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1
Balasubramanian, Sriram, Susanne Steggerda, Mint Sirisawad, Marshall Schreeder, Luke Doiron, and Joseph J. Buggy. "The Histone Deacetylase-8 (HDAC8) Selective Inhibitor PCI-34051 Decreases Interleukin-1 Beta Secretion in Vitro and Reduces Inflammation in Vivo." Blood 112, no. 11 (November 16, 2008): 2581. http://dx.doi.org/10.1182/blood.v112.11.2581.2581.
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Abstract Inhibitors of histone deacetylases (HDACs) which are currently in clinical testing for treating various cancers typically inhibit multiple isoforms of the 11-member HDAC family. We have developed an isoform-selective HDAC inhibitor, PCI-34051, that inhibits HDAC8 with a Ki of 10 nM and greater than 200-fold selectivity over other HDAC isoforms (Balasubramanian et al. (2008) Leukemia,22:1026–34). We have shown that PCI-34051 selectively induced apoptosis in cell lines derived from T-cell lymphomas and leukemias, but not in other tumor or normal cell types. Here we show that it potently inhibits the secretion of the pro-inflammatory cytokine interleukin-1 beta (IL-1b) in lipopolysaccharide (LPS)-stimulated peripheral mononuclear blood cells (PBMC) and isolated monocytes. PCI-34051 inhibited IL-1b secretion (by 80% compared to control) from LPS-stimulated human PBMC with an IC50 of 0.6 uM, which is much lower than the growth inhibitory concentrations of 2.4–4 uM required in T-cell lymphomas. We found that PCI-34051 also inhibited the secretion of interleukin-18 (IL-18) to a similar extent as IL-1b, but secretion of other pro-inflammatory cytokines, including MIP-1b, MCP-1, TNFa, and IL-6, was inhibited to a smaller extent. Interestingly, IL-18, like IL-1b, is synthesized without a signal peptide, and also utilizes the same non-classical endosomal secretory pathway as IL-1b including cleavage of the pro-form by caspase-1. Thus, we theorized that the modulatory effect of PCI-34051 is likely to involve modulation of the post-translational secretory process. In accordance, we found that the IL-1b mRNA levels were reduced by only 20% compared to control, but the intracellular protein levels of the pro-form was increased by >50% in primary monocytes after treatment with PCI-34051, indicating that the mechanism was due to inhibition of the processing from the pro- to the mature form of the cytokine. We showed that this was not due to a direct inhibition of caspase-1 or TACE (TNF-alpha converting enzyme), but is likely due to an as-yet unidentified substrate of HDAC8. In vivo, PCI-34051 inhibited ear swelling induced by oxazolone in a model of contact hypersensitivity in mice, and we showed that this was accompanied by a reduction in IL-1b at both the protein and mRNA levels. Based on this result, we examined the effect of PCI-34051 on IL-1b secretion in human keratinocytes, as well as in PBMC from psoriasis patients, and found that it could reduce IL-1b secretion in both. We found that PCI-34051 decreased IL-1b by 60% in LPS-stimulated PBMC from rheumatoid arthritis (RA) patients, but the pan-HDAC inhibitors which were only weakly inhibitory to HDAC8 did not have this effect, indicating a specific role for HDAC8 in the secretory process. Finally, we found that in unstimulated PBMC from RA patients that had basal production of IL-1b that this could be decreased by 90% by treatment with PCI-34051. Taken together, these findings indicate that PCI-34051 is an active drug that could be useful for the treatment of T-cell lymphoma as well as for autoinflammatory diseases such as RA and psoriasis.
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Kim, Ji Yoon, Seung Yoon Han, Jung Yoo, Go Woon Kim, Yu Hyun Jeon, Sang Wu Lee, Jongsun Park, and So Hee Kwon. "HDAC8-Selective Inhibition by PCI-34051 Enhances the Anticancer Effects of ACY-241 in Ovarian Cancer Cells." International Journal of Molecular Sciences 23, no. 15 (August 3, 2022): 8645. http://dx.doi.org/10.3390/ijms23158645.
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HDAC6 is overexpressed in ovarian cancer and is known to be correlated with tumorigenesis. Accordingly, ACY-241, a selective HDAC6 inhibitor, is currently under clinical trial and has been tested in combination with various drugs. HDAC8, another member of the HDAC family, has recently gained attention as a novel target for cancer therapy. Here, we evaluated the synergistic anticancer effects of PCI-34051 and ACY-241 in ovarian cancer. Among various ovarian cancer cells, PCI-34051 effectively suppresses cell proliferation in wild-type p53 ovarian cancer cells compared with mutant p53 ovarian cancer cells. In ovarian cancer cells harboring wild-type p53, PCI-34051 in combination with ACY-241 synergistically represses cell proliferation, enhances apoptosis, and suppresses cell migration. The expression of pro-apoptotic proteins is synergistically upregulated, whereas the expressions of anti-apoptotic proteins and metastasis-associated proteins are significantly downregulated in combination treatment. Furthermore, the level of acetyl-p53 at K381 is synergistically upregulated upon combination treatment. Overall, co-inhibition of HDAC6 and HDAC8 through selective inhibitors synergistically suppresses cancer cell proliferation and metastasis in p53 wild-type ovarian cancer cells. These results suggest a novel approach to treating ovarian cancer patients and the therapeutic potential in developing HDAC6/8 dual inhibitors.
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Cheng, Alfred Sze, Yu Feng, Jingying Zhou, Weiqin Yang, Hangyong Sun, Zhiwei Chen, Tyler Goodwin, Rihe Liu, and Leaf Huang. "HDAC8 inhibition modulates liver tumor microenvironment: Rationale for combined epigenetic and immunotherapy." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 76.26. http://dx.doi.org/10.4049/jimmunol.198.supp.76.26.
