Journal articles: 'Inhibitor of HDAC6'

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Stubbs, Matthew C., Won-Il Kim, Tina Davis, Jun Qi, James Bradner, Andrew L. Kung, and Scott A. Armstrong. "Selective Inhibition of HDAC1 and HDAC2 Is a Potential Therapeutic Option for B-All." Blood 116, no. 21 (November 19, 2010): 2900. http://dx.doi.org/10.1182/blood.v116.21.2900.2900.

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Abstract Abstract 2900 Histone deacetylase inhibitors (HDACi) have emerged as potent anticancer agents, and could open the door for future epigenetic therapies. As our understanding of the importance of epigenetic histone modifications in B-cell acute lymphoblastic leukemia (B-ALL) increases, we hypothesized that HDACi could potentially be a useful therapeutic option. The pan-HDAC inhibitor LAQ824 (Novartis) was toxic to B-ALLs in low nM concentrations in vitro, and treated cells had increased p21 and DNA damage response as indicated by increased γH2A.X protein levels. Additionally, the related compound panobinostat (Novartis) reduced leukemic burden from B-ALL patient samples in primary xenograft models, indicating that pan-HDAC inhibition is a putative B-ALL therapeutic option. To determine HDAC isoform-specific effects, we used a high throughput assay that exposed B-ALL cell lines to a panel of HDAC inhibitors. This screen indicated that tubacin, an HDAC6 specific inhibitor, cannot inhibit B-ALL cell growth within a dose range where HDAC6 is the only HDAC targeted. This finding was further validated using another HDAC6 specific inhibitor, WT-161. The screen also indicated that benzimide compounds such as MGCD-0103 (MethylGene) and MS-275 (Entinostat, Syndax) which only target class I HDACs (HDAC1-3) effectively inhibited growth in the cell lines. These data indicate that inhibiting the class I HDACs is sufficient to suppress B-ALL cell line growth. To determine which HDACs are necessary for cell viability, we lentivirally introduced isoform-specific shRNAs into our ALL cell lines. Knockdown of HDAC1 or HDAC2 resulted in p21 induction, slowed growth rate and resulted in a modest increase in apoptosis. Knockdown of HDAC3 lead to increased p21 and γH2A.X protein levels, along with induction of apoptosis, closely mimicking the results of pan-HDAC inhibitor treatment of the cells. Although depletion of HDAC3 had a more immediate impact on B-ALL viability by comparison to HDAC1/2, concerns about the contribution of HDAC3 inhibition to toxicity led us to further investigate whether specific inhibition of HDAC1/2 might be efficacious in B-ALL. Treatment of B-ALL cells with Merck 60, a tool compound with selectivity for HDAC1/2, was efficacious against was effective against B-ALL lines in the low to mid nM range. The kinetics of growth suppression were slower with this compound than with the pan-HDAC inhibitors. Using this compound, the ALL lines required 72 hours of exposure before cell growth was diminished, and apoptosis ensued. This may be due to the increased time necessary to accumulate acetylated histone marks as observable by western blot (18 hours for Merck 60 vs. 2–4 hours for LAQ824). Increased levels of p21 and γH2A.X were also observed. Interestingly, AML cell lines were much less sensitive to the HDAC1/2 specific inhibitor than were the B-ALL lines (roughly 5–10 fold), whereas pan-HDAC inhibitors were equally effective against AML and ALL. Additionally, non-hematopoietic tumor derived cell lines were insensitive to Merck 60, with EC50 values exceeding 20μM. Our findings indicate that pan-HDAC and class I specific HDAC inhibitors are possible therapeutic options for B-ALL. In contrast to most other cancer cell types studied, selective inhibition of HDAC1 and HDAC2 was sufficient to induce apoptosis in B-ALL lines. Together, these results suggest that small molecules specifically targeting HDAC1/2 may have therapeutic utility in B-ALL, and may provide improved therapeutic index by comparison to pan-HDAC or class I HDAC inhibitors that also target HDAC3. Disclosures: No relevant conflicts of interest to declare.

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Khan, Nagma, Michael Jeffers, Sampath Kumar, Craig Hackett, Ferenc Boldog, Nicholai Khramtsov, Xiaozhong Qian, et al. "Determination of the class and isoform selectivity of small-molecule histone deacetylase inhibitors." Biochemical Journal 409, no. 2 (December 21, 2007): 581–89. http://dx.doi.org/10.1042/bj20070779.

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The human HDAC (histone deacetylase) family, a well-validated anticancer target, plays a key role in the control of gene expression through regulation of transcription. While HDACs can be subdivided into three main classes, the class I, class II and class III HDACs (sirtuins), it is presently unclear whether inhibiting multiple HDACs using pan-HDAC inhibitors, or targeting specific isoforms that show aberrant levels in tumours, will prove more effective as an anticancer strategy in the clinic. To address the above issues, we have tested a number of clinically relevant HDACis (HDAC inhibitors) against a panel of rhHDAC (recombinant human HDAC) isoforms. Eight rhHDACs were expressed using a baculoviral system, and a Fluor de Lys™ (Biomol International) HDAC assay was optimized for each purified isoform. The potency and selectivity of ten HDACs on class I isoforms (rhHDAC1, rhHDAC2, rhHDAC3 and rhHDAC8) and class II HDAC isoforms (rhHDAC4, rhHDAC6, rhHDAC7 and rhHDAC9) was determined. MS-275 was HDAC1-selective, MGCD0103 was HDAC1- and HDAC2-selective, apicidin was HDAC2- and HDAC3-selective and valproic acid was a specific inhibitor of class I HDACs. The hydroxamic acid-derived compounds (trichostatin A, NVP-LAQ824, panobinostat, ITF2357, vorinostat and belinostat) were potent pan-HDAC inhibitors. The growth-inhibitory effect of the HDACis on HeLa cells showed that both pan-HDAC and class-I-specific inhibitors inhibited cell growth. The results also showed that both pan-HDAC and class-I-specific inhibitor treatment resulted in increased acetylation of histones, but only pan-HDAC inhibitor treatment resulted in increased tubulin acetylation, which is in agreement with their activity towards the HDAC6 isoform.

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Cao, Jiangying, Wei Zhao, Chunlong Zhao, Qian Liu, Shunda Li, Guozhen Zhang, C. James Chou, and Yingjie Zhang. "Development of a Bestatin-SAHA Hybrid with Dual Inhibitory Activity against APN and HDAC." Molecules 25, no. 21 (October 28, 2020): 4991. http://dx.doi.org/10.3390/molecules25214991.

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With five histone deacetylase (HDAC) inhibitors approved for cancer treatment, proteolysis-targeting chimeras (PROTACs) for degradation of HDAC are emerging as an alternative strategy for HDAC-targeted therapeutic intervention. Herein, three bestatin-based hydroxamic acids (P1, P2 and P3) were designed, synthesized and biologically evaluated to see if they could work as HDAC degrader by recruiting cellular inhibitor of apoptosis protein 1 (cIAP1) E3 ubiquitin ligase. Among the three compounds, the bestatin-SAHA hybrid P1 exhibited comparable even more potent inhibitory activity against HDAC1, HDAC6 and HDAC8 relative to the approved HDAC inhibitor SAHA. It is worth noting that although P1 could not lead to intracellular HDAC degradation after 6 h of treatment, it could dramatically decrease the intracellular levels of HDAC1, HDAC6 and HDAC8 after 24 h of treatment. Intriguingly, the similar phenomenon was also observed in the HDAC inhibitor SAHA. Cotreatment with proteasome inhibitor bortezomib could not reverse the HDAC decreasing effects of P1 and SAHA, confirming that their HDAC decreasing effects were not due to protein degradation. Moreover, all three bestatin-based hydroxamic acids P1, P2 and P3 exhibited more potent aminopeptidase N (APN, CD13) inhibitory activities than the approved APN inhibitor bestatin, which translated to their superior anti-angiogenic activities. Taken together, a novel bestatin-SAHA hybrid was developed, which worked as a potent APN and HDAC dual inhibitor instead of a PROTAC.

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Skov, Vibe, Thomas Stauffer Larsen, Mads Thomassen, Caroline Riley, Morten Krogh Jensen, Ole Weis Bjerrum, Torben A. Kruse, and Hans Carl Hasselbalch. "Increased Gene Expression of Histone Deacetylases In Patients with Philadelphia-Negative Chronic Myeloproliferative Neoplasms." Blood 116, no. 21 (November 19, 2010): 4119. http://dx.doi.org/10.1182/blood.v116.21.4119.4119.

