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.

RETRACTED: Inhibition of HDACs reduces Ewing sarcoma tumor growth through EWS-FLI1 protein destabilization

“…, 51. , 52 , 53. Clinical trials have been testing HDAC inhibitors for the treatment of young patients with recurrent or refractory solid tumors.…”

Drug Targeting of Chromosomal Translocations in Fusion-Positive Sarcoma

“…In Ewing sarcomas, the EWSR1-FLI1 protein upregulates EZH2 by binding to its promoter, thereby blocking its endothelial and neuronal differentiation capabilities [90]. Recent data show that in this process EZH2-containing PRC2 complexes interact with HDAC1, 2 and this HDAC activity mediates the immature, tumorigenic phenotype of Ewing sarcoma [91]. However, in the alveolar RMS HDAC1,2,3 also appears to serve an essential function of P3F-driven super-enhancers, as appropriate inhibitors disrupt the activity of these tumor-specific super-enhancers [92].…”

“…But, chemical inhibitors of EZH2 activity cannot reproduce the results after RNA interference (unpublished). Yet, recent data show that EZH2-containing PRC2 complexes interact with HDAC1, 2 and this HDAC activity mediates the immature, tumorigenic phenotype of Ewing sarcoma [91].…”

“…RNA analyses showed that treatment with single HDAC inhibitors (HDACi) blocked an EWSR1-FLI1-specific expression profile, and EwS cells in the presence of HDAC inhibitors (HDACi) such as entinostat and romidepsin had increased susceptibility to treatment with chemotherapeutic agents including doxorubicin. HDACi acted synergistically with the EED inhibitor A-395 and together inhibited tumor growth of Ewing sarcoma xenografts [91]. Similarly, the dual HDAC and phosphatidylinositol 3-kinase (PI3K) inhibitor Fimepinostat can thus also provide simultaneous and sustained inhibition of multiple oncogenic pathways in Ewing sarcoma and reduce EWSR1-FLI1 levels and transcriptional activity [97].…”

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)(155)(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).…”