Introduction: Recent evidence demonstrated that a low mutation rate seems a general feature of pediatric cancers. Ewing sarcoma (EwS) is defined by balanced chromosomal EWS/ETS translocations, which give rise to oncogenic chimeric proteins (EWS-ETS). Previously, we identified the histone methyl-transferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), the enzymatic subunit of the polycomb PRC2 complex, to be over-expressed in EwS. RNA interference of EZH2 suppressed tumor development and metastasis in vivo and microarray analysis of EZH2 knock down revealed an EZH2-maintained, undifferentiated, reversible phenotype in EwS. Microarray analysis further revealed that treatment with MS-275 or TSA resulted in the induction of a similar pattern of differentiation genes as was previously observed after EZH2 blockade and seems to be dependent on histone deacetylase (HDAC) activity.
Experimental procedures: The role of class I histone deacetylases (HDACs) was determined using different potent inhibitors like Trichostatin A (TSA), Romidepsin (FK228), Entinostat (MS-275) and PCI-34051 as well as a CRISPR/Cas9 approach to knock out class I HDACs. To analyze resulting changes microarray analysis, gene set enrichment analysis (GSEA), qRT-PCR, western blotting, proliferation, apoptosis and invasion assays as well as immunofluorescence experiments were deployed.
Results: Interestingly, treatment with enzymatic inhibitors blocking EZH2 activity could not mimic the results observed after EZH2 RNA interference indicating that EZH2-containing PRC2 complexes may serve as a building block of class I HDAC activity in ES. Consequently, in proliferation assays EwS cells were more susceptible to treatment with HDAC inhibitors than other small round blue cell tumors such as neuroblastoma or pediatric cALL cells. Microarray and GSEA analysis demonstrated that treatment with Romidepsin resulted in an inhibition of an EwS specific expression profile while Entinostat suppressed metastasis relevant genes. In addition, EwS cells demonstrated increased susceptibility to treatment with chemotherapeutics such as Doxorubicin and Vincristine in the presence of HDACi. Consequently, CRISPR/Cas9 knock outs of HDAC1 to HDAC3 in EwS cell lines resulted in a reduction of their proliferative ability and greater sensitivity to treatment with chemotherapeutics.
Conclusion: Class I HDAC proteins seem to be important mediators of the pathognomonic EWS-ETS-mediated transcription program in EwS and thus interesting new treatment opportunities for this malignant disease.
Citation Format: Oxana Schmidt, Nadja Nehls, Kristina von Heyking, Tim Hensel, Stefan Burdach, Günther H. Richter. Class I histone deacetylases (HDAC) critically contribute to Ewing sarcoma pathogenesis [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 3181.