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Abstract Abstract 4119 Introduction: Several new treatment strategies within the Philadelphia-negative chronic myeloproliferative neoplasms CMPNs are beeing explored, among these, agents belonging to the class of HDAC-inhibitors, including givinostat (ITF2357) and vorinostat (suberoylanilide hydroxamic acid (SAHA)). These agents are inhibitors of class I and II HDAC enzymes, promoting cell-cycle arrest and apoptosis of cancer cells. Recently, enhanced histone deacetylase (HDAC) enzyme activity has been found in CD34+ cells from patients with primary myelofibrosis (PMF), enzyme activity levels highly exceeding those recorded in essential thrombocythemia (ET) and polycythemia vera (PV). The raised levels correlated to the degree of splenomegaly, suggesting that HDAC might be recruited as ET or PV progresses into myelofibrosis or PMF progresses into a more advanced stage. In order to further describe HDACs in CMPNs, we have assessed gene expression of several HDACs in a larger cohort of patients with ET, PV and PMF. Patients and Methods: Gene expression microarray studies have been performed on control subjects (n=21) and patients with ET (n =19), PV (n=41), and PMF (n=9). Most patients were studied on cytoreductive therapy, which for the large majority included hydroxyurea. Gene expression profiles were generated using Affymetrix HG-U133 2.0 Plus microarrays recognizing 54675 probe sets (38.500 genes). Total RNA was purified from whole blood and amplified to biotin-labeled aRNA and hybridized to microarray chips. Results: We identified 20439, 25307, 17417, and 25421 probe sets which were differentially expressed between controls and patients with ET, PV, PMF, and CPMNs as a whole, respectively (false discovery rate (FDR) adjusted p values < 0.05). Several HDAC-genes were significantly deregulated. In ET patients upregulated genes included HDAC5, HDAC9 and downregulated genes HDAC1, HDAC4, HDAC7. In PV-patients HDAC4, HDAC5, HDAC6, HDAC9, and HDAC11 genes were upregulated, and HDAC1, HDAC7, HDAC9, and HDAC11 genes were downregulated. In PMF -patients HDAC4, HDAC6, HDAC9, and HDAC11 genes were upregulated, and HDAC1 and HDAC7 were downregulated. Compared to controls the CMPN-patients as a group exhibited upregulation of HDAC4, HDAC5, HDAC6, HDAC9, and HDAC11 genes. The HDAC genes 9 and 11 were significantly upregulated in both ET, PV, PMF, and CMPNs as a whole, the highest values being recorded in patients with ET, PMF, and CMPNs as compared to controls. In regard to HDAC9 gene expression, the fold changes (FC) were 1.3, 1.2, 1.3, and 1.3 for ET, PV, PMF, and CMPNs, respectively; for HDAC11 the highest values were recorded in patients with ET with the following FCs 1.2, 1.1, 1.1, 1.1 for ET, PV, PMF, and CMPNs, respectively; FDR adjusted p values < 0.05). Within patients, the HDAC6-gene was also differentially expressed with the highest levels being recorded in patients with PMF (FC 1.2, FDR adjusted p values < 0.01). When comparing non-PMF-patients with PMF-patients, a significant upregulation of the HDAC2-gene was found in PMF patients (FC 1.5, FDR adjusted p-value=0.007), whereas the HDAC7-gene was significantly downregulated (FC -1.3, FDR adjusted p-value=0,001. Discussion and Conclusions: Using global gene expression profiling of whole-blood from patients with CMPNs, we have found a pronounced deregulation of HDAC-genes, involving significant upregulation of the HDAC-genes 9 and 11 with the highest expression levels being found in patients with ET, in PMF (HDAC9) and in CMPNs both HDAC9 and HDAC11. Furthermore, we have identified that the HDAC-6 gene is progressively expressed in patients with ET, PV, and PMF reflecting a steady accumulation of abnormally expressed HDAC-6 during disease evolution. Our results lend further support to HDACs as important epigenetic targets in the future treatment of patients with CMPNs. Since the highest expression levels of HDAC-genes was recorded in ET (HDACs 9 and 11), in PMF (HDAC9) and in the whole CMPN-group both HDACs 9 and 11, their downregulation by HDAC-inhibitors might be associated with decreased disease activity including reduction of splenomegaly in MF. Preliminary data indicate that enlarged spleens are diminished in subsets of patients during treatment with HDAC-inhibitors. Studies are in progress on the impact of vorinostat on global gene expression profiling, including HDAC- gene expression subclasses in patients with CMPNs. Disclosures: No relevant conflicts of interest to declare.

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Bui, Hue Thi Buu, Phuong Hong Nguyen, Quan Minh Pham, Hoa Phuong Tran, De Quang Tran, Hosun Jung, Quang Vinh Hong, et al. "Target Design of Novel Histone Deacetylase 6 Selective Inhibitors with 2-Mercaptoquinazolinone as the Cap Moiety." Molecules 27, no. 7 (March 28, 2022): 2204. http://dx.doi.org/10.3390/molecules27072204.

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Epigenetic alterations found in all human cancers are promising targets for anticancer therapy. In this sense, histone deacetylase inhibitors (HDACIs) are interesting anticancer agents that play an important role in the epigenetic regulation of cancer cells. Here, we report 15 novel hydroxamic acid-based histone deacetylase inhibitors with quinazolinone core structures. Five compounds exhibited antiproliferative activity with IC50 values of 3.4–37.8 µM. Compound 8 with a 2-mercaptoquinazolinone cap moiety displayed the highest antiproliferative efficacy against MCF-7 cells. For the HDAC6 target selectivity study, compound 8 displayed an IC50 value of 2.3 µM, which is 29.3 times higher than those of HDAC3, HDAC4, HDAC8, and HDAC11. Western blot assay proved that compound 8 strongly inhibited tubulin acetylation, a substrate of HDAC6. Compound 8 also displayed stronger inhibition activity against HDAC11 than the control drug Belinostat. The inhibitory mechanism of action of compound 8 on HDAC enzymes was then explored using molecular docking study. The data revealed a high binding affinity (−7.92 kcal/mol) of compound 8 toward HDAC6. In addition, dock pose analysis also proved that compound 8 might serve as a potent inhibitor of HDAC11.

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Varricchio, Lilian, Carmela Dell'Aversana, Angela Nebbioso, Giovanni Migliaccio, Lucia Altucci, James J. Bieker, and Anna Rita F. Migliaccio. "Identification of a New Functional HDAC Complex Composed by HDAC5, GATA1 and EKLF in Human Erythroid Cells." Blood 120, no. 21 (November 16, 2012): 979. http://dx.doi.org/10.1182/blood.v120.21.979.979.

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Abstract Abstract 979 Histone deacetylation, the reaction that maintains chromatin in a condensed configuration preventing gene expression, is catalyzed by the histone deacetylase (HDAC) superfamily. The human HDAC family includes 18 different isoforms classified on the basis of their sequence homology to HDACs from Saccharomyces Cerevisiae into class I (HDAC1, −2, −3, and −8), IIa (HDAC4, −5, −7, and −9), IIb (HDAC6 and −10) and IV (HDAC11). Class I HDACs bind the DNA directly while class IIa HDACs shuffles other proteins between nucleus and cytoplasm. While the role of individual class I HDACs in erythropoiesis is starting to emerge, that of class IIa and b HDACs is still largely unknown. To clarify the role played by class IIa HDACs in the control of human erythropoiesis, an extensive analysis of expression, activity, and function of different classes of HDACs during the maturation of erythroblasts derived in vitro from adult blood or cord blood was performed. HDACs expression/activity. Erythroid maturation was associated with increased expression of class I HDACs (both mRNA and protein) which, in the case of HDAC1, was also associated with increased enzymatic activity and association with its NuRD partner GATA1. By contrast, reductions either in expression (HDAC4) or activity (HDAC5) of class IIa HDACs were observed with maturation. In addition, GATA1 and EKLF were consistently found associated in human erythroblasts but EKLF was not found associated with HDAC1. The extent of nuclear-cytoplasmic trafficking of class I (HDAC1 and 2) and IIa (HDAC4 and 5) and of the transcription factors EKLF and GATA1 in response to EPO was determined. HDAC2/EKLF/GATA1 and HDAC4 were found constitutively present in the nucleus and in the cytoplasm, respectively. By contrast, the nuclear concentration of HDAC1 increased while that of HDAC5 and of GATA1fl decreased upon stimulation with EPO. The last two observations suggested that HDAC5, GATA1 and EKLF might be associated in a complex. Identification of the HDAC5/EKLF/GATA1 complex. A series of IPs followed by WB experiments showed that HDAC5 was consistently associated with EKLF and GATA1 and conversely, both GATA1 (preferentially GATA1fl over GATA1s) and EKLF were consistently associated with HDAC5 (Fig 1A and not shown). Interestingly also pERK was detected in IPs with HDAC5, EKLF and GATA1 antibodies. These results indicate that in erythroid cells HDAC5 forms a complex with GATA1, EKLF and pERK. Identification of the biological activity of the HDAC5/GATA1/EKLF/pERK complex. The association between GATA1/EKLF was greater in cells generated with cord blood (which express high HbF levels) than in those derived from adult blood and their association decreased with maturation, suggesting that the complex may regulate HbF expression. To confirm this hypothesis, HDAC5/GATA1 association and γ/(γ+ β) mRNA ratios were determined in erythroid cells induced to mature in the presence of a pan-class II-specific (APHA9, ID50=20 μM for HDAC4) HDAC inhibitor (HDACi) (Fig 1B)1. Cells exposed in parallel to the class I/IIa-specific (UBHA24, ID50 =0.2 and 0.6 μM for HDAC1 and HDAC4, respectively) HDACi, were used as control. Exposure to APHA9 reduced the association between GATA1 and HDAC5 and increased γ/(γ + β) mRNA expression ratio, while this association was not affected by exposure to the class I/II HDACi which, as expected, also increased γ/(γ+ β) mRNA ratio. Conclusions. These data identify a new HDAC complex formed by HDAC5, EKLF and GATA1 that regulates γ/(γ + β) ratio. We hypothesize that the biological role of this new complex is to shuffle GATA1 and EKLF from the cytoplasm to the nucleus, making them able to engage into the NuRD and Sin3A complex respectively, and that inhibition of the activity of this complex affects γ-globin expression indirectly by limiting the amount of GATA1and EKLF available to associate with NuRD and Sin3A. Disclosures: No relevant conflicts of interest to declare.

