates various cellular functions, the use of EZH2 inhibitors as sensitizers for CAR T cell targeting requires consideration of additional effects both on tumor cells and on T cells. Due to its role in cell differentiation and tumorigenicity in EwS, EZH2 was suggested as a therapeutic target.24, 30 As a single agent in a preclinical in vivo model, the inhibitor tazemetostat had only low activity against EwS xenografts. Still, the antitumor effects of epigenetic therapy and GD2-specific immune targeting could add up to a potent combination strategy. In T cells, EZH2 inhibition was found to increase the cytotoxic activity of effector T cells and reprogram regulatory functional profiles of suppressive T cell populations, further encouraging the combination of EZH2 inhibitors with adoptive T cell therapies. Importantly, pharmacological EZH2 inhibition may reverse the limited T cell trafficking into the tumor microenvironment of solid cancers caused by epigenetic silencing of chemokines CXCL9 and CXCL10, and, thereby, it may overcome one of the most significant barriers to CAR T cell targeting of solid tumors.