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Abstract The tumor microenvironment plays an instrumental role in cancer development and treatment response/resistance. Accumulating evidence is underscoring the fundamental importance of epigenetic regulation in tumor immune evasion. Epigenetic modification agents represent a ‘double-edged sword’, as they can exert divergent effects on cancer cells and the milieu of immune and stromal cells. Our integrative epigenomics analysis has elucidated previously unexplored functions of histone deacetylase 8 (HDAC8) in promoting β-catenin-dependent hepatocarcinogenesis through interacting with another critical chromatin regulator enhancer of zeste homology 2 (EZH2). Given the strong oncogenicity of HDAC8, we further investigated the therapeutic potential of a potent and highly-selective HDAC8-specific inhibitor PCI-34051. Using a hepatocellular carcinoma (HCC) orthotopic model in immunocompetent mice, we demonstrated that HDAC8 inhibition exerted a strong anti-tumorigenic effect (Ctrl vs. PCI: 6.32 vs. 0.33*107 ROI intensity; p<0.01), which was comparable with programmed death-ligand 1 (PD-L1) blockade. Intriguingly, PCI-34051 significantly increased tumor-infiltrating multi-functional CD8+ T cells (~3-fold) and specifically reduced regulatory T cells (~2-fold). However, the level of PD-1+CD8+ T cells also increased, implicating potential T cell exhaustion. Our data suggest that selective chromatin modifications by HDAC8 and EZH2 alter the tumor immune surveillance program. By understanding the impact of epigenetic control on the liver tumor microenvironment, rational combinatorial epigenetic and immune checkpoint targeting has the potential to fully unleash T cell responses against HCC.
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Hendrix, Sven, Selien Sanchez, Elissia Ventriglia, and Stefanie Lemmens. "HDAC8 Inhibition Reduces Lesional Iba-1+ Cell Infiltration after Spinal Cord Injury without Effects on Functional Recovery." International Journal of Molecular Sciences 21, no. 12 (June 25, 2020): 4539. http://dx.doi.org/10.3390/ijms21124539.
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Pan-histone deacetylase (HDAC) inhibition with valproic acid (VPA) has beneficial effects after spinal cord injury (SCI), although with side effects. We focused on specific HDAC8 inhibition, because it is known to reduce anti-inflammatory mediators produced by macrophages (Mφ). We hypothesized that HDAC8 inhibition improves functional recovery after SCI by reducing pro-inflammatory classically activated Mφ. Specific HDAC8 inhibition with PCI-34051 reduced the numbers of perilesional Mφ as measured by histological analyses, but did not improve functional recovery (Basso Mouse Scale). We could not reproduce the published improvement of functional recovery described in contusion SCI models using VPA in our T-cut hemisection SCI model. The presence of spared fibers might be the underlying reason for the conflicting data in different SCI models.
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Balasubramanian, S., J. Ramos, W. Luo, M. Sirisawad, E. Verner, and J. J. Buggy. "A novel histone deacetylase 8 (HDAC8)-specific inhibitor PCI-34051 induces apoptosis in T-cell lymphomas." Leukemia 22, no. 5 (February 7, 2008): 1026–34. http://dx.doi.org/10.1038/leu.2008.9.
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Ramos, Teresa L., Luis Ignacio Sánchez-Abarca, Rosón Beatriz, Concepción Rodríguez Serrano, Alba Redondo, Rebeca Ortega, Ángel Hernández-Hernández, et al. "HDAC8 Overexpression in Mesenchymal Stromal Cells from JAK2+ myeloproliferative Neoplasms: A New Therapeutic Target?" Blood 126, no. 23 (December 3, 2015): 2831. http://dx.doi.org/10.1182/blood.v126.23.2831.2831.
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Abstract JAK2-V617F mutation in hematopoietic stem cells (HSC) is a common finding in myeloproliferative neoplasms (MPNs). Although alterations in the hematopoietic microenvironment have been described in these entities, information on the functional and genetic characteristics of bone marrow (BM) derived mesenchymal stromal cells (BM-MSC) from JAK2+ MPNs patients is scarce. The aim of the current study was to characterize and compare BM-MSC from 24 MPNs patients with JAK2V617F mutation (14 BM-MSC from essential thrombocythemia-ET and 10 BM-MSC from polycythemia vera-PV) with those from 14 healthy donors-HD. For this purpose BM-MSC expansion, multilineage differentiation, apoptosis, inmunophenotyping, gene expression profiling, RT-PCR and Western Blot analysis were performed. Compared with HD, BM-MSC from MPNs patients showed similar morphology and differentiation capacity, but an increased proliferation rate with less apoptosis cells. BM-MSC from MPNs expressed comparable levels of CD73, CD44, CD90 and CD166, whereas they were negative for hematopoietic markers. The median expression of CD105 was lower in BM-MSC from MPNs patients (p <.05) when compared with BM-MSC from HD. Gene expression profile of BM-MSCs from 8 JAK2V617F (4 PV/4 TE) patients, and from 10 HD showed a total of 169 genes that were differentially expressed in BM-MSC from MPNs patients compared to HD. RT-PCR was performed in two genes to confirm these results, demonstrating that HDAC8 and CXCL12 genes were up-regulated. To analyze whether these changes in MPNs-MSC conferred an alteration in their functional capacity, co-cultures with CD34+ cells from MPNs and BM-MSC were performed. A significant increase in the CFU-GM clonogenic supporting capacity of MPNs-MSC when compared with HD-MSC was observed. To evaluate whether a Histone deacetylase (HDAC) inhibitor could modify the behavior of MPNs-MSC an HDAC8 specific inhibitor, PCI-34051 was used. A decrease in HDAC8 gene (RT-PCR) and protein (WB analysis) expression was observed in BM-MSC from MPNs treated with PCI-34051 at a concentration of 25µM for 48 hours. HDAC8-selective inhibition also induced a cell cycle arrest in the MPNs BM-MSC with an increase of the proportion of apoptotic cells. To assess the impact of this inhibition on the capacity of MPNs-MSC to support hematopoiesis, BM mononuclear cells (BM-MNC) were co-cultured in transwell for 48 hours with PCI-34051-treated and non-treated BM-MSC. After co-culture, cell viability, clonogenic (CFU-GM) assays and TP53 expression were analyzed. A decrease in cell viability (p=0.028) and CFU-GM (p=0.018) was demonstrated when BM-MNC from MPNs had been in culture with MPNs BM-MSC treated with the HDAC8 inhibitor, as well as an increase in TP53 expression. These results suggest that MPNs-MSC display different proliferative rate, MSC markers, gene expression profile and HDAC8 overexpression compared to HD-MSC. The inhibition of HDAC8 expression by its specific inhibitor decreases the capacity of the stroma to support hematopoietic cells from MPNs patients, suggesting that HDAC8 may be a potential therapeutic target in this setting. Disclosures Sánchez-Guijo: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau.
