Project description:Background: Ewing sarcoma (EwS) are characterized by oncogenic chimeric EWS-ETS proteins. EZH2 is upregulated via predominant EWS-FLI1 in EwS. RNAi of EZH2 revealed an EZH2-maintained, undifferentiated, stemness phenotype. Herein, microarray analysis demonstrated that treatment with HDAC inhibitors (HDACi) entinostat or TSA resulted in the induction of a similar pattern of differentiation genes as observed after EZH2 RNAi. This indicates that EZH2-containing PRC2 complexes may serve as a building block of class I HDAC activity in EwS. Methods: The role of class I HDACs was determined using different inhibitors including TSA, romidepsin (FK228), entinostat (MS-275) and PCI-34051 as well as CRISPR/Cas9 class I HDAC knock outs and HDAC RNAi. To analyze resulting changes microarray and gene set enrichment analysis (GSEA), qRT-PCR, western blotting, Co-IP, proliferation, apoptosis, differentiation, invasion assays and xenograft-mouse models were used. Results: Class I HDACs are constitutively expressed in EwS. They seem regulated via EWS-FLI1. Interestingly, patients with high levels of individual class I HDAC expression show decreased overall survival. CRISPR/Cas9 class I HDAC knock out of individual HDACs such as HDAC1 and HDAC2 inhibited growth, invasiveness, and blocked local tumor growth in xenograft mice. Microarray and GSEA analysis demonstrated that treatment with individual HDAC inhibitors (HDACi) blocked an EWS-FLI1 specific expression profile, while entinostat in addition suppressed metastasis relevant genes. EwS cells demonstrated increased susceptibility to treatment with first line chemotherapeutics including doxorubicin in the presence of HDACi. Furthermore, HDACi treatment mimicked RNAi of EZH2 in EwS. Treated cells showed diminished growth capacity, but an increased endothelial as well as neuronal differentiation ability. HDACi synergizes with EED inhibitor (EEDi) in vitro and together inhibited tumor growth in xenograft mice. Co-IP experiments identified HDAC class I family members as part of a regulatory complex together with PRC2. Conclusion: Class I HDAC proteins seem to be important mediators of the pathognomonic EWS-ETS-mediated transcription program in EwS and in combination therapy, co-treatment with HDACi are interesting new treatment opportunities for this malignant disease.

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