Class I histone deacetylases (HDAC) critically contribute to Ewing sarcoma pathogenesis

Oxana Schmidt,Kristina Von Heyking,Angelika Eggert,Nadja Nehls,Tim Hensel,Katja Steiger,Dennis Gürgen,Carolin Prexler,Heathcliff D Garcia,Tanja Groll,Günther H S Richter,Katharina Pardon,Stefan Burdach,Anton G Henssen

doi:10.1186/s13046-021-02125-z

Abstract

BackgroundHistone acetylation and deacetylation seem processes involved in the pathogenesis of Ewing sarcoma (EwS). Here histone deacetylases (HDAC) class I were investigated.MethodsTheir role was determined using different inhibitors including TSA, Romidepsin, Entinostat and PCI-34051 as well as CRISPR/Cas9 class I HDAC knockouts and HDAC RNAi. To analyze resulting changes microarray analysis, qRT-PCR, western blotting, Co-IP, proliferation, apoptosis, differentiation, invasion assays and xenograft-mouse models were used.ResultsClass I HDACs are constitutively expressed in EwS. Patients with high levels of individual class I HDAC expression show decreased overall survival. CRISPR/Cas9 class I HDAC knockout of individual HDACs such as HDAC1 and HDAC2 inhibited invasiveness, and blocked local tumor growth in xenograft mice. Microarray 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 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.ConclusionsClass 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 is an interesting new treatment opportunity for this malignant disease.

Highlights

Histone acetylation and deacetylation seem processes involved in the pathogenesis of Ewing sarcoma (EwS)
Expression of histone deacetylases (HDAC) class I genes in EwS Histone deacetylase (HDAC) class I genes HDAC1, 2, 3, and 8 are widely expressed in different pediatric sarcomas (Fig. 1a) and other pediatric and adult tumor entities (Additional file 2: Fig. S1a)
To analyze whether the oncogenic fusion protein EWS-FLI1 may influence HDAC class I gene expression in EwS, we investigated RNA interference-mediated EWS-FLI1 silencing and observed a significant suppression of HDAC class I expression levels in two different EwS cell lines while one (EW7) seemed less affected (Fig. 1c)

Summary

Introduction

Histone acetylation and deacetylation seem processes involved in the pathogenesis of Ewing sarcoma (EwS). EWS-FLI1 expression leads to widespread epigenetic changes in promoters, enhancers, and super-enhancers. Such global alterations of histone H3K27-acetylation as well as H3K27-trimethylation associated with an altered HDAC activity seem a general feature of EwS [10, 11]. RNA interference of EZH2 revealed an EZH2-maintained, undifferentiated reversible, highly malignant, stemness phenotype in EwS [12, 13]. The effects of gene silencing after RNA interference of EZH2 were mimicked by treatment of EwS with different HDAC inhibitors including Trichostatin A (TSA) or Entinostat (MS-275) [12]

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Discussion

Conclusion