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Kraft, Fabian B., Maria Hanl, Felix Feller, Linda Schäker-Hübner, and Finn K. Hansen. "Photocaged Histone Deacetylase Inhibitors as Prodrugs in Targeted Cancer Therapy." Pharmaceuticals 16, no. 3 (February 25, 2023): 356. http://dx.doi.org/10.3390/ph16030356.

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Histone deacetylases (HDACs) play a key role in the control of transcription, cell proliferation, and migration. FDA-approved histone deacetylase inhibitors (HDACi) demonstrate clinical efficacy in the treatment of different T-cell lymphomas and multiple myeloma. However, due to unselective inhibition, they display a wide range of adverse effects. One approach to avoiding off-target effects is the use of prodrugs enabling a controlled release of the inhibitor in the target tissue. Herein, we describe the synthesis and biological evaluation of HDACi prodrugs with photo-cleavable protecting groups masking the zinc-binding group of the established HDACi DDK137 (I) and VK1 (II). Initial decaging experiments confirmed that the photocaged HDACi pc-I could be deprotected to its parent inhibitor I. In HDAC inhibition assays, pc-I displayed only low inhibitory activity against HDAC1 and HDAC6. After irradiation with light, the inhibitory activity of pc-I strongly increased. Subsequent MTT viability assays, whole-cell HDAC inhibition assays, and immunoblot analysis confirmed the inactivity of pc-I at the cellular level. Upon irradiation, pc-I demonstrated pronounced HDAC inhibitory and antiproliferative activities which were comparable to the parent inhibitor I. Additionally, only phototreated pc-I was able to induce apoptosis in Annexin V/PI and caspase-Glo 3/7 assays, making pc-I a valuable tool for the development of light-activatable HDACi.

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Perez-Salvia, Montserrat, Aldaba Eneko, Vara Yosu, Fabre Myriam, Ferrer Cristina, Masdeu Carme, Zubia Aizpea, et al. "Efficacy of a New Small-Molecule Inhibitor of Histone Deacetylase 6 (HDAC6) in Preclinical Models of B-Cell Lymphoma and Acute Myeloid Leukemia." Blood 132, Supplement 1 (November 29, 2018): 5383. http://dx.doi.org/10.1182/blood-2018-99-111578.

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Abstract Histone deacetylase 6 (HDAC6) is a protein modifier that is an increasingly attractive pharmacological target. Interestingly, the observation that the HDAC6 knock-out mouse is not lethal, in contrast to those undergoing complete loss of class I, II and III HDACs, suggests that specific HDAC6 inhibitors may be better tolerated than pan-HDAC inhibitors or drugs that target the other HDAC classes. In this regard, the compound ACY-1215 (Rocilinostat), the described selective HDAC6 inhibitors, is undergoing clinical trials for the treatment of multiple myeloma. Taking into account the previous information about HDAC6 inhibitor structures, the structural differences between HDAC6 and other HDAC isoforms and also the structural information of other developed HDAC inhibitors, we have previously designed and synthesized a new potential HDAC6 selective inhibitor, QTX125 with growth inhibitory effects in mantle cell lymphoma (MCL) cell lines, mouse models and ex vivo treatment of primary samples obtained from patients with MCL. Herein, we have extended these findings to show that the newly identified HDAC6 inhibitor QTX125 is also able to inhibit the growth of preclinical models of other B-cell lymphomas such as follicular lymphoma and Burkitt's cell lymphoma, but also of acute acute myeloid leukemia. In addition beyond a-tubulin, a well known HDAC6 target, we have developed a pharmacological and proteomic screening to identify other proteins modified by HDAC6 that can contribute to the described lymphoma and leukemia phenotypes. Disclosures Eneko: Quimatryx: Employment. Yosu:Quimatryx: Employment. Myriam:Oncomatryx: Employment. Cristina:Oncomatryx: Employment. González-Barca:Roche: Speakers Bureau; Celtrion: Consultancy; Gilead: Consultancy; janssen: Consultancy, Speakers Bureau. Fernando:Quimatryx: Consultancy.

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Lodish, Harvey F., and Peng Ji. "Enucleation." Blood 114, no. 22 (November 20, 2009): SCI—18—SCI—18. http://dx.doi.org/10.1182/blood.v114.22.sci-18.sci-18.

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Abstract Abstract SCI-18 Late erythroblasts undergo terminal cell cycle exit, chromatin condensation, and extrusion of the pycnotic nucleus via an asymmetric cell division, but the signaling pathways and genes involved in these last steps of erythropoiesis are not known. Last year we showed that enucleation of mouse fetal erythroblasts requires both Rac GTPases and mDia2, a downstream effector of Rho GTPases and a formin protein required for nucleation of unbranched actin filaments. Rac and mDia2 are required for formation of the cortical actin ring that apparently powers the separation between the membrane- enveloped nucleus and forming reticulocyte. In collaboration with Drs. Tzutzuy Ramirez Hernandez and Maki Murata-Hori of the Temasek Life Sciences Laboratory in Singapore we showed by time-lapse microscopy that extrusion of the nucleus begins with microtubule- dependent “pushing” of the nucleus to one pole of the cell. Final budding off of the nucleus happens quickly – requiring only about 5 minutes. By analyzing fractionated erythroblasts at all stages of differentiation we confirmed that the nucleus undergoes gradual condensation before being extruded out of the cytoplasm. To investigate how chromatin condensation affects condensation and enucleation, we treated cultured erythroblasts with trichostatin A, a pan-histone deacetylase inhibitor, and discovered that enucleation is completely blocked. We further demonstrated that HDAC1, HDAC2 and HDAC3 are highly expressed in erythroid cells. Using a retroviral system to express shRNAs in developing erythroblasts, we found that downregulation of HDAC2 partially blocked enucleation. We also focused on the role of HDAC6 on enucleation since previous reports showed that HDAC6 forms a complex with mDia2. We confirmed the interaction between HDAC6 and mDia2 and demonstrated that HDAC6 can deacetylate mDia2. This may activate mDia2 and eventually promote enucleation. Our hypothesis is that HDACs play two distinct roles in enucleation: HDAC1, HDAC2 and HDAC3 (class I HDACs) are necessary for the condensation of the chromatin which is required for enucleation; on the other hand, HDAC6 (class II HDAC) interacts with and deacetylates mDia2 to promote enucleation. Disclosures Lodish: Amgen: Consultancy, Research Funding.

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Hancock, Wayne W. "Epigenetic Regulation of Regulatory T-Cells: Impact on Autoimmunity and Graft Rejection." Blood 116, no. 21 (November 19, 2010): SCI—23—SCI—23. http://dx.doi.org/10.1182/blood.v116.21.sci-23.sci-23.