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Bai, Shi-Yao, Meng-Lu Li, Yuan Ren, and Xin-Ming Su. "HDAC8-inhibitor PCI-34051-induced exosomes inhibit human bronchial smooth muscle cell proliferation via miR-381-3p mediated TGFB3." Pulmonary Pharmacology & Therapeutics 71 (December 2021): 102096. http://dx.doi.org/10.1016/j.pupt.2021.102096.
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Heppt, Markus V., Anja Wessely, Eva Hornig, Claudia Kammerbauer, Saskia A. Graf, Robert Besch, Lars E. French, et al. "HDAC2 Is Involved in the Regulation of BRN3A in Melanocytes and Melanoma." International Journal of Molecular Sciences 23, no. 2 (January 13, 2022): 849. http://dx.doi.org/10.3390/ijms23020849.
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The neural crest transcription factor BRN3A is essential for the proliferation and survival of melanoma cells. It is frequently expressed in melanoma but not in normal melanocytes or benign nevi. The mechanisms underlying the aberrant expression of BRN3A are unknown. Here, we investigated the epigenetic regulation of BRN3A in melanocytes and melanoma cell lines treated with DNA methyltransferase (DNMT), histone acetyltransferase (HAT), and histone deacetylase (HDAC) inhibitors. DNMT and HAT inhibition did not significantly alter BRN3A expression levels, whereas panHDAC inhibition by trichostatin A led to increased expression. Treatment with the isoform-specific HDAC inhibitor mocetinostat, but not with PCI-34051, also increased BRN3A expression levels, suggesting that class I HDACs HDAC1, HDAC2, and HDAC3, and class IV HDAC11, were involved in the regulation of BRN3A expression. Transient silencing of HDACs 1, 2, 3, and 11 by siRNAs revealed that, specifically, HDAC2 inhibition was able to increase BRN3A expression. ChIP-Seq analysis uncovered that HDAC2 inhibition specifically increased H3K27ac levels at a distal enhancer region of the BRN3A gene. Altogether, our data suggest that HDAC2 is a key epigenetic regulator of BRN3A in melanocytes and melanoma cells. These results highlight the importance of epigenetic mechanisms in regulating melanoma oncogenes.
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Qi, Jing, Sandeep Singh, Qi Cai, Hongjun Liu, Hieu Vu, Sriram Balasubramanian, and Ya-Huei Kuo. "CBFβ-SMMHC Inactivates p53 Tumor Suppressor Through Aberrant Protein Interaction and Recruitment of HDAC8." Blood 120, no. 21 (November 16, 2012): 772. http://dx.doi.org/10.1182/blood.v120.21.772.772.
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Abstract Abstract 772 Chromosomal inversion inv(16)(p13.1q22) is found in approximately 12% of acute myeloid leukemia (AML) patients, and leads to the fusion of the transcription factor gene CBFb and the MYH11 gene, and encodes a fusion protein CBFβ-SMMHC. Previous studies revealed that CBFβ-SMMHC is a dominant inhibitor of core-binding factor (CBF) function, and impairs hematopoietic differentiation. Expression of CBFβ-SMMHC predisposes for leukemia transformation, however, the molecular mechanism underlying the leukemogenic function of CBFβ-SMMHC remains elusive. The tumor suppressor p53 is considered the master genomic guardian that is frequently mutated in a wide variety of tumors but is rarely mutated in inv(16) AML. Thus, we examined whether CBFβ-SMMHC fusion protein might impair p53 function. We found that p53 acetylation (Ac-p53) level was reduced in the presence of CBFβ-SMMHC fusion protein in the myeloid progenitor 32D cell line as well as in primary pre-leukemic bone marrow progenitor cells isolated from our conditional Cbfb-MYH11 knock-in (Cbfb56M/+/Mx1-Cre) mice (Kuo et al, Cancer Cell 2006, 9:1,57-68). We assessed the effect of CBFβ-SMMHC on p53 transcriptional activity by quantitative RT-PCR analysis of p53 target genes including TP53 and p21 Cdkn1a, Mdm2, Bid, Bax, Stag1, LincRNA-p21, Gadd45b in 32D cells. The result showed that expression of these p53 target genes are reduced in the presence of CBFβ-SMMHC fusion protein, consistent with the impaired Ac-p53 by CBFβ-SMMHC. To understand how CBFβ-SMMHC impairs p53 function, we tested whether CBFβ-SMMHC fusion protein might interact with the p53 protein by co-immunoprecipitation (co-IP) assays. We found that CBFβ-SMMHC fusion protein interacts with p53 both in 32D cells and primary bone marrow cells. Although CBFβ-SMMHC fusion protein is detected both in the nucleus and the cytoplasm, the complex with p53 is present exclusively in the nucleus. It has been reported that CBFβ-SMMHC interacts with histone deacetylase 8 (HDAC8) through the C-terminal SMMHC region. Therefore, we assessed the interaction between CBFβ-SMMHC, p53 and HDAC8 in 32D cell line by co-IP and sequential co-IP. We were able to detect a multimeric protein complex containing CBFβ-SMMHC, p53, and Hdac8. To access whether HDAC8 contributes to the deacetylation of p53, we used two independent small-hairpin (sh)-RNA to knock-down Hdac8 in 32D-CBFβ-SMMHC cells. Hdac8 knock-down led to robust increase in Ac-p53 levels