EZH2 Inhibition in Ewing Sarcoma Upregulates G(D2) Expression for Targeting with Gene-Modified T Cells

Chimeric antigen receptor (CAR) engineering of T cells allows one to specifically target tumor cells via cell surface antigens. A candidate target in Ewing sarcoma is the ganglioside G(D2), but heterogeneic expression limits its value. Here we report that pharmacological inhibition of Enhancer of Zeste Homolog 2 (EZH2) at doses reducing H3K27 trimethylation, but not cell viability, selectively and reversibly induces G(D2) surface expression in Ewing sarcoma cells. EZH2 in Ewing sarcoma cells directly binds to the promoter regions of genes encoding for two key enzymes of G(D2) biosynthesis, and EZH2 inhibition enhances expression of these genes. G(D2) surface expression in Ewing sarcoma cells is not associated with distinct in vitro proliferation, colony formation, chemosensitivity, or in vivo tumorigenicity. Moreover, disruption of G(D2) synthesis by gene editing does not affect its in vitro behavior. EZH2 inhibitor treatment sensitizes Ewing sarcoma cells to effective cytolysis by G(D2)-specific CAR gene-modified T cells. In conclusion, we report a clinically applicable pharmacological approach for enhancing efficacy of adoptively transferred G(D2)-redirected T cells against Ewing sarcoma, by enabling recognition of tumor cells with low or negative target expression.