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Abstract Abstract SCI-23 Mutations of Foxp3, a transcription factor characteristic of T regulatory (Treg) cells, often cause lethal autoimmunity, leading to much research into how Foxp3+ Tregs control inflammatory and immune responses. This presentation will emphasize therapeutic aspects of our ongoing studies showing that the functions of Foxp3 are regulated by histone/protein deacetylases (HDAC), histone acetyltransferases (HAT), and DNA methyltransferases (DNMT), and for the sake of time will focus on the effects of HDAC inhibitors (HDACi). We have found that Foxp3 acetylation promotes DNA binding and can induce or suppress expression of multiple Foxp3-dependent genes in Tregs. Acetylation is catalyzed by specific HATs whose neutralization can diminish Treg function, with relevance to cancer and HIV, whereas use of selected HDAC inhibitors (HDACi) can increase acetylation and Treg suppression, with relevance to control of autoimmunity and transplant rejection. Many HDAC inhibitors (HDACi) were evaluated for their effects on Treg function. As class I-selective HDACi agents (e.g. benzamides) had little or no effect on Treg function, but pan-HDACi (e.g. hydroxymates) enhanced Treg function, we focused on class II HDACs. There are 2 class II subfamilies; class IIa members are thought to largely function in a tissue-specific manner through recruitment of other proteins since they display only weak catalytic activity, whereas class IIb family members display bona fide catalytic activity. We therefore analyzed class IIb members, of which HDAC6 is the best established and for which selective HDACi are available. HDAC6 exists in the cytoplasm and regulates acetylation of alpha-tubulin and other proteins, including HSP90. Blocking HDAC6 through the use of an HDAC6-specific inhibitor promotes HSP90 acetylation and release of HSP90 client proteins. Use of HDAC6 or HSP90 inhibitors increased Foxp3 expression and enhanced Treg function in vitro and in vivo, and could prevent, or treat pre-existing, autoimmunity in a Treg-dependent manner. We have also investigated the various class IIa family members, of which HDAC9 is of particular interest since its expression is increased 30-fold in Treg vs. regular T cells. HDAC9 decreases Foxp3 expression and function, and its neutralization promotes Treg survival by regulating expression of HSP70 and related HSPs. Our ongoing studies show that HDAC6 neutralization leads to acetylation of HSP90, release of HSF-1 and induction of HSP70, and also suggest that HDAC9 may regulate the acetylation and stabilization of HSF-1. Once produced, HSP70 can chaperone and promote Foxp3 nuclear translocation and function, such that the HDAC6 and HDAC9 pathways are closely intertwined with regard to control of Treg biology. In summary, acetylation, methylation and other epigenetic mechanisms in Tregs are being probed using genetic and pharmacologic approaches. Various currently approved drugs influence Foxp3-dependent Treg functions by affecting epigenetic mechanisms, and while additional HDAC-specific regulators are needed, a rationale is now in place for use of HDAC inhibitors as powerful tools to promote the development and functions of Foxp3+ Tregs in vitro and in vivo. Disclosures: No relevant conflicts of interest to declare.

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Marlow, Laura A., Ilah Bok, Robert C. Smallridge, and John A. Copland. "RhoB upregulation leads to either apoptosis or cytostasis through differential target selection." Endocrine-Related Cancer 22, no. 5 (July 23, 2015): 777–92. http://dx.doi.org/10.1530/erc-14-0302.

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Anaplastic thyroid carcinoma is a highly aggressive undifferentiated carcinoma with a mortality rate near 100% due to an assortment of genomic abnormalities which impede the success of therapeutic options. Our laboratory has previously identified that RhoB upregulation serves as a novel molecular therapeutic target and agents upregulating RhoB combined with paclitaxel lead to antitumor synergy. Knowing that histone deacetylase 1 (HDAC1) transcriptionally suppresses RhoB, we sought to extend our findings to other HDACs and to identify the HDAC inhibitor (HDACi) that optimally synergize with paclitaxel. Here we identify HDAC6 as a newly discovered RhoB repressor. By using isoform selective HDAC inhibitors (HDACi) and shRNAs, we show that RhoB has divergent downstream signaling partners, which are dependent on the HDAC isoform that is inhibited. When RhoB upregulates only p21 (cyclin kinase inhibitor) using a class I HDACi (romidepsin), cells undergo cytostasis. When RhoB upregulates BIMEL using class II/(I) HDACi (belinostat or vorinostat), apoptosis occurs. Combinatorial synergy with paclitaxel is dependent upon RhoB and BIMEL while upregulation of RhoB and only p21 blocks synergy. This bifurcated regulation of the cell cycle by RhoB is novel and silencing either p21 or BIMEL turns the previously silenced pathway on, leading to phenotypic reversal. This study intimates that the combination of belinostat/vorinostat with paclitaxel may prove to be an effective therapeutic strategy via the novel observation that class II/(I) HDACi antagonize HDAC6-mediated suppression of RhoB and subsequent BIMEL, thereby promoting antitumor synergy. These overall observations may provide a mechanistic understanding of optimal therapeutic response.

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Ariffin, Juliana K., Kaustav das Gupta, Ronan Kapetanovic, Abishek Iyer, Robert C. Reid, David P. Fairlie, and Matthew J. Sweet. "Histone Deacetylase Inhibitors Promote Mitochondrial Reactive Oxygen Species Production and Bacterial Clearance by Human Macrophages." Antimicrobial Agents and Chemotherapy 60, no. 3 (December 28, 2015): 1521–29. http://dx.doi.org/10.1128/aac.01876-15.

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Broad-spectrum histone deacetylase inhibitors (HDACi) are used clinically as anticancer agents, and more isoform-selective HDACi have been sought to modulate other conditions, including chronic inflammatory diseases. Mouse studies suggest that HDACi downregulate immune responses and may compromise host defense. However, their effects on human macrophage antimicrobial responses are largely unknown. Here, we show that overnight pretreatment of human macrophages with HDACi prior to challenge withSalmonella entericaserovar Typhimurium orEscherichia coliresults in significantly reduced intramacrophage bacterial loads, which likely reflect the fact that this treatment regime impairs phagocytosis. In contrast, cotreatment of human macrophages with HDACi at the time of bacterial challenge did not impair phagocytosis; instead, HDACi cotreatment actually promoted clearance of intracellularS. Typhimurium andE. coli. Mechanistically, treatment of human macrophages with HDACi at the time of bacterial infection enhanced mitochondrial reactive oxygen species generation by these cells. The capacity of HDACi to promote the clearance of intracellular bacteria from human macrophages was abrogated when cells were pretreated with MitoTracker Red CMXRos, which perturbs mitochondrial function. The HDAC6-selective inhibitor tubastatin A promoted bacterial clearance from human macrophages, whereas the class I HDAC inhibitor MS-275, which inhibits HDAC1 to -3, had no effect on intracellular bacterial loads. These data are consistent with HDAC6 and/or related HDACs constraining mitochondrial reactive oxygen species production from human macrophages during bacterial challenge. Our findings suggest that, whereas long-term HDACi treatment regimes may potentially compromise host defense, selective HDAC inhibitors may have applications in treating acute bacterial infections.

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Hackanson, Bjorn W., Leander Rimmele, Manfred Jung, and Michael Lübbert. "Selective HDAC6 Inhibition and Antileukemic Activity of the Novel HDAC Inhibitor ST80 in Myeloid Leukemia Cell Lines." Blood 114, no. 22 (November 20, 2009): 4808. http://dx.doi.org/10.1182/blood.v114.22.4808.4808.

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Abstract Abstract 4808 The antileukemic activity of histone deacetylase inhibitors (HDACi) has driven the search for epigenetic drugs with higher substrate specificity. Most of the currently used HDACi target class I, II and IV, with some bearing a class preference but only very few being selective in inhibiting specific HDACs, such as the HDAC6-selective inhibitor tubacin. HDAC6 inhibition leads to acetylation of non-histone proteins such as Hsp90 and alpha-tubulin. As it was recently demonstrated that HDAC6 is overexpressed in AML cells, we sought to investigate the effect of selective HDAC6 inhibition by the novel hydroxamic acid derivate ST80 in myeloid cell lines. Methods ST80 (selective HDAC6 inhibitor, 30-fold higher enzyme inhibition as compared to HDAC1) and the non-selective novel hydroxamic acid derivates ST13 (pan-HDACi) and ST34 (pan-HDACi with preference for class I) had previously been tested in enzymatic assays for their HDAC inhibitory potential (Scott et al. Mol Cancer Res. 6:1250-8, 2008). Cell lines HL60, Kasumi-1, NB4, THP1, U937 and K562 were treated with 10 nM to 30 μM of these three drugs. Viability and growth inhibition were determined using trypan blue staining. Acetylation of histone H3, H4 and alpha-tubulin and HDAC6 expression were determined by Western blot and quantified by densitometry. Tubulin-selective acetylation was calculated as the ratio of tubulin acetylation vs. H4 acetylation (ac-tubulin:ac-H4 quotient). Results At 1 μm, ST80, ST13 and ST34 all acetylated tubulin (8-, 8- and 2-fold in NB4, 11-, 14- and 3.4-fold in HL60, respectively, after a 12 h treatment). However, the calculated ac-tubulin:ac-H4 ratio of ST80 was 15- and 8-fold higher in NB4 and 9-fold higher in HL60 when compared to ST13 and ST34. The inhibitory concentration (IC) 50 (cell growth) of ST80 in the six myeloid cell lines ranged from 2.8 μM (NB4) to 5.1 μM (Kasumi-1) after 48 h treatment. Median cell viability of all 6 cell lines at 48 h was 93.7 % (range: 87.0 - 96.8 %) at 1 μm and 90.3 % (65.7 - 95.7 %) at 5 μm of ST80. HDAC6 protein levels were strongly variable between cell lines; however, growth inhibition by ST80 was independent of HDAC6 expression. Conclusion The novel hydroxamic acid derivate ST80 shows antileukemic activity in myeloid cell lines at low micromolar concentrations, which affect cell viability only modestly. The degree of relative tubulin acetylation by ST80 indicates a selective HDAC6 inhibitory activity in myeloid leukemias. The favorable ratio of ST80 growth inhibition vs. cytotoxicity warrants combination studies of this drug with other compounds. Disclosures: No relevant conflicts of interest to declare.

