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

Abstract

Background Histone acetylation and deacetylation seem processes involved in the pathogenesis of Ewing sarcoma (EwS). Here histone deacetylases (HDAC) class I were investigated. Methods Their 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. Results Class 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. Conclusions 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 is an interesting new treatment opportunity for this malignant disease.

Novel Targeting of DNA Methyltransferase Activity Inhibits Ewing Sarcoma Cell Proliferation and Enhances Tumor Cell Sensitivity to DNA Damaging Drugs by Activating the DNA Damage Response

“…The unique epigenetic signature of EWS could potentially be reverted by the identification of novel agents targeting epigenetic mechanisms. Several promising agents for the treatment of this disease have been reported at the preclinical level, including inhibitors of histone deacetylases (HDACi) ( 15 – 17 ), lysine-specific histone demethylase 1A (LSD1i) ( 14 , 18 ), DNA methyltransferases (DNMTi) ( 9 , 19 – 21 ) and combinations of these agents ( 22 , 23 ). DNA methylation is essential for crucial biological processes, such as maintaining genome stability, embryonic development and cell differentiation ( 24 , 25 ).…”

The importance of fusion protein activity in Ewing sarcoma and the cell intrinsic and extrinsic factors that regulate it: A review

“…Due to the reliance of EWS::FLI1 on chromatin remodeling complexes, there is great interest in targeting Ewing sarcoma and the fusion specifically using epigenetic drugs including agents that inhibit HDACs (HDACi) ( 149 ), bromodomain proteins (BETi) ( 73 , 150 – 152 ), LSD1 (LSD1i) ( 49 , 72 ), and KDM3A (JIB-04) ( 153 ). Use of HDACi in vitro and in vivo inhibits Ewing sarcoma viability, proliferation, and tumor growth ( 154 – 156 ). HDACi can directly alter expression of the fusion protein ( 58 , 155 , 156 ) and indirectly affect transcriptional function by reactivating expression of repressed target genes ( 37 , 154 , 155 ).…”

Ewing Sarcoma Meets Epigenetics, Immunology and Nanomedicine: Moving Forward into Novel Therapeutic Strategies

“…Furthermore, the combination of the HDAC6 inhibitor ACY-1215 with doxorubicin reduced tumor growth in EWS xenografts [ 48 ]. On the other hand, HDAC1 and HDAC2 knockouts demonstrated a reduction in invasiveness and tumor growth in xenografts [ 49 ]. Since the effect in tumor growth resembled EZH2 inhibition [ 37 ], the HDACi romidepsin was combined with the embryonic ectoderm development (EED) inhibitor (A-395), which inactivates the PRC2 complex.…”

“…Since the effect in tumor growth resembled EZH2 inhibition [ 37 ], the HDACi romidepsin was combined with the embryonic ectoderm development (EED) inhibitor (A-395), which inactivates the PRC2 complex. This combination treatment was superior to monotherapy blocking the proliferation and tumor growth of SK-N-MC or EW7 xenograft models [ 49 ]. In addition, the combination of SAHA with HCI-2509 decreased cell proliferation, triggering cell cycle arrest and apoptosis, reducing EWSR1-FLI1 expression by regulation of the EWSR1 promoter and altering tumor growth [ 50 ].…”