Ewing Tumors (ET) are highly malignant, localized in bone or soft tissue and are molecularly defined by ews/ets translocations. DNA microarray analysis revealed a relationship of ET to both endothelium and fetal neural crest. We identified expression of histone methyl-transferase Enhancer of Zeste, Drosophila, Homolog 2 (EZH2) to be increased in ET. EZH2’s suppressive activity maintains stemness in normal and malignant cells.

Here, we found EWS/FLI1 bound to the EZH2 promoter in vivo and induced EZH2 expression in ET and mesenchymal stem cells. Down-regulation of EZH2 by RNA interference in ET suppressed oncogenic transformation by inhibiting clonogenicity in vitro. Similarly, tumor development and metastasis was suppressed in immunodeficient Rag2−/−γC−/− mice. EZH2-mediated gene silencing was shown to be dependent on histone deacetylase (HDAC) activity. Subsequent microarray analysis of EZH2 knock down, HDAC-inhibitor treatment and confirmation in independent assays revealed an undifferentiated phenotype maintained by EZH2 in ET. EZH2 regulated stemness genes such as nerve growth factor receptor (NGFR) as well as genes involved in neuroectodermal and endothelial differentiation (EMP1, EPHB2, GFAP, GAP43).

These data suggest that EZH2 might play a central role in Ewing Tumor pathology by shaping the oncogenicity and stem cell phenotype of this tumor.

The rationale of adoptive T cell therapy is based on the attempt to circumvent pre-existing tolerance mechanisms by stimulating potentially tumor-reactive T cells ex vivo. Efforts to eradicate cancer by adoptive T cell transfer have been limited due to the difficulty of isolating tumor-reactive T cells present in low numbers in peripheral blood of tumor patients. Furthermore, the development of an effective immunotherapy in the autologous context is hampered by the deficit of an effective T cell repertoire against tumor antigens.

We have optimized the techniques for isolating and expanding antigen-specific allogeneic T cells. Following repetitive peptide-driven stimulations with HLA-A*0201 positive dendritic cells the responding HLA-A*0201 negative CD8+ T cells were stained with HLA-A*0201/peptide pentamers. Multimer-positive T cells were sorted and directly cloned by limiting dilution. Using this technique we have succeeded in establishing T cell clones directed against several HLA-A*0201-resticted peptides derived from Ewing Tumor (ET) specific antigens identified via previous DNA microarray analysis and supposed to play a central role in the pathogenesis of this tumor. These T cells not only specifically recognized peptide-pulsed target cells or antigen transfected cells in the context of HLA-A*0201 but also killed HLA-A*0201+ ET expressing the antigen while HLA-A*0201– ET were not affected.

Allogeneic, tumor specific T cells can be easily isolated via Peptide/HLA-multimer technology and may benefit therapeutic strategies in allogeneic stem cell transplantation.