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.

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Class I Histone Deacetylases (HDAC) Critically Contribute to Ewing Sarcoma Pathogenesis

10.21203/rs.3.rs-614360/v1 ◽

2021 ◽

Author(s):

Oxana Schmidt ◽

Nadja Nehls ◽

Carolin Prexler ◽

Kristina von Heyking ◽

Tanja Groll ◽

...

Keyword(s):

Tumor Growth ◽

Microarray Analysis ◽

Ewing Sarcoma ◽

Histone Deacetylases ◽

Hdac Inhibitors ◽

Class I ◽

Specific Expression ◽

Regulatory Complex ◽

Xenograft Mice

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.

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Profile of Class I Histone Deacetylases (HDAC) by Human Dendritic Cells after Alcohol Consumption and In Vitro Alcohol Treatment and Their Implication in Oxidative Stress: Role of HDAC Inhibitors Trichostatin A and Mocetinostat

PLoS ONE ◽

10.1371/journal.pone.0156421 ◽

2016 ◽

Vol 11 (6) ◽

pp. e0156421 ◽

Cited By ~ 7

Author(s):

Marisela Agudelo ◽

Gloria Figueroa ◽

Tiyash Parira ◽

Adriana Yndart ◽

Karla Muñoz ◽

...

Keyword(s):

Oxidative Stress ◽

Dendritic Cells ◽

Alcohol Consumption ◽

Histone Deacetylases ◽

Trichostatin A ◽

Hdac Inhibitors ◽

Class I ◽

Human Dendritic Cells

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Synthesis, Molecular Docking and Biological Characterization of Pyrazine Linked 2-Aminobenzamides as New Class I Selective Histone Deacetylase (HDAC) Inhibitors with Anti-Leukemic Activity

International Journal of Molecular Sciences ◽

10.3390/ijms23010369 ◽

2021 ◽

Vol 23 (1) ◽

pp. 369

Author(s):

Hany S. Ibrahim ◽

Mohamed Abdelsalam ◽

Yanira Zeyn ◽

Matthes Zessin ◽

Al-Hassan M. Mustafa ◽

...

Keyword(s):

Molecular Docking ◽

Cancer Cells ◽

Histone Deacetylases ◽

Hdac Inhibitors ◽

Md Simulations ◽

Docking Studies ◽

Class I ◽

Hek293 Cells ◽

Human Acute Myeloid Leukemia

Class I histone deacetylases (HDACs) are key regulators of cell proliferation and they are frequently dysregulated in cancer cells. We report here the synthesis of a novel series of class-I selective HDAC inhibitors (HDACi) containing a 2-aminobenzamide moiety as a zinc-binding group connected with a central (piperazin-1-yl)pyrazine or (piperazin-1-yl)pyrimidine moiety. Some of the compounds were additionally substituted with an aromatic capping group. Compounds were tested in vitro against human HDAC1, 2, 3, and 8 enzymes and compared to reference class I HDACi (Entinostat (MS-275), Mocetinostat, CI994 and RGFP-966). The most promising compounds were found to be highly selective against HDAC1, 2 and 3 over the remaining HDAC subtypes from other classes. Molecular docking studies and MD simulations were performed to rationalize the in vitro data and to deduce a complete structure activity relationship (SAR) analysis of this novel series of class-I HDACi. The most potent compounds, including 19f, which blocks HDAC1, HDAC2, and HDAC3, as well as the selective HDAC1/HDAC2 inhibitors 21a and 29b, were selected for further cellular testing against human acute myeloid leukemia (AML) and erythroleukemic cancer (HEL) cells, taking into consideration their low toxicity against human embryonic HEK293 cells. We found that 19f is superior to the clinically tested class-I HDACi Entinostat (MS-275). Thus, 19f is a new and specific HDACi with the potential to eliminate blood cancer cells of various origins.

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Histone deacetylases are critical targets of bortezomib-induced cytotoxicity in multiple myeloma

Blood ◽

10.1182/blood-2009-07-235663 ◽

2010 ◽

Vol 116 (3) ◽

pp. 406-417 ◽

Cited By ~ 90

Author(s):

Jiro Kikuchi ◽

Taeko Wada ◽

Rumi Shimizu ◽

Tohru Izumi ◽

Miyuki Akutsu ◽

...