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Laschanzky, Richard S., Lisa E. Humphrey, Jihyun Ma, Lynette M. Smith, Thomas J. Enke, Surendra K. Shukla, Aneesha Dasgupta, et al. "Selective Inhibition of Histone Deacetylases 1/2/6 in Combination with Gemcitabine: A Promising Combination for Pancreatic Cancer Therapy." Cancers 11, no. 9 (September 7, 2019): 1327. http://dx.doi.org/10.3390/cancers11091327.

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Pancreatic ductal adenocarcinoma (PDAC) has a five-year survival rate of <10% due in part to a lack of effective therapies. Pan-histone deacetylase (HDAC) inhibitors have shown preclinical efficacy against PDAC but have failed in the clinic due to toxicity. Selective HDAC inhibitors may reduce toxicity while retaining therapeutic efficacy. However, their use requires identification of the specific HDACs that mediate the therapeutic effects of HDAC inhibitors in PDAC. We determined that the HDAC1/2/3 inhibitor Mocetinostat synergizes with the HDAC4/5/6 inhibitor LMK-235 in a panel of PDAC cell lines. Furthermore, while neither drug alone synergizes with gemcitabine, the combination of Mocetinostat, LMK-235, and gemcitabine showed strong synergy. Using small interfering (si)RNA-mediated knockdown, this synergy was attributed to inhibition of HDACs 1, 2, and 6. Pharmacological inhibition of HDACs 1 and 2 with Romidepsin and HDAC6 with ACY-1215 also potently synergized with gemcitabine in a panel of PDAC cell lines, and this drug combination potentiated the antitumor effects of gemcitabine against PDAC xenografts in vivo. Collectively, our data show that inhibition of multiple HDACs is required for therapeutic effects of HDAC inhibitors and support the development of novel strategies to inhibit HDACs 1, 2, and 6 for PDAC therapy.

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Clauß, Oliver, Linda Schäker-Hübner, Barbara Wenzel, Magali Toussaint, Winnie Deuther-Conrad, Daniel Gündel, Rodrigo Teodoro, et al. "Development and Biological Evaluation of the First Highly Potent and Specific Benzamide-Based Radiotracer [18F]BA3 for Imaging of Histone Deacetylases 1 and 2 in Brain." Pharmaceuticals 15, no. 3 (March 8, 2022): 324. http://dx.doi.org/10.3390/ph15030324.

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The degree of acetylation of lysine residues on histones influences the accessibility of DNA and, furthermore, the gene expression. Histone deacetylases (HDACs) are overexpressed in various tumour diseases, resulting in the interest in HDAC inhibitors for cancer therapy. The aim of this work is the development of a novel 18F-labelled HDAC1/2-specific inhibitor with a benzamide-based zinc-binding group to visualize these enzymes in brain tumours by positron emission tomography (PET). BA3, exhibiting high inhibitory potency for HDAC1 (IC50 = 4.8 nM) and HDAC2 (IC50 = 39.9 nM), and specificity towards HDAC3 and HDAC6 (specificity ratios >230 and >2080, respectively), was selected for radiofluorination. The two-step one-pot radiosynthesis of [18F]BA3 was performed in a TRACERlab FX2 N radiosynthesizer by a nucleophilic aliphatic substitution reaction. The automated radiosynthesis of [18F]BA3 resulted in a radiochemical yield of 1%, a radiochemical purity of >96% and a molar activity between 21 and 51 GBq/µmol (n = 5, EOS). For the characterization of BA3, in vitro and in vivo experiments were carried out. The results of these pharmacological and pharmacokinetic studies indicate a suitable inhibitory potency of BA3, whereas the applicability for non-invasive imaging of HDAC1/2 by PET requires further optimization of the properties of this compound.

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Knox, Tessa, Maritza Lienlaf, Patricio Perez-Villarroel, Calvin Lee, Eva Sahakian, John J. Powers, Fengdong Cheng, et al. "Selective Inhibition of HDAC6 Decreases Viability of Cutaneous T-Cell Lymphoma and Improves Immune Recognition." Blood 124, no. 21 (December 6, 2014): 5423. http://dx.doi.org/10.1182/blood.v124.21.5423.5423.

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Abstract Cutaneous T-cell lymphoma (CTCL) includes a spectrum of non-Hodgkin’s T-cell lymphomas. While most patients with CTCL experience non-life threatening skin symptoms, those who do progress to later stages of the disease may develop serious complications. For patients with tumor stage or lymph node involvement there is a significant decline in treatment response & few treatment options are available. Current available therapeutic options include the use of histone deacetylase (HDAC) inhibitors as Romidepsin, Panobinostat and Vorinostat. However, their mechanism of action is still unknown. The role of histone deacetylases (HDACs) in cell biology, initially limited to their effects upon histones, now encompasses more complex regulatory functions that are dependent on their tissue expression, cellular compartment distribution, pathophysiological conditions and stage of cellular differentiation. Although major advances have been made in understanding the role of specific HDACs in cell proliferation and the survival of cancer cells, their individual participation in specific intracellular pathways is not completely understood. Here we present data demonstrating the changes in several immune-related pathways in two CTCL cell lines after exposure to both pan-HDAC inhibitors and selective HDAC6 inhibitors. We compared the pharmacological effects of the pan-HDAC inhibitor LBH589 (Panbinostat), the class I selective Romidepsin, and the selective HDAC6 inhibitors such as TubastatinA and NexturstatB, on two CTCL cell lines (HuT78, HuT102). Our findings thus far demonstrate the following: First, we observed a marked inhibition of proliferation capacity of both cell lines when treated with either pan-HDACi or the more selective HDAC6 inhibitors. However, the selective HDAC6 inhibitors showed less cytotoxicity. Second, we observed important changes in the expression of the co-inhibitory molecules, such as PD-L1. Given our results, we conclude that the selective targeting of HDAC6 could recapitulate the anti-tumor effects of pan-HDAC inhibitors without having the non-target cytotoxic effects often encountered when using pan-HDAC inhibitors. Disclosures No relevant conflicts of interest to declare.

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Mensah, Afua Adjeiwaa, Sergio Valente, Milos Matkovic, Giulio Sartori, Chiara Falzarano, Chiara Tarantelli, Luciano Cascione, et al. "Abstract 3279: Dual inhibition of EZH2 and histone deacetylases for the treatment of lymphomas with epigenetic aberrations." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3279. http://dx.doi.org/10.1158/1538-7445.am2022-3279.

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Abstract Background: Lymphomas are characterized by aberrations in epigenetic proteins that contribute to establishing and maintaining the malignant phenotype. Gain of function mutations in EZH2 histone methyltransferase and inactivating mutations in CREBBP acetyltransferase occur in up to 30% of diffuse large B cell lymphomas (DLBCLs). Half of DLBCLs with mutated EZH2 have also mutated CREBBP. In vitro, mutated EZH2 DLBCLs have a lower sensitivity to HDAC inhibitors, HDACi (Mensah et al, 2021). Here, we explored the dual pharmacological inhibition of EZH2 and HDAC for an improved anti-lymphoma activity. Methods: Biochemical evaluation and characterization of target engagement were done using fluorescence polarization, thermal shift, surface plasmon resonance, isothermal titration calorimetry and microscale thermophoresis. Computer modelling was performed using available EZH2 and HDACs crystal structures. Anti-proliferative activity was assessed after 7 days (d) using MTT and live imaging in DLBCL cells (n = 5). For cell cycle analysis by flow cytometry, cells were treated, fixed then stained with 7-AAD. Results: We designed and synthesized 2 dual EZH2/HDAC inhibitors, MC4343 and MC4355, starting from the structures of EZH2 inhibitor tazemetostat (taz) and HDACi vorinostat. In biochemical assays, MC4343 and MC4355 had equal potencies towards EZH2 (0.032 nM) but different specificities towards class I and class II HDACs: MC4355 showed 7.5-fold greater inhibition of HDAC3 compared to MC4343 (0.38 µM and 2.85 µM, respectively) and more potently inhibited HDACs 6 and 8 (0.016, 0.17 µM and no activity, respectively). Computational modelling showed that the coordinative functional group of MC4343, but not of MC4355, caused steric clashes with several HDACs in increasing order: HDAC1 = HDAC3 > HDAC8 >> HDAC4 = HDAC6. These results closely mirrored those obtained from the biochemical analysis. Both compounds inhibited proliferation in DLBCL cell lines irrespective of EZH2 or CREBBP mutational status but EZH2 and/or CREBBP mutants were more sensitive. MC4355 (IC50 range = 0.17 - 1.68 µM; median = 0.2 µM) was more potent than MC4343 (IC50 range = 0.17-2.72 µM; median = 1.78 µM). This was confirmed by live imaging analyses. SUDHL4, with EZH2 Y666N, showed poorer sensitivity to both inhibitors compared to DLBCLs with EZH2 Y646N/S. MC4343 and MC4355 induced cell death and G1 arrest in a dose-dependent manner. Pfeiffer, KARPAS422, WSUDLCL2, most sensitive to taz alone (IC50 = 4, 16, 77 nM), were most sensitive to MC4355 (IC50 = 200, 174, 171 nM). Notably, dual inhibitor treatment of Toledo and SUDHL4 with low sensitivity to taz (IC50 = 9, 14 µM), partially rescued sensitivity (IC50 = 1.7, 1.6 µM). Conclusions: We designed and synthesized 2 novel dual EZH2/HDAC inhibitors, MC4343 and MC4355, with robust anti-proliferative effects in DLBCL. Our data show the efficacy of this novel class of epigenetic agents in lymphomas. Citation Format: Afua Adjeiwaa Mensah, Sergio Valente, Milos Matkovic, Giulio Sartori, Chiara Falzarano, Chiara Tarantelli, Luciano Cascione, Stefano A. Pileri, Andrea Cavalli, Antonello Mai, Francesco Bertoni. Dual inhibition of EZH2 and histone deacetylases for the treatment of lymphomas with epigenetic aberrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3279.