while total p53 levels were modestly stabilized. To test whether this effect is dependent on the deacetylase function of HDAC8, we used HDAC8 selective pharmacological inhibitors (HDAC8i including PCI-34051 and PCI-48012) directed against its catalytic sites (Balasubramanian et al Leukemia 2008, 22:5,1026-34). Treatment with HDAC8i remarkably increased Ac-p53 in both control and CBFβ-SMMHC cells. Since p53 protein levels were also increased upon HDAC8i treatment, we included Mdm2 inhibitor Nutlin-3 to stabilize p53. HDAC8i treatment alone or in combination with Nutlin-3 was able to enhance Ac-p53 compared to Nutlin-3 treatment, confirming its effect in restoring p53 acetylation. Collectively, our study shows that the CBFβ-SMMHC fusion protein forms an aberrant complex with p53 and HDAC8, leading to the aberrant deacetylation and impaired activity of p53. In addition, this deacetylation of p53 conferred by CBFβ-SMMHC is mediated by HDAC8. Our study reveals a novel leukemogenic mechanism in which CBFβ-SMMHC disrupts p53 activation through aberrant protein-protein interaction and recruitment of HDAC8. Disclosures: No relevant conflicts of interest to declare.
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Chyra, Zuzana, Maria Gkotzamanidou, Masood A. Shammas, Vassilis L. Souliotis, Yan Xu, Mehmet K. Samur, Aaron B. Beeler, Roman Hajek, Mariateresa Fulciniti, and Nikhil C. Munshi. "HDAC8 Maintain Cytoskeleton Integrity Via Homologous Recombination and Represent a Novel Therapeutic Target in Multiple Myeloma." Blood 134, Supplement_1 (November 13, 2019): 4385. http://dx.doi.org/10.1182/blood-2019-130700.
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Multiple Myeloma (MM) is a plasma cell malignancy vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets in combination with current anti-myeloma agents. Pan-HDAC inhibitors are effective as therapeutic agents both in preclinical and clinical setting; however, there is an increasing emphasis on understanding the biological and molecular roles of individual HDACs to limit toxicities observed with pan-HDAC inhibitors. Based on correlation with patient outcome in three independent myeloma datasets, we have evaluated the functional role of HDAC8, a member of Class I HDAC isoenzymes, in MM. Unlike other isoforms, there is limited information about molecular and epigenomic functions of HDAC8. We have previously confirmed expression of HDAC8 in a large panel of MM cell lines, where it is localized predominantly to cytoplasm. Moreover, genetic and pharmacological modulation of HDAC8 with RNAi and specific inhibitor PCI-34051 resulted in a significant inhibition of myeloma cell proliferation and decrease in colony formation (p<.001). HDAC8 inhibition led to an increase in the ongoing spontaneous and radiation-induced DNA damage in MM cells by affecting DNA repair via the homologous recombination (HR) pathway, suggesting a novel function of HDAC8 in promoting HR and DNA repair in MM cells. Using laser micro-irradiation in MM1S and U2OS cells, we observed HDAC8 recruitment to DSBs sites and its co-localization with Rad51 and Scm3, a member of cohesin complex. A transcriptomic analysis of HDAC8 knock-down cells also shows perturbation of number of cytoskeleton-related genes confirming significant role of HAD8 in cytoskeleton rearrangement in MM. Mass-spectrometry analysis to identify the HDAC8 substrates in MM cells is currently ongoing. Classical pan-HDACi, such as SAHA (vorinostat), bind to HDAC8 with substantially diminished activity (IC50 = 2 μM), reflecting a unique binding site of this isoform. To discover and validate new small molecules with HDAC8 subtype selectivity, we have explored the efficacy of OJI-1, a novel selective and potent HDAC8 inhibitor (IC50 = 0.8 nM) with modest inhibition of HDAC6 (1200 nM). Treatment with OJI-1 selectively impact cell viability of a large panel of MM cell lines (n=20) in a time and dose dependent manner, while sparing healthy donors PBMC both in resting and activated state (n=3). The significantly higher IC50 observed in PBMCs suggests a favorable therapeutic index. Western blotting analysis confirmed target selectivity with significant time and dose dependent decrease in H3 and H4 acetylation in MM cells treated with OJI-1. Moreover, pharmacological inhibition of HDAC8 specifically inhibited HR but not non-homologous end joining. These data suggest that targeting of HDAC8 using OJI-1 could be effective treatment approach in MM. Based on molecular data combination studies and in vivo evaluation are ongoing. In conclusion, our results provide insight into the role of HDAC8 in DNA stability and cell growth and viability which can be exploited in future for therapeutic application alone and in combination in MM. Disclosures Munshi: Takeda: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Abbvie: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Adaptive: Consultancy; Abbvie: Consultancy; Oncopep: Consultancy; Oncopep: Consultancy; Celgene: Consultancy.