Keyword(s):

Multiple Myeloma ◽

Transcriptional Repression ◽

Histone Deacetylases ◽

Hdac Inhibitors ◽

Class I ◽

Transcriptional Level ◽

Critical Pathways ◽

Histone Hyperacetylation

Abstract Bortezomib is now widely used for the treatment of multiple myeloma (MM); however, its action mechanisms are not fully understood. Despite the initial results, recent investigations have indicated that bortezomib does not inactivate nuclear factor-κB activity in MM cells, suggesting the presence of other critical pathways leading to cytotoxicity. In this study, we show that histone deacetylases (HDACs) are critical targets of bortezomib, which specifically down-regulated the expression of class I HDACs (HDAC1, HDAC2, and HDAC3) in MM cell lines and primary MM cells at the transcriptional level, accompanied by reciprocal histone hyperacetylation. Transcriptional repression of HDACs was mediated by caspase-8–dependent degradation of Sp1 protein, the most potent transactivator of class I HDAC genes. Short-interfering RNA-mediated knockdown of HDAC1 enhanced bortezomib-induced apoptosis and histone hyperacetylation, whereas HDAC1 overexpression inhibited them. HDAC1 overexpression conferred resistance to bortezomib in MM cells, and administration of the HDAC inhibitor romidepsin restored sensitivity to bortezomib in HDAC1-overexpressing cells both in vitro and in vivo. These results suggest that bortezomib targets HDACs via distinct mechanisms from conventional HDAC inhibitors. Our findings provide a novel molecular basis and rationale for the use of bortezomib in MM treatment.

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Abstract 238: Class I HDAC inhibition prevents Ncx1 upregulation by stabilizing an HDAC5-HDAC1/Sin3a Repressor Complex during cardiac hypertrophy.

Circulation Research ◽

10.1161/res.113.suppl_1.a238 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Sabina Wang ◽

Lillianne G Harris ◽

Santhosh Mani ◽

Donald Menick

Keyword(s):

Cardiac Hypertrophy ◽

Hdac Inhibitor ◽

Histone Deacetylases ◽

Hdac Inhibitors ◽

Class I ◽

Proximal Promoter ◽

Hdac Inhibition ◽

Repressor Complex

Cardiac hypertrophy is often associated with the activation of signaling pathways that perpetuate altered calcium efflux and influx. One gene that is upregulated and contributes to altered intracellular calcium concentrations and worsening contractility during cardiac hypertrophy is the Sodium Calcium Exchanger ( Ncx1 ). Molecular studies implicate histone deacetylases (HDACs) in possibly regulating the expression of this gene. Our recent work reveals that HDAC1, HDAC5 and Sin3a interact and are recruited to the Ncx1 promoter through the Nkx2.5 transcription factor. Interestingly, we observed greater associated/interaction of the HDAC1-HDAC5/Sin3a repressor complex upon broad HDAC inhibition. Taken together, we hypothesized that HDAC inhibition, stabilizes an HDAC1-HDAC5/Sin3a repressor complex during cardiac hypertrophy. We addressed this hypothesis by treating isolated adult cardiomyocytes with class specific HDAC inhibitors since HDAC1 is a Class I HDAC and HDAC5 is a Class IIa HDAC. Co-Immunoprecipitation (Co-IP) revealed a greater association of repressor complex molecules in the presence of Entinostat, a Class I HDAC inhibitor compared to both non-treated control and TSA, a broad HDAC inhibitor (n=3). These works show enhanced recruitment Sin3a (co-repressor) at the proximal promoter of NCX1 as demonstrated by Chromatin-Immunoprecipitation (ChIP) (n=3). To test whether these observations translated into in vivo models, we subjected mice to transaortic constriction (TAC) to induce hypertrophy. In this model, Co-IP revealed results that similar to our in vitro studies with greater immuno- detection of repressor complex component, Sin3a after immune-precipitation with HDAC1. Furthermore, our ChIP data showed a greater PCR product amplification of proximal Ncx1 promoter, from experimental groups that were subjected to Entinostat (n=3). Our cumulative data suggests that Class I HDAC inhibition stabilizes a repressor complex on the Ncx1 promoter that hinders hypertrophy- mediated Ncx1 upregulation. Class specific HDAC inhibition may be useful in the stabilization and repression of aberrantly expressed genes that contribute to poor clinical outcomes in cardiac hypertrophy.