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Freese, Kim, Tatjana Seitz, Peter Dietrich, Serene M. L. Lee, Wolfgang E. Thasler, Anja Bosserhoff, and Claus Hellerbrand. "Histone Deacetylase Expressions in Hepatocellular Carcinoma and Functional Effects of Histone Deacetylase Inhibitors on Liver Cancer Cells In Vitro." Cancers 11, no. 10 (October 18, 2019): 1587. http://dx.doi.org/10.3390/cancers11101587.

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Hepatocellular carcinoma (HCC) is a leading cause for deaths worldwide. Histone deacetylase (HDAC) inhibition (HDACi) is emerging as a promising therapeutic strategy. However, most pharmacological HDACi unselectively block different HDAC classes and their molecular mechanisms of action are only incompletely understood. The aim of this study was to systematically analyze expressions of different HDAC classes in HCC cells and tissues and to functionally analyze the effect of the HDACi suberanilohydroxamic acid (SAHA) and trichostatin A (TSA) on the tumorigenicity of HCC cells. The gene expression of all HDAC classes was significantly increased in human HCC cell lines (Hep3B, HepG2, PLC, HuH7) compared to primary human hepatocytes (PHH). The analysis of HCC patient data showed the increased expression of several HDACs in HCC tissues compared to non-tumorous liver. However, there was no unified picture of regulation in three different HCC patient datasets and we observed a strong variation in the gene expression of different HDACs in tumorous as well as non-tumorous liver. Still, there was a strong correlation in the expression of HDAC class IIa (HDAC4, 5, 7, 9) as well as HDAC2 and 8 (class I) and HDAC10 (class IIb) and HDAC11 (class IV) in HCC tissues of individual patients. This might indicate a common mechanism of the regulation of these HDACs in HCC. The Cancer Genome Atlas (TCGA) dataset analysis revealed that HDAC4, HDAC7 and HDAC9 as well as HDAC class I members HDAC1 and HDAC2 is significantly correlated with patient survival. Furthermore, we observed that SAHA and TSA reduced the proliferation, clonogenicity and migratory potential of HCC cells. SAHA but not TSA induced features of senescence in HCC cells. Additionally, HDACi enhanced the efficacy of sorafenib in killing sorafenib-susceptible cells. Moreover, HDACi reestablished sorafenib sensitivity in resistant HCC cells. In summary, HDACs are significantly but differently increased in HCC, which may be exploited to develop more targeted therapeutic approaches. HDACi affect different facets of the tumorigenicity of HCC cells and appears to be a promising therapeutic approach alone or in combination with sorafenib.

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Cheng, Fengdong, Bing Xia, Eva Sahakian, Tint Lwin, Hongwei Wang, Limin Xing, Bijal D. Shah, et al. "Selective Inhibition of Histone Deacetylase 6 (HDAC6) and HDAC3 As a Novel Therapeutic Strategy in Mantle Cell Lymphoma (MCL)." Blood 124, no. 21 (December 6, 2014): 5397. http://dx.doi.org/10.1182/blood.v124.21.5397.5397.

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Abstract In recent years, the deeper understanding of the molecular alterations that occurs in MCL has provided the foundation for the development of new targeted therapeutic approaches. Histone deacetylases (HDACs), represent ideal targets due to their dual role not only in supporting lymphoma survival and resistance to apoptosis1,2, but also in immunologic escape and autocrine cytokine signaling which maintains the microenvironmental niche and facilitates drug resistance3,4. Recently, we have identified two members of the HDAC family, HDAC6 and HDAC3, which play integral roles in regulation of 1) survival/apoptosis, 2) microenvironment-mediated drug resistance, and 3) immunogenicity of MCL cells. Our studies to date have shown the following: First, HDAC6 and HDAC3 are overexpressed in MCL cell lines and primary cells from MCL patients. This expression is further up-regulated following adhesion of MCL cells to stromal cells, consequently increasing survival and acquisition of a drug-resistance phenotype. Second, genetic or pharmacologic inhibition of HDAC6 with isotype-selective inhibitor(s) induced cell cycle arrest, apoptosis and overcome stroma-mediated drug resistance. Of note, MCL cells lacking HDAC6 are more immunogenic and able of inducing stronger antitumor responses. This increased immunogenicity is due to a novel mechanism involving HDAC6 regulation of the immunosuppressive STAT3 signaling pathway and PD-L1 expression. Strikingly, mice lacking HDAC6 (germ-line HDAC6KO) displayed significantly reduced tumor growth kinetics when compared to the wild-type controls. Third, Genetic or pharmacologic disruption of HDAC3 resulted in decreased cell proliferation, increased apoptosis and reversal of the drug-resistance phenotype of MCL cells. This is due to HDAC3-mediated regulation of a group of small non-coding tumor suppressor microRNAs (miR-15a/16-1, miR-26 and miR29). The importance of HDAC3 as a promising therapeutic target in MCL is further highlighted by our recent findings using the HDAC3-selective inhibitor RGFP966 in human MCL cells, Four,strategies targeting both, HDAC6 and HDAC3 with selective inhibitors represent a promising strategy in MCL specially when these novel agents are combined with the BTK inhibitor Ibrutinib. Although inhibition of HDACs are extensively known for their ability to induce cell cycle arrest and apoptosis of malignant cells, their immuneregulatory effects upon tumor cell and/or immune cells are yet to be elucidated. In this study we present data that can convincingly provide a proper framework for combinatorial therapy using HDAC inhibitors in MCL. 1. Gupta M, et alLeukemia 2012; 26(6): 1356-64. 2. Kawamata N, et alBlood 2007; 110(7): 2667-73. 3. Lwin T, et alLeukemia 2007; 21(7): 1521-31. 4. Meads MB, et alNat Rev Cancer 2009; 9(9): 665-74. Disclosures Shah: Seattle Genetics, Inc.: Research Funding; NCCN: Consultancy; Celgene: Consultancy, Speakers Bureau; SWOG: Consultancy; Pharmacyclics: Consultancy; Janssen: Consultancy. Quayle:Acetylon Pharmaceuticals Inc.: Employment. Jones:Acetylon Pharmaceuticals Inc.: Employment.

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Baumann, Philipp, Carmen Junghanns, Strobl Stefan, Fuat Oduncu, and Ralf Schmidmaier. "The Novel Pan-HDAC Inhibitor CR2408 Inhibits Multiple Myeloma Cell Growth and Proliferation." Blood 118, no. 21 (November 18, 2011): 5133. http://dx.doi.org/10.1182/blood.v118.21.5133.5133.