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Gkotzamanidou, Maria, Masood Shammas, Jesus Martin Sanchez, Lai Ding, Stephane Minvielle, Florence Magrangeas, Herve Avet-Loiseau, Kenneth C. Anderson, and Nikhil C. Munshi. "HDAC8 Mediates Homologous Recombination and Cytoskeleton Integrity in Myeloma with Potential Impact on Cell Growth and Survival." Blood 124, no. 21 (December 6, 2014): 416. http://dx.doi.org/10.1182/blood.v124.21.416.416.
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Abstract Epigenomic changes have become an important component of cellular regulation and ultimately, of our understanding of oncogenomics in Multiple Myeloma (MM) as well as in other cancers. In recent years, both clinical and preclinical studies have confirmed that MM is vulnerable to epigenetic intervention, with histone deacetylases (HDACs) emerging as the most promising epigenetic targets. Although Pan-HDAC inhibitors are effective as therapeutic agents, there is increasing emphasis on understanding the biological and molecular roles of individual HDACs. Here we have evaluated the role of HDAC8, a member of Class I HDAC isoenzymes in MM. First, we evaluated the expression of HDAC8 in 172 newly-diagnosed MM patients from the IFM myeloma dataset and observed HDAC8 overexpression as well as its significant correlation with poor survival outcome (p<0.0015). We further evaluated the expression of HDAC8 in HMCLs (probe ID_223909-s_at, 223345_at) and confirmed the high expression and its cytoplasmic and nuclear localization in all six MM cells lines studied (MM1S, OPM2, RPMI8226, U266, MOLP8 and NCI-H929) and in primary bone marrow plasma cells (CD138+) from newly diagnosed MM patients (N=3). To address the functional role of HDAC8 in MM biology and to evaluate its potency as therapeutic target, we used a lentiviral-shRNA delivery system for HDAC8-knockdown in MM1S and OPM2 myeloma cells. The HDAC8 depletion in HMCLs resulted in significant inhibition of proliferation of MM at 1 week as measured by 3[H]-thymidine assay, and as decrease in colony formation evaluated after 3 weeks post transfection (p<.001). We observed similar cell growth inhibition using PCI-34051, a small molecule HDAC8 inhibitor. Interestingly, the combination of HDAC8 inhibitor with melphalan or bendamustine enhanced the anti-MM effects of the DNA damaging agents (all p<0.01) and was confirmed to be synergistic using Calcusyn software. Immunoblotting using a panel of 15 antibodies for DNA damage response (DDR) pathway proteins (including γH2Ax, pATM, pATR, pBRCA1, pBRCA2, pCHK2, pCHK1, ku70, RPA70, 53BP1, DNA-PKs, pP53) confirmed increased levels of DNA damage in OPM2 and MM1S cells with HDAC8 depletion. In consistence with this observation HDAC8 knockdown led to decreased homologous recombination (HR) activity as measured by a transient direct repeat DsRED-GFP/I-SceI plasmid-based assay. We performed singe cell electrophoresis under neutral conditions (comet-assay) in OPM2 and MM1S after HDAC8 depletion with or without exposure to gamma irradiation (γ-IR), and in OPM2 and MM1S cells treated and untreated with HDAC8 inhibitor in combination with γ-IR and observed decreased repair of DSBs after γ-IR measured following HDAC8 knockdown as well as following treatment of the cells with HDAC8 inhibitor. Importantly, using laser micro-irradiation in myeloma and U2OS cells, we observed HDAC8 recruitment to DSBs sites. Moreover, the HDAC8 protein was co-localized and co-immunoprecipitated with Rad51 after IR, and with Scm3, member of cohesion complex after mitotic sychronization, suggesting its relation with cytoskeleton. We confirmed the significant alteration in expression of cohesion complex members SMC1 and RAD21 after HDAC8 depletion and re-overexpression in MM cells. In MM1s cells containing a stably integrated Rad51-luciferase reporter construct, the addition of HDAC8 inhibitor resulted in a decrease in Rad51 promoter activity, confirming the immunoblotting findings. An ongoing mass spectromentry-based analysis is expected to identify thoroughly the HDAC8-interacting proteins. In conclusion, our results demonstrate an impact of aberrant epigenome on DNA integrity through connection between HDAC8 and DDR pathway, and provide insights into the effect of HDAC8 on cellular growth and survival with potent therapeutic implications in MM. Disclosures Anderson: Celgene: Consultancy; Sanofi-Aventis: Consultancy; Onyx: Consultancy; Acetylon: Scientific Founder, Scientific Founder Other; Oncoprep: Scientific Founder Other; Gilead Sciences: Consultancy.
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Kitadate, Akihiro, Sho Ikeda, Fumito Abe, Naoto Takahashi, Norio Shimizu, Kosei Matsue, and Hiroyuki Tagawa. "Histone Deacetylase Inhibitors Downregulate CCR4 Expression and Decrease Mogamulizumab Efficacy in CCR4-Positive Mature T-Cell Lymphomas." Blood 130, Suppl_1 (December 7, 2017): 720. http://dx.doi.org/10.1182/blood.v130.suppl_1.720.720.