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HDAC inhibitors impair Fshb subunit expression in murine gonadotrope cells

Journal of Molecular Endocrinology ◽

10.1530/jme-18-0145 ◽

2019 ◽

Vol 62 (2) ◽

pp. 67-78 ◽

Cited By ~ 1

Author(s):

Gauthier Schang ◽

Chirine Toufaily ◽

Daniel J Bernard

Keyword(s):

Hdac Inhibitor ◽

Histone Deacetylases ◽

Trichostatin A ◽

Hdac Inhibitors ◽

Class I ◽

Subunit Expression ◽

Beta Subunit Gene ◽

Underlying Mechanisms

Fertility is dependent on follicle-stimulating hormone (FSH), a product of gonadotrope cells of the anterior pituitary gland. Hypothalamic gonadotropin-releasing hormone (GnRH) and intra-pituitary activins are regarded as the primary drivers of FSH synthesis and secretion. Both stimulate expression of the FSH beta subunit gene (Fshb), although the underlying mechanisms of GnRH action are poorly described relative to those of the activins. There is currently no consensus on how GnRH regulates Fshb transcription, as results vary across species and between in vivo and in vitro approaches. One of the more fully developed models suggests that the murine Fshb promoter is tonically repressed by histone deacetylases (HDACs) and that GnRH relieves this repression, at least in immortalized murine gonadotrope-like cells (LβT2 and αT3-1). In contrast, we observed that the class I/II HDAC inhibitor trichostatin A (TSA) robustly inhibited basal, activin A-, and GnRH-induced Fshb mRNA expression in LβT2 cells and in primary murine pituitary cultures. Similar results were obtained with the class I specific HDAC inhibitor, entinostat, whereas two class II-specific inhibitors, MC1568 and TMP269, had no effects on Fshb expression. Collectively, these data suggest that class I HDACs are positive, not negative, regulators of Fshb expression in vitro and that, contrary to earlier reports, GnRH may not stimulate Fshb by inhibiting HDAC-mediated repression of the gene.

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First Fluorescent Acetylspermidine Deacetylation Assay for HDAC10 Identifies Inhibitors of Neuroblastoma Cell Colony Growth That Increase Lysosome Accumulation

10.26434/chemrxiv.12440096.v1 ◽

2020 ◽

Author(s):

Daniel Herp ◽

Johannes Ridinger ◽

Dina Robaa ◽

Stephen A. Shinsky ◽

Karin Schmidtkunz ◽

...

Keyword(s):

Histone Deacetylases ◽

High Throughput Screening ◽

Neuroblastoma Cell ◽

Hdac Inhibitors ◽

Anticancer Therapy ◽

Colony Growth ◽

Autophagic Flux ◽

Enzymatic Conversion ◽

Lysine Deacetylase

Histone deacetylases (HDACs) are important epigenetic regulators involved in many diseases, esp. cancer. First HDAC inhibitors have been approved for anticancer therapy and many are in clinical trials. Among the 11 zinc-dependent HDACs, HDAC10 has received relatively little attention by drug discovery campaigns, despite its involvement e.g. in the pathogenesis of neuroblastoma. This is due in part to a lack of robust enzymatic conversion assays. In contrast to the protein lysine deacetylase and deacylase activity of the other HDAC subtypes, it has recently been shown that HDAC10 has strong preferences for deacetylation of oligoamine substrates like spermine or spermidine. Hence, it also termed a polyamine deacetylase (PDAC). Here, we present the first fluorescent enzymatic conversion assay for HDAC10 using an aminocoumarin labelled acetyl spermidine derivative to measure its PDAC activity, which is suitable for high-throughput screening. Using this assay, we identified potent inhibitors of HDAC10 mediated spermidine deacetylation in-vitro. Among those are potent inhibitors of neuroblastoma colony growth in culture that show accumulation of lysosomes, implicating disturbance of autophagic flux.