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Abstract Abstract 5133 Background: Myeloma (MM) is still an incurable disease, and new cytotoxic drugs are urgently needed. CR2408 is a novel pan-histon deacetylases inhibitor with promising properties and effects in MM cells. Methods: The effect of CR2408 in MM cells was characterized by multiple assays. HDAC inhibition was shown by western blotting as well as HDAC enzyme inhibition assays. Cell growth and viability was shown by the common WST-1 assay. Induction of apoptosis was detected using flow cytometry after annexin-V-FITC staining as well as caspase cleavage detected by western blotting. Basal and cytokine stimulated cell growth rates of myeloma cells were measured by the WST-1 assay. Myeloma cell proliferation was determined by the BrdU assay. Alterations of the cell cycle were determined by flow cytometry after staining with propidium iodide. Modulation of intracellular signalling was shown by western blotting. Results: We have found CR2408 to induce profound hyperacetylation of histone H4 in MM cells. Our experiments revealed that nanomolar concentrations of CR2408 abrogate HDAC activity in 11 HDAC enzymes. Comparison with SAHA shows lower IC50 values for CR2408 (HDAC1 27nM, HDAC2 76nM, HDAC3 28nM, HDAC4 151nM, HDAC5 51nM, HDAC6 13nM, HDAC7 360nM, HDAC8 522nM, HDAC9 92nM, HDAC10 77nM and HDAC11 56nM). CR2408 abrogated myeloma cell growth at nanomolar concentrations (250nM: NCI-H929: −93%; OPM-2: −85%; U266: −87%; RPMI-8226: −86%) and induced apoptosis in multiple myeloma cell lines and primary cells, as shown by the annexin V assay (500nM: NCI-H929: 75%, OPM-2: 65%, RPMI-8226: 80%, U266: 18%, primary cells: 50%). Induction of apoptosis was confirmed by showing cleavage of caspase 3, 8 and 9. Furthermore, increased cell growth induced by conditioned medium obtained from bone marrow stromal cells was abrogated by CR2408. The BrdU assay revealed that inhibition of cell growth was due to inhibition of myeloma cell proliferation (500nM: OPM-2: −50%; RPMI-8226: −58%, U266: −55%). Furthermore, we analysed cell cycle distribution and found that in contrast to other HDAC inhibitors, CD2408 does not provoke a G0/G1 cell cycle arrest but leads to immediate DNA and cell fragmentation, resulting in an accumulation of cell fragments in the subG1 phase. Inhibition of cell proliferation was accompanied by a strong downregulation of the proteins cdc25A, cdk4 and hypophosphorylation of RB. Incubation of myeloma cells with CR2408 did not alter the phosphorylation of 4E-BP-1, P70S6k, but the mitochondrial proteins Bad and Bcl-Xl were downregulated and Bim and pJNK were upregulated. Finally, CR2408 shows significant synergistic effects when combined with doxorubicin and bortezomib. Conclusions: The HDAC inhibitor CR2408 inhibits MM cell proliferation and induces apoptosis. This is accompanied by a strong perturbation of mitochondrial proteins. Since CR2408 inhibit myeloma growth and proliferation as low nanomolar levels, this study provides the rationale for the further in vivo evaluation of CR2408 in order to find a more efficient and less toxic member of this group of compounds. Disclosures: Stefan: 4SC AG: Employment.

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Verbeek, Tamara C. A. I., Susan Arentsen-Peters, Patricia Garrido Castro, Sandra Pinhancos, Kirsten Vrenken, Aida Varela-Moreira, M. Emmy Dolman, Bianca Koopmans, Rob Pieters, and Ronald W. Stam. "Selective Inhibition of Class II HDAC Isoforms 4 and 5 Provides a Promising Therapeutic Intervention for MLL-Rearranged Acute Lymphoblastic Leukemia in Infants." Blood 138, Supplement 1 (November 5, 2021): 2206. http://dx.doi.org/10.1182/blood-2021-147164.

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Abstract MLL-rearranged acute lymphoblastic leukemia (ALL) is characterized by deregulation of the epigenome and shows susceptibility towards epigenetic perturbators such as histone deacetylase (HDAC) inhibitors. Hence, HDACs represent attractive drug targets and a variety of small molecule HDAC inhibitors have been developed and evaluated for the treatment of hematological malignancies. However, most broad-spectrum inhibitors, which simultaneously target the majority of human HDAC isoforms, often induce toxicity, especially in combination with other therapeutic agents. Therefore, selective inhibition of only one or two HDAC isoforms may represent a better alternative, provided that disease-specific dependency on specific HDACs has been identified. We examined the effects of shRNA-mediated knock-down of the class II HDACs (i.e. HDAC4, HDAC5, HDAC6, HDAC7 and HDAC9) in the MLL-rearranged ALL cell lines SEM and ALL-PO. Except for HDAC9, loss of expression (both on the mRNA and protein level) of all HDACs led to strong reductions in viable cells (0.70 to 0.19-fold; p=0.02-0.0016) in both models due to apoptosis, cell cycle arrest, or a combination thereof. Next, we evaluated the in vitro efficacy of a variety of class II HDAC-specific inhibitors on a panel of MLL-rearranged ALL (n=5) using 4-day viability MTT assays. This revealed that the selective HDAC4/5 inhibitor LMK-235 was able to recapitulate the loss-of-function phenotype of HDAC4 and HDAC5. Dose response curves showed complete growth inhibition in MLL-rearranged ALL cell lines (n=5), as well as in primary MLL-rearranged infant ALL patient samples (n=4), with IC 50 values of ~100 nM and 40-100 nM, respectively. Importantly, at these concentrations, LMK-235 hardly affected whole bone marrow samples derived from healthy individuals (n=2), for which IC 50 values were ~1 µM. To further explore the potential of class II HDAC inhibitor-based therapeutic strategies, we performed a combinatorial drug screen to identify compounds that synergize with LMK-235. For this, a compound library (comprising >200 unique agents) was screened in the absence and presence of varying concentrations of LMK235 in the MLL-rearranged cell line models SEM and ALL-PO. This, and subsequent validation experiments in additional cell line models, revealed that Venetoclax (BCL2 inhibitor), Trametinib (MEK/ERK inhibitor), Ponatinib (multi-tyrosine kinase inhibitor) and Omipalisib (a PI3K/mTOR inhibitor) strongly synergized with LMK-235. Average ZIP synergy scores ranged from 10-30, with peak ZIP scores up to 40. Importantly, synergistic effects were consistent over all concentration combinations tested. The addition of 50-100 nM LMK-235 strongly reduced IC 50 values for Omipalisib, Ponatinib and Venetoclax (0.27-fold p=0.003, 0.11-fold p=0.0005, 0.75-fold p=0.0004, respectively) in both models. In preparation to assess the in vivo efficacy of LMK-235 in patient-derived xenograft (PDX) mouse models of MLL-rearranged infant ALL, pharmacokinetic/pharmacodynamic (PK/PD) analysis was performed in immunodeficient NSG mice (n=5). For this, mice were treated with 20 mg/kg of LMK-235, daily administered via intraperitoneal injections for a total of 29 days. While none of the mice showed signs of toxicity or weight loss, LMK-235 plasma levels were stably maintained at concentrations that are highly effective against MLL-rearranged ALL cells in vitro. Taken together, these data demonstrate that various class II HDAC isoforms are targetable vulnerabilities in MLL-rearranged ALL and that pharmaceutical inhibition of HDAC4/5 by LMK-235 represents an attractive therapeutic option. Moreover, high levels of synergy observed between this HDAC inhibitor and various agents belonging to drug classes already reported to be effective against MLL-rearranged ALL, warrants pre-clinical evaluation in vivo. Currently, the assessment of the in vivo efficacy of LMK-235 monotherapy in MLL-rearranged infant ALL PDX models is in progress, after which promising synergistic HDAC inhibitor-based drug combinations will be evaluated. To determine the additional therapeutic value, the efficacy of LMK-235 and promising synergistic combinations will be evaluated in the background of conventional combination chemotherapy, where PDX models will receive a mouse-adapted version of induction therapy currently applied for treatment of MLL-rearranged infant ALL patients. Disclosures No relevant conflicts of interest to declare.

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Zhang, Bingbing, Zhu-Wei Ruan, Dongdong Luo, Yueyue Zhu, Tingbo Ding, Qiang Sui, and Xinsheng Lei. "Unexpected Enhancement of HDACs Inhibition by MeS Substitution at C-2 Position of Fluoro Largazole." Marine Drugs 18, no. 7 (June 30, 2020): 344. http://dx.doi.org/10.3390/md18070344.

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Given our previous finding that fluorination at the C18 position of largazole showed reasonably good tolerance towards inhibitory activity and selectivity of histone deacetylases (HDACs), further modification on the valine residue in the fluoro-largazole’s macrocyclic moiety with S-Me l-Cysteine or Glycine residue was performed. While the Glycine-modified fluoro analog showed poor activity, the S-Me l-Cysteine-modified analog emerged to be a very potent HDAC inhibitor. Unlike all previously reported C2-modified compounds in the largazole family (including our recent fluoro-largazole analogs) where replacement of the Val residue has failed to provide any potency improvement, the S-Me l-Cysteine-modified analog displayed significantly enhanced (five–nine-fold) inhibition of all the tested HDACs while maintaining the selectivity of HDAC1 over HDAC6, as compared to largazole thiol. A molecular modeling study provided rational explanation and structural evidence for the enhanced inhibitory activity. This new finding will aid the design of novel potent HDAC inhibitors.

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Joshi, Atul D., Nektarios Barabutis, Charalampos Birmpas, Christiana Dimitropoulou, Gagan Thangjam, Mary Cherian-Shaw, John Dennison, and John D. Catravas. "Histone deacetylase inhibitors prevent pulmonary endothelial hyperpermeability and acute lung injury by regulating heat shock protein 90 function." American Journal of Physiology-Lung Cellular and Molecular Physiology 309, no. 12 (December 15, 2015): L1410—L1419. http://dx.doi.org/10.1152/ajplung.00180.2015.