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Abstract Background: Histone deacetylase inhibitors (HDACis) are promising agents for various T-cell lymphomas, including cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), and adult T-cell lymphoma/leukemia (ATLL). CCR4 is an important therapeutic target molecule because mogamulizumab, an anti-CCR4 antibody, has shown promising efficacy against CTCL, PTCL, and ATLL. However, their combined effects and interactions have not been examined thus far. We previously showed that CCR6, a chemokine receptor, is overexpressed in cutaneous T-cell lymphomas (Ito et al., 2014 Blood). Moreover, we recently demonstrated that HDACis downregulate CCR6 expression in advanced cutaneous T-cell lymphomas (Abe et al., 2017 Oncotarget). These reports lead us to hypothesize that HDACis might also downregulate CCR4 in various T-cell lymphomas. In this study, we clarify the effect of the combined use of mogamulizumab and HDACis on various T-cell and NK-cell lymphomas. Based on our findings, we discuss what benefits or adverse effects might be assumed for patients if these molecular targeting agents are used in clinical practice. Methods: We evaluated changes in CCR4 expression and antibody-dependent cell-mediated cytotoxicity (ADCC) activities against mogamulizumab- and HDACi-treated T-cell and NK-cell lymphoma lines and primary cases. To determine which HDAC mainly regulated CCR4 expression, we used isoform-specific HDACis and induced knockdown of respective HDACs for T-cell lymphoma cell lines. To examine the effect of CCR4 downregulation by HDACis in clinical cases, we examined the CCR4 expression of CTCL skin samples, which were obtained from the same patients before and after HDACi treatment (n = 6). Results: We first examined the expression of CCR4 for 15 T-cell and NK-cell lymphoma cell lines and a peripheral blood mononuclear cell (PBMC) sample derived from healthy donors to investigate the effect of vorinostat, a pan-HDACi, on CCR4 expression. The expression of CCR4 was mostly expressed in the (11 out of 15) cell lines: ATLL (MT-1, MT-2, MT-4, and TL-Su), CTCL (My-La, HH, and MJ), and NK/T-cell lymphoma cell lines (Kai3, SNK6, HANK1, and SNK10). We found that vorinostat decreases mRNA expression and surface expression of CCR4 except for the cell lines without CCR4 expression. Next, we used isoform-specific HDACis to examine which isoform of HDAC is involved in the regulation of CCR4. We used the following class-specific HDACis: romidepsin as a class I selective HDACi, CI-994 as an HDAC1/HDAC2-selective inhibitor, RGFP966 as an HDAC3-selective inhibitor, ricolinostat as an HDAC6-selective inhibitor, and PCI-34051 as an HDAC8-selective inhibitor. When these drugs were exposed to T-cell lymphoma cells, romidepsin and CI-994 strongly suppressed CCR4 expression. These results suggest that class I HDACs might controls CCR4 expression. We further performed knockdown experiments using siRNAs against HDAC1, HDAC2, and HDAC3. When we compared the expression change of CCR4 in HDAC-knockdown cells, HDAC2 knockdown cells showed the most significantly decreased expression of CCR4. These results suggest that class I HDACs, especially HDAC2, might be deeply involved in CCR4 expression regulation. When we examined the CCR4 expression in skin samples from primary CTCL, obtained from the same patients before and after vorinostat treatment, we found that CCR4 expression was greatly reduced after vorinostat treatment. Finally, when we conducted an ADCC assay with mogamulizumab by using various lymphoma cell lines and primary T-cell lymphoma samples, we found that the efficacy of mogamulizumab was significantly reduced by pre-treatment with vorinostat. Conclusion: Our results suggest that the primary use of HDACis before treatment of mogamulizumab might not be suitable to obtain synergistic effects. Moreover, these results provide potential implications for optimal therapeutic sequences in various CCR4 positive T-cell and NK-cell lymphomas. Disclosures Kitadate: Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Eisai: Research Funding; Otsuka: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Asahi Kasei: Research Funding; Chugai: Research Funding; Toyama kagaku: Research Funding. Abe: Kyowa Kirin: Research Funding; Fujimoto: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Otsuka: Research Funding; Toyama Kagaku: Research Funding; Chugai: Research Funding; Asahi Kasei: Research Funding; Eisai: Research Funding. Tagawa: TaNeDS (Daiichi Sankyo): Research Funding.
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Goncalves, Kevin A., Megan D. Hoban, Jennifer L. Proctor, Hillary L. Adams, Sharon L. Hyzy, Anthony E. Boitano, and Michael P. Cooke. "Phenotype Does Not Always Equal Function: HDAC Inhibitors and UM171, but Not SR1, Lead to Rapid Upregulation of CD90 on Non-Engrafting CD34+CD90-Negative Human Cells." Blood 130, Suppl_1 (December 7, 2017): 659. http://dx.doi.org/10.1182/blood.v130.suppl_1.659.659.