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HDAC4 promotes nasopharyngeal carcinoma progression and serves as a therapeutic target

Cell Death and Disease ◽

10.1038/s41419-021-03417-0 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Chun Cheng ◽

Jun Yang ◽

Si-Wei Li ◽

Guofu Huang ◽

Chenxi Li ◽

...

Keyword(s):

Nasopharyngeal Carcinoma ◽

Tumor Growth ◽

Therapeutic Target ◽

Histone Deacetylases ◽

Migration And Invasion ◽

Poor Overall Survival ◽

Mesenchymal Transition ◽

E Cadherin

AbstractHistone deacetylases (HDACs) are involved in tumor progression, and some have been successfully targeted for cancer therapy. The expression of histone deacetylase 4 (HDAC4), a class IIa HDAC, was upregulated in our previous microarray screen. However, the role of HDAC4 dysregulation and mechanisms underlying tumor growth and metastasis in nasopharyngeal carcinoma (NPC) remain elusive. Here, we first confirmed that the HDAC4 levels in primary and metastatic NPC tissues were significantly increased compared with those in normal nasopharyngeal epithelial tissues and found that high HDAC4 expression predicted a poor overall survival (OS) and progression-free survival (PFS). Functionally, HDAC4 accelerated cell cycle G1/S transition and induced the epithelial-to-mesenchymal transition to promote NPC cell proliferation, migration, and invasion in vitro, as well as tumor growth and lung metastasis in vivo. Intriguingly, knockdown of N-CoR abolished the effects of HDAC4 on the invasion and migration abilities of NPC cells. Mechanistically, HDAC3/4 binds to the E-cadherin promoter to repress E-cadherin transcription. We also showed that the HDAC4 inhibitor tasquinimod suppresses tumor growth in NPC. Thus, HDAC4 may be a potential diagnostic marker and therapeutic target in patients with NPC.

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Inhibition of EZH2 by chidamide exerts antileukemia activity and increases chemosensitivity through Smo/Gli-1 pathway in acute myeloid leukemia

Journal of Translational Medicine ◽

10.1186/s12967-021-02789-3 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Xuejie Jiang ◽

Ling Jiang ◽

Jiaying Cheng ◽

Fang Chen ◽

Jinle Ni ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Histone Deacetylases ◽

Myeloid Leukemia ◽

Hdac Inhibitors ◽

Annexin V ◽

Fluorescein Isothiocyanate ◽

Gli 1 ◽

Acute Myeloid

Abstract Background Epigenetic dysregulation plays important roles in leukemogenesis and the progression of acute myeloid leukemia (AML). Histone acetyltransferases (HATs) and histone deacetylases (HDACs) reciprocally regulate the acetylation and deacetylation of nuclear histones. Aberrant activation of HDACs results in uncontrolled proliferation and blockade of differentiation, and HDAC inhibition has been investigated as epigenetic therapeutic strategy against AML. Methods Cell growth was assessed with CCK-8 assay, and apoptosis was evaluated by flow cytometry in AML cell lines and CD45 + and CD34 + CD38- cells from patient samples after staining with Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI). EZH2 was silenced with short hairpin RNA (shRNA) or overexpressed by lentiviral transfection. Changes in signaling pathways were detected by western blotting. The effect of chidamide or EZH2-specific shRNA (shEZH2) in combination with adriamycin was studied in vivo in leukemia-bearing nude mouse models. Results In this study, we investigated the antileukemia effects of HDAC inhibitor chidamide and its combinatorial activity with cytotoxic agent adriamycin in AML cells. We demonstrated that chidamide suppressed the levels of EZH2, H3K27me3 and DNMT3A, exerted potential antileukemia activity and increased the sensitivity to adriamycin through disruption of Smo/Gli-1 pathway and downstream signaling target p-AKT in AML cells and stem/progenitor cells. In addition to decreasing the levels of H3K27me3 and DNMT3A, inhibition of EZH2 either pharmacologically by chidamide or genetically by shEZH2 suppressed the activity of Smo/Gli-1 pathway and increased the antileukemia activity of adriamycin against AML in vitro and in vivo. Conclusions Inhibition of EZH2 by chidamide has antileukemia activity and increases the chemosensitivity to adriamycin through Smo/Gli-1 pathway in AML cells (Fig. 5). These findings support the rational combination of HDAC inhibitors and chemotherapy for the treatment of AML.

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