Ganglioside GD2 as a target for novel combined therapies with chimeric antigen receptor-modified T cells in Ewing sarcomas

• Applicant: Professorin Dr. Claudia Rössig
• Subject Area: Pediatric and Adolescent Medicine ; Hematology, Oncology
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 256560714

Ewing sarcomas are aggressive cancers of bone and soft tissues associated with a characteristic chromosomal translocation. To increase cure rates, novel therapies are needed that eradicate micrometastatic cells capable to reinitiate tumor growth. In previous work, we found that the ganglioside GD2, a mesenchymal stroma cell marker, is overexpressed at variable densities on the surface of Ewing sarcoma cells. Surface GD2 allowed to specifically eliminate Ewing sarcoma cells by GD2-redirected immune effector cells and thus opens a new avenue for therapeutic targeting. Functional comparisons of GD2hi and GD2low Ewing sarcoma subpopulations revealed that GD2 expression in this cancer is dynamic and associated with a characteristic cellular phenotype. We further obtained evidence that GD2 expression in Ewing sarcoma cells is subject to manipulation by agents interfering with epigenetic gene regulation. Here, we explore the potential of GD2 to serve as a specific target for innovative therapies in this cancer. To substantiate our finding that GD2 expression in Ewing sarcoma is subject to epigenetic regulation, we will systematically assess the effect of HDAC inhibitors and demethylating agents on expression of GD2 and GD3S in Ewing sarcoma cultures in vitro and in vivo. Moreover, we will investigate the biological role and relevance of GD2 expression in Ewing sarcoma. GD2low and GD2hi subpopulations will be isolated by cell sorting from tumor cell cultures newly initated from biopsies. In vitro studies will address expression of markers and transcription factors associated with genetic stem cell reprogramming, generation of colonies under anchorage-independent conditions, and self-renewal by sequential replating of single cells from colonies. Tumorigenicity will be compared by xenografting limiting cell doses of each subpopulation into immunodeficient mice. The pattern and extent of disease dissemination will be compared in a systemic disease xenograft model. To establish the direct effects of GD2 expression on sarcoma cell functionality, key experiments will be repeated following inhibition of the rate-limiting enzyme of GD2 synthesis, GD3S. Clinical correlations will aim to establish GD2hi expression as a biomarker of distinct Ewing sarcoma subtypes. To explore the hypothesis that epigenetic modulators and GD2 targeting strategies have synergistic activity against Ewing sarcomas, the antitumor activity of the combination strategy will be assessed in our systemic disease model. Together, these experiments will explore the functional significance of GD2 expression in a mesenchymal malignancy and establish the preclinical prerequisites for clinical translation of combined cellular and epigenetic therapy in Ewing sarcoma and other GD2-expressing malignancies.

• DFG Programme: Research Grants