Abstract 4631: Expression of ganglioside G_D2 in Ewing sarcoma cells is not associated with specific functional characteristics or stem cell features

Safe and effective targeting of cancer with chimeric antigen receptor (CAR) engineered T cells relies on the presence of adequate tumor-associated surface antigens. We have found that Ewing sarcomas (EwS) express ganglioside G_D2 and are recognized by T cells engineered with G_D2-specific CARs. In an extended series of 97 EwS patient samples, 52 were G_D2^pos by immunohistochemistry. Based on reports that G_D2 in breast cancer defines a malignant population with stem cell characteristics, we hypothesized that G_D2 expression in EwS is associated with high capacity to self-renew and reinitiate tumor growth. Among 15 individual EwS cell lines with variable surface expression of G_D2 by flow cytometry, G_D2 expression levels were not associated with the capacities to proliferate and expand in vitro, form colonies in semi-solid medium, nor with their chemosensitivity, assessed by cell viabilities in the presence of increasing concentrations of the cytotoxic drug doxorubicin. Subpopulations with G_D2^hi phenotype selected from G_D2^pos and from G_D2^low EwS cell lines by cell sorting maintained their G_D2^hi and G_D2^low expression status during subsequent cultures over several weeks. The two subpopulations did not have different in vitro growth, colony-formation capacity, or chemosensitivity. In xenografting experiments, G_D2^hi and G_D2^low subpopulations of two EwS cell lines initiated tumors with comparable efficacies. To obtain direct evidence that G_D2 surface expression is irrelevant for the biology of EwS cells, we performed genetic knockdown of the GD3S gene which drives biosynthesis of G_D2 by CRISPR/Cas9 gene editing. GD3S gene editing resulted in effective elimination of G_D2 surface expression in the G_D2hi EwS cell lines TC-71, VH-64 and A4573. The knockdown did not affect the capacity of the cells to proliferate, form colonies in soft agar in vitro, nor their chemosensitivity compared to wild-type EwS cells from the individual cell lines. We conclude that G_D2 expression in EwS cells, other than reported in breast cancer, is not associated with distinct functional features. Specifically, G_D2 does not affect the growth characteristics, clono- and tumorigenicity and chemosensitivity of the tumor cells. Elimination of G_D2^pos subpopulations from heterogeneous tumors by CAR T cell targeting is therefore unlikely to eradicate the disease and will have to be combined with additional targeting strategies.

Citation Format: Sareetha Kailayangiri, Bianca Altvater, Silke Jamitzky, Stefanie Lesch, Jan-Henrik Mikesch, Sonja Schelhaas, Schaefers Michael, Jutta Meltzer, Nicole Farwick, Wolfgang Hartmann, Eva Wardelmann, Petra Fischer, Jendrik Hardes, Claudia Rossig. Expression of ganglioside G_D2 in Ewing sarcoma cells is not associated with specific functional characteristics or stem cell features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4631.