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Transendothelial hyperpermeability caused by numerous agonists is dependent on heat shock protein 90 (Hsp90) and leads to endothelial barrier dysfunction (EBD). Inhibition of Hsp90 protects and restores transendothelial permeability. Hyperacetylation of Hsp90, as by inhibitors of histone deacetylase (HDAC), suppresses its chaperone function and mimics the effects of Hsp90 inhibitors. In this study we assessed the role of HDAC in mediating lipopolysaccharide (LPS)-induced transendothelial hyperpermeability and acute lung injury (ALI). We demonstrate that HDAC inhibition protects against LPS-mediated EBD. Inhibition of multiple HDAC by the general inhibitors panobinostat or trichostatin provided protection against LPS-induced transendothelial hyperpermeability, acetylated and suppressed Hsp90 chaperone function, and attenuated RhoA activity and signaling crucial to endothelial barrier function. Treatment with the HDAC3-selective inhibitor RGFP-966 or the HDAC6-selective inhibitor tubastatin A provided partial protection against LPS-mediated transendothelial hyperpermeability. Similarly, knock down of HDAC3 and HDAC6 by specific small-interfering RNAs provided significant protection against LPS-induced EBD. Furthermore, combined pharmacological inhibition of both HDAC3 and -6 attenuated the inflammation, capillary permeability, and structural abnormalities associated with LPS-induced ALI in mice. Together these data indicate that HDAC mediate increased transendothelial hyperpermeability caused by LPS and that inhibition of HDAC protects against LPS-mediated EBD and ALI by suppressing Hsp90-dependent RhoA activity and signaling.

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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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S, Dhanalakshmi, Sridharan Rajagopal, Naveen Sadhu, Chandru G, Amir Siddiqui, Saif Wahid, Basava Prabhu, et al. "Novel Dual Inhibitor of LSD1-HDAC6/8 for Treatment of Cancer." Blood 136, Supplement 1 (November 5, 2020): 29. http://dx.doi.org/10.1182/blood-2020-142685.

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Introduction: Lysine specific demethylase 1 (LSD1) and histone deacetylases (HDACs) are known to modulate the expression of several disease specific genes as part of repressor complexes, including CoREST. In addition, they also have complex mutually exclusive roles in cancer cells. Accordingly, several studies have shown that combined inhibition of these proteins to have a profound effect in inhibiting tumor growth. In this regard, although class I HDAC inhibition has been well studied, dose limiting toxicities associated with these inhibitors is still a challenge in the clinic, it has been hypothesized that isoform selective HDAC6 inhibition could provide desired efficacy with minimal safety concerns. To study the effect of dual inhibition of LSD1 and HDAC6, we used JBI-802, a novel, dual LSD1 and HDAC6 isoform selective inhibitor by rational design. JBI-802 shows superior efficacy in select AML models as compared to LSD1 or HDAC6 selective inhibitors and also has a better tolerability profile. We also performed several mechanistic studies with JBI-802 to understand the interaction of LSD1 and HDAC6 and to dissect out the molecular mechanism of LSD1/HDAC6 inhibition. Methods: To assess in vitro LSD1 and HDAC6 potency, TR-FRET and fluorescence based activity assays were performed. Western blotting, co-immunoprecipitation and qRT-PCR studies were used to assess biomarkers of LSD1 and HDAC inhibition. Transcriptome studies and AI based analysis was performed to delineate the mechanism of single agents (LSD1 or HDAC6) vs. LSD1/HDAC6 dual inhibition. Xenograft and syngeneic disease models were used to assess the in vivo efficacy. Results: JBI-802 shows an IC50 of ~0.05 µM for LSD1 and ~ 0.01 µM for HDAC6 and a strong dose-dependent modulation of biomarkers specific for both these targets. JBI-802 showed anti-proliferative activity against a panel of haematological cancers with EC50 ranging from 0.01 to 0.3 µM. Co-immunoprecipitation studies clearly showed that in addition to HDAC1, HDAC6 also co-immuno-precipitated with LSD1 suggesting that HDAC6 is a part of the CoREST complex. These findings also correlated well with stronger biomarker modulation of some key proteins by JBI-802, in comparison with the single agent LSD1 or HDAC6 selective inhibitors. Additional genomic and transcriptome based studies analyses led to the identification of a biomarker that is specific for dual inhibition. Interestingly, neither LSD1 nor HDAC6 inhibition alone lead to the modulation of this specific biomarker, which was observed only in cell lines that were sensitive to the dual inhibitor. These studies not only pave way for patient stratification in the clinic, but also could be a robust biomarker for treatment response. Data generated so far highlight the promise of this dual inhibitor in several heamatological cancers and more specifically in neoplasms with certain gain-of-function mutations. JBI-802 has optimal oral exposure and has been tested in multiple animal models by oral administration. Consistently, it showed superior efficacy in these models as compared to single agent inhibitors. Exploratory toxicity studies have clearly demonstrated that JBI-802 has an excellent safety profile. Conclusion: JBI-802 is currently being evaluated in IND-enabling studies to be progressed into clinical trials and such inhibitors could serve as powerful therapeutic agents for the treatment of specific cancers. Disclosures No relevant conflicts of interest to declare.

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Angiolilli, Chiara, Pawel A. Kabala, Aleksander M. Grabiec, Iris M. Van Baarsen, Bradley S. Ferguson, Samuel García, Beatriz Malvar Fernandez, et al. "Histone deacetylase 3 regulates the inflammatory gene expression programme of rheumatoid arthritis fibroblast-like synoviocytes." Annals of the Rheumatic Diseases 76, no. 1 (July 25, 2016): 277–85. http://dx.doi.org/10.1136/annrheumdis-2015-209064.

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ObjectivesNon-selective histone deacetylase (HDAC) inhibitors (HDACi) have demonstrated anti-inflammatory properties in both in vitro and in vivo models of rheumatoid arthritis (RA). Here, we investigated the potential contribution of specific class I and class IIb HDACs to inflammatory gene expression in RA fibroblast-like synoviocytes (FLS).MethodsRA FLS were incubated with pan-HDACi (ITF2357, givinostat) or selective HDAC1/2i, HDAC3/6i, HDAC6i and HDAC8i. Alternatively, FLS were transfected with HDAC3, HDAC6 or interferon (IFN)-α/β receptor alpha chain (IFNAR1) siRNA. mRNA expression of interleukin (IL)-1β-inducible genes was measured by quantitative PCR (qPCR) array and signalling pathway activation by immunoblotting and DNA-binding assays.ResultsHDAC3/6i, but not HDAC1/2i and HDAC8i, significantly suppressed the majority of IL-1β-inducible genes targeted by pan-HDACi in RA FLS. Silencing of HDAC3 expression reproduced the effects of HDAC3/6i on gene regulation, contrary to HDAC6-specific inhibition and HDAC6 silencing. Screening of the candidate signal transducers and activators of transcription (STAT)1 transcription factor revealed that HDAC3/6i abrogated STAT1 Tyr701 phosphorylation and DNA binding, but did not affect STAT1 acetylation. HDAC3 activity was required for type I IFN production and subsequent STAT1 activation in FLS. Suppression of type I IFN release by HDAC3/6i resulted in reduced expression of a subset of IFN-dependent genes, including the chemokines CXCL9 and CXCL11.ConclusionsInhibition of HDAC3 in RA FLS largely recapitulates the effects of pan-HDACi in suppressing inflammatory gene expression, including type I IFN production in RA FLS. Our results identify HDAC3 as a potential therapeutic target in the treatment of RA and type I IFN-driven autoimmune diseases.

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Porter, Nicholas J., Adaickapillai Mahendran, Ronald Breslow, and David W. Christianson. "Unusual zinc-binding mode of HDAC6-selective hydroxamate inhibitors." Proceedings of the National Academy of Sciences 114, no. 51 (December 4, 2017): 13459–64. http://dx.doi.org/10.1073/pnas.1718823114.

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Histone deacetylases (HDACs) regulate myriad cellular processes by catalyzing the hydrolysis of acetyl–l-lysine residues in histone and nonhistone proteins. The Zn2+-dependent class IIb enzyme HDAC6 regulates microtubule function by deacetylating α-tubulin, which suppresses microtubule dynamics and leads to cell cycle arrest and apoptosis. Accordingly, HDAC6 is a target for the development of selective inhibitors that might be useful in new therapeutic approaches for the treatment of cancer, neurodegenerative diseases, and other disorders. Here, we present high-resolution structures of catalytic domain 2 from Danio rerio HDAC6 (henceforth simply “HDAC6”) complexed with compounds that selectively inhibit HDAC6 while maintaining nanomolar inhibitory potency: N-hydroxy-4-[(N(2-hydroxyethyl)-2-phenylacetamido)methyl)-benzamide)] (HPB), ACY-1215 (Ricolinostat), and ACY-1083. These structures reveal that