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Abstract Background. The ability to expand human hematopoietic stem cells (HSCs) has the potential to improve outcomes in HSC transplantation and increase the dose of gene-modified HSCs. While many approaches have been reported to expand HSCs, a direct comparison of the various methods to expand transplantable HSCs has not been published and clinical outcome data for the various methods is incomplete. In the present study, we compared several small molecule approaches reported to expand human HSCs including HDAC inhibitors, the aryl hydrocarbon antagonist, SR1, and UM171, a small molecule with unknown mechanism, for the ability to expand phenotypic HSC during in vitro culture and to expand cells that engraft NSG mice. Although all strategies increased the number of phenotypic HSC (CD34+CD90+CD45RA-) in vitro, SR1 was the most effective method to increase the number of NOD-SCID engrafting cells. Importantly, we found that HDAC inhibitors and UM171 upregulated phenotypic stem cell markers on downstream progenitors, suggesting that these compounds do not expand true HSCs. Methods. Small-molecules, SR1, HDAC inhibitors (BG45, CAY10398, CAY10433, CAY10603, Entinostat, HC Toxin, LMK235, PCI-34051, Pyroxamide, Romidepsin, SAHA, Scriptaid, TMP269, Trichostatin A, or Valproic Acid) and UM171 were titrated and then evaluated at their optimal concentrations in the presence of cytokines (TPO, SCF, FLT3L, and IL6) for the ability to expand human mobilized peripheral blood (mPB)-derived CD34+ cells ex vivo . Immunophenotype and cell numbers were assessed by flow cytometry following a 7-day expansion assay in 10-point dose-response (10 µM to 0.5 nM). HSC function was evaluated by enumeration of colony forming units in methylcellulose and a subset of the compounds were evaluated by transplanting expanded cells into sub-lethally irradiated NSG mice to assess engraftment potential in vivo . All cells expanded with compounds were compared to uncultured or vehicle-cultured cells. Results. Following 7 days of expansion, SR1 (5-fold), UM171 (4-fold), or HDAC inhibitors (>3-35-fold) resulted in an increase in CD34+CD90+CD45RA- number relative to cells cultured with cytokines alone; however, only SR1 (18-fold) and UM171 (8-fold) demonstrated enhanced engraftment in NSG mice. Interestingly, while HDAC inhibitors and UM171 gave the most robust increase in the number and frequency of CD34+CD90+CD45RA- cells during in vitro culture, these methods were inferior to SR1 at increasing NSG engrafting cells. The increase in CD34+CD90+CD45RA- cells observed during in vitro culture suggested that these compounds may be generating a false phenotype by upregulating CD90 and down-regulating CD45RA on progenitors that were originally CD34+CD90-CD45RA+. We tested this hypothesis by sorting CD34+CD90-CD45RA+ cells and culturing these with the various compounds. These experiments confirmed that both HDAC inhibitors (33-100 fold) and UM171 (28-fold) led to upregulation of CD90 on CD34+CD90-CD45RA+ cells after 4 days in culture. Since approximately 90% of the starting CD34+ cells were CD90-, these data suggest that most of the CD34+CD90+CD45RA- cells in cultures with HDAC inhibitors and UM171 arise from upregulation of CD90 rather than expansion of true CD34+CD90+CD45RA- cells and may explain the disconnect between in vitro HSC phenotype and NSG engraftment in vivo . This was further confirmed by evaluation of colony forming unit frequency of CD34+CD90-CD45RA+ cells after culture with compounds. Conclusions. We have showed that AHR antagonism is optimal for expanding functional human HSCs using the NSG engraftment model. We also demonstrated that UM171 and HDAC inhibitors upregulate phenotypic HSC markers on downstream progenitors. This could explain the discrepancy between impressive in vitro phenotypic expansion and insufficient functional activity in the NSG mouse model. Therefore, these data suggest caution when interpreting in vitro expansion phenotypes without confirmatory functional transplantation data, especially as these approaches move into clinical trials in patients. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership. Hoban: Magenta Therapeutics: Employment, Equity Ownership. Proctor: Magenta Therapeutics: Employment, Equity Ownership. Adams: Magenta Therapeutics: Employment, Equity Ownership. Hyzy: Magenta Therapeutics: Employment, Equity Ownership. Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.
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Zhang, Pu, Lindsey Brinton, James S. Blachly, John C. Byrd, and Rosa Lapalombella. "Identification of Novel Synthetic Lethal Partners of NAMPT Inhibitor By CRISPR-Cas9 Screens in Acute Myeloid Leukemia." Blood 134, Supplement_1 (November 13, 2019): 2072. http://dx.doi.org/10.1182/blood-2019-129036.
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Acute myeloid leukemia (AML) is a hematopoietic neoplasm arising from the clonal expansion of myeloid progenitors resulting in bone marrow failure. Nicotinamide phosphoribosyltransferase (NAMPT) is a rate‐limiting enzyme which regulates the generation of the metabolic enzyme substrate NAD+. NAD+ is key in several survival pathways in AML such as DNA repair and metabolic pathways. AML subtypes differ vastly in their inherent DNA repair and metabolic capacities. Since NAMPT is essential in single and double strand DNA repair, NAMPT inhibitors (NAMPTis) were used to block DNA repair as single agents or in combinational therapies in solid tumors. However the clinical experience with the first generation of NAMPTis suggests that higher doses will be needed in order to achieve efficacy, which are associated with high toxicity. We hypothesize that identification of critical synthetic lethal partners for NAMPTi will increase efficacy with lower doses. To this end, we conducted genome-wide CRISPR KO (loss-of-function) screens by using two libraries, GeCKO and Brunello, and identified, along with genes reported essential for AML survival, 16 genes whose knockout displays synergism or resistance, after a NAMPTi (KPT-9274) treatment in AML cells. According to their gene ontology (GO) functions, these co-essential candidates are involved in DNA damage repair and metabolism. Genetic depletion of co-essential genes, SIRT6, HDAC8 and DCPS, sensitizes AML cell lines, MOLM13, Kasumi-1 and MV4-11, to KPT-9274 treatment. In addition, preclinical inhibitor for HDAC8 (PCI-34051), in combination with KPT-9274, synergistically decreased AML primary cell survival in a dose-dependent manner and decreased colony formation in AML patient samples across multiple genotypes with a minimal decrease in colony formation of normal CD34+ hematopoietic cells. In addition, drug combo treatment of AML cells decreased re-plating capacity, implying attenuated self-renewal capacity. Drug combo also increased apoptotic cells as measured by Annexin/PI staining and induced myeloid differentiation as detected by CD45+CD34- staining. In DNA homologous recombination (HR) pathway, SIRT6 and HDAC8 deacetylate CtIP and Rad51 to promote DNA end resection at double strand breaks. Since NAMPT inhibition blocks the generation of NAD+ thus compromising downstream PARP1 mediated base excision repair (BER) as well as the Sirtuin1 (Sirt1) mediated, KU- dependent NHEJ and atypical Rad51-independent HR repair, we expect that inhibiting these HR pathway components (like SIRT6 and HDAC8) may shut down these compensatory repair pathways and sensitize AML subtypes to NAMPT inhibition. Indeed, Western blotting shows drug combo increased acetyl-P53, acetyl-Rad51, phosphor-γH2Ax and Chk1 compared NAMPT inhibition alone. The in vivo efficacy of these combinatorial therapies has been investigated on PDX mouse models established in our laboratory. We demonstrated that the dug combo successfully eliminated engrafted AML cells and conferred survival advantages to mice. Our study provides evidence that co-essential genes, SIRT6, HDAC8 and DCPS, are synthetic lethal partners for NAMPT inhibition to target drug-resistant AML cells, with minimal toxicity towards normal cells. Disclosures Byrd: BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Acerta: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau.
