EZH2 Inhibition in Ewing Sarcoma Upregulates GD2 Expression for Targeting with Gene-Modified T Cells

Abstract

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.

Prognostic profiling of the immune cell microenvironment in Ewing´s Sarcoma Family of Tumors

“…8 Recently, it was shown that EZH2 inhibition together with chimeric antigen receptor (CAR) T cell therapy is an interesting novel immunotherapeutic treatment approach in EWST, as it leads to upregulation of the immune target ganglioside G D2 . 38 Similar to adult tumor types, the relative contribution of total T cells correlates directly with a better OS and EFS (training cohort: p = .044, p = .032, respectively; validation cohort: p = .005, p = .02, respectively) in ESFT. Previously, it has been demonstrated that HLA-G, a non-classical, immuneinhibitory MHC class 1 molecule is expressed in tumor cells and/or lymphocytes in ESFT primary tumor samples.…”

Design and Characterization of an “All-in-One” Lentiviral Vector System Combining Constitutive Anti-GD2 CAR Expression and Inducible Cytokines

“…We first reassessed the well-established second generation CAR targeting the cell-surface ganglioside G D2 with respect to specific target recognition. The G D2 CAR contains a single-chain variable fragment (scFv) derived from the monoclonal antibody 14.G2a that has been approved for therapeutic use in children with neuroblastoma and has been used in previous clinical trials [33], an IgG1 (HCH 2 CH 3 ) hinge domain followed by the CD28 transmembrane (TM) domain, and the intracellular 4-1BB and CD3ζ signaling domains [25] ( Figure 1A). To verify the specificity and function of the G D2 CAR, the CAR was initially cloned into a third generation lentiviral self-inactivating (SIN) vector [34] under the control of the human phosphoglycerate kinase (hPGK) promoter (LV-G D2 CAR) and tested in a murine T cell hybridoma reporter cell line.…”

“…The amplified and sequenced fragment was cloned via three-fragment ligation into vector pRRL.PPT.PGK.newMCS.EBFP2.i2.Zeo.PRE using the XhoI and NheI sites between the cPPT and the hPGK promoter sequences. The human codon-usage optimized ORF second generation CAR containing scFv (14.G2a), HCH 2 CH 3 hinge, CD28, CD137 (4-1BB), and CD3ζ domains [25] was flanked by the restriction enzymes AgeI and SalI were cloned into the lentiviral "all-in-one" SIN vector driven by an hPGK promoter. The restriction sites MluI and EcoRI, or AfeI and EcoRI, respectively, were inserted to exchange the iGOI within the "all-in-one" vector construct (Figure 2A).…”

“…As proof-of-concept, we generated a modular "all-in-one" vector system based on clinically applied third generation lentiviral self-inactivating (SIN) vectors designed to constitutively express a CAR in combination with NFAT-inducible cytokine expression, for example, IL12 or IL18. Therefore, we further codon-optimized a well-established second generation CAR that targets the cell-surface disialoganglioside G D2 [25], which has restricted expression in normal tissues, but is expressed on several solid tumors, including glioblastoma [26], neuroblastoma [27], and sarcomas [28]. Clinical trials already support the safety and efficacy of G D2 -redirected CAR T cells without undesirable adverse effects [29][30][31].…”

Epigenetic Inhibition Puts Target Antigen in the Crosshairs of CAR T Cells

“…The successes of chimeric antigen receptor (CAR) T cell therapies targeting hematologic malignancies have not yet translated to solid tumors due to challenges with antigen selection, tumor trafficking, T cell persistence, and the tumor microenvironment (TME). In this issue of Molecular Therapy, Kailayangiri et al 1 report that inhibiting Enhancer of Zeste Homolog 2 (EZH2) to upregulate tumor antigen GD2 synthase (GD2) expression in Ewing sarcoma cells circumvents its variable expression. The combination of EZH2 inhibition therapy with CAR T cell therapies may therefore improve outcomes in Ewing sarcoma and potentially other malignancies.…”