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Transcriptomic and genomic studies classify NKL54 as a histone deacetylase inhibitor with indirect influences on MEF2-dependent transcription (RNA-Seq)

ABSTRACT: In leiomyosarcoma class IIa HDACs bind MEF2 and convert these transcription factors into repressors to sustain proliferation. Theoretically, disruption of this complex should reverse the transformed phenotype. NKL54, a PAOA (pimeloylanilide o-aminoanilide) derivative, binds a hydrophobic groove of MEF2, that is used as a docking site by class IIa HDACs. However, NKL54 could also act as an HDAC inhibitor (HDACI). Therefore, it is not clear which activity is predominant. Here, we show that NKL54 and similar compounds are unable to release MEF2 from binding to class IIa HDACs. Comparative transcriptomic analysis classifies these compounds as strongly related to SAHA/vorinostat. Low expressed genes are upregulated by HDACI, while abundant genes are repressed. This transcriptional resetting correlates with a reorganization of H3K27 acetylation around the TSS. Among the upregulated genes there are several BH3-only family members, thus explaining the induction of apoptosis. Moreover, NKL54 triggers the upregulation of MEF2 and the downregulation of class IIa HDACs. NKL54 also increases the binding of MEF2D to promoters of genes that are upregulated after treatment. In summary, although NKL54 cannot relieve MEF2 from binding to class IIa HDACs binding, it supports MEF2-dependent transcription through several actions, including potentiation of chromatin binding.

ORGANISM(S): Homo sapiens

PROVIDER: GSE180804 | GEO | 2022/01/25

REPOSITORIES: GEO

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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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Transcriptomic and genomic studies classify NKL54 as a histone deacetylase inhibitor with indirect influences on MEF2-dependent transcription (RNA-Seq)

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