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Zhou, Xun, Hui Chen, Yingfeng Shi, Jinqing Li, Xiaoyan Ma, Lin Du, Yan Hu, et al. "Histone deacetylase 8 inhibition prevents the progression of peritoneal fibrosis by counteracting the epithelial-mesenchymal transition and blockade of M2 macrophage polarization." Frontiers in Immunology 14 (February 23, 2023). http://dx.doi.org/10.3389/fimmu.2023.1137332.
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BackgroundPeritoneal dialysis (PD) is an effective replacement therapy for end-stage renal disease patients. However, long-term exposure to peritoneal dialysate will lead to the development of peritoneal fibrosis. Epigenetics has been shown to play an important role in peritoneal fibrosis, but the role of histone deacetylases 8 (HDAC8) in peritoneal fibrosis have not been elucidated. In this research, we focused on the role and mechanisms of HDAC8 in peritoneal fibrosis and discussed the mechanisms involved.MethodsWe examined the expression of HDAC8 in the peritoneum and dialysis effluent of continuous PD patients. Then we assessed the role and mechanism of HDAC8 in peritoneal fibrosis progression in mouse model of peritoneal fibrosis induced by high glucose peritoneal dialysis fluid by using PCI-34051. In vitro, TGF-β1 or IL-4 were used to stimulate human peritoneal mesothelial cells (HPMCs) or RAW264.7 cells to establish two cell injury models to further explore the role and mechanism of HDAC8 in epithelial-mesenchymal transition (EMT) and macrophage polarization.ResultsWe found that HDAC8 expressed highly in the peritoneum from patients with PD-related peritonitis. We further revealed that the level of HDAC8 in the dialysate increased over time, and HDAC8 was positively correlated with TGF-β1 and vascular endothelial growth factor (VEGF), and negatively correlated with cancer antigen 125. In mouse model of peritoneal fibrosis induced by high glucose dialysate, administration of PCI-34051 (a selective HDAC8 inhibitor) significantly prevented the progression of peritoneal fibrosis. Treatment with PCI-34051 blocked the phosphorylation of epidermal growth factor receptor (EGFR) and the activation of its downstream signaling pathways ERK1/2 and STAT3/HIF-1α. Inhibition of HDAC8 also reduced apoptosis. In vitro, HDAC8 silencing with PCI-34051 or siRNA inhibited TGF-β1-induced EMT and apoptosis in HPMCs. In addition, continuous high glucose dialysate or IL-4 stimulation induced M2 macrophage polarization. Blockade of HDAC8 reduced M2 macrophage polarization by inhibiting the activation of STAT6 and PI3K/Akt signaling pathways. ConclusionsWe demonstrated that HDAC8 promoted the EMT of HPMCs via EGFR/ERK1/2/STAT3/HIF-1α, induced M2 macrophage polarization via STAT6 and PI3K/Akt signaling pathways, and ultimately accelerated the process of peritoneal fibrosis.
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Kurzawa, Nils, Isabelle Becher, Sindhuja Sridharan, Holger Franken, André Mateus, Simon Anders, Marcus Bantscheff, Wolfgang Huber, and Mikhail M. Savitski. "A computational method for detection of ligand-binding proteins from dose range thermal proteome profiles." Nature Communications 11, no. 1 (November 13, 2020). http://dx.doi.org/10.1038/s41467-020-19529-8.
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AbstractDetecting ligand-protein interactions in living cells is a fundamental challenge in molecular biology and drug research. Proteome-wide profiling of thermal stability as a function of ligand concentration promises to tackle this challenge. However, current data analysis strategies use preset thresholds that can lead to suboptimal sensitivity/specificity tradeoffs and limited comparability across datasets. Here, we present a method based on statistical hypothesis testing on curves, which provides control of the false discovery rate. We apply it to several datasets probing epigenetic drugs and a metabolite. This leads us to detect off-target drug engagement, including the finding that the HDAC8 inhibitor PCI-34051 and its analog BRD-3811 bind to and inhibit leucine aminopeptidase 3. An implementation is available as an R package from Bioconductor (https://bioconductor.org/packages/TPP2D). We hope that our method will facilitate prioritizing targets from thermal profiling experiments.
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"THERAPETIC EFFECTS OF HDAC8 SPECIFIC INHIBITOR PCI-34051 IN ACUTE ASTHMATIC MICE AND ITS EFFECT ON GALECTIN-3." Respirology 22 (November 2017): 89. http://dx.doi.org/10.1111/resp.13207_5.
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Schmidt, Oxana, Nadja Nehls, Carolin Prexler, Kristina von Heyking, Tanja Groll, Katharina Pardon, Heathcliff D. Garcia, et al. "Class I histone deacetylases (HDAC) critically contribute to Ewing sarcoma pathogenesis." Journal of Experimental & Clinical Cancer Research 40, no. 1 (October 15, 2021). http://dx.doi.org/10.1186/s13046-021-02125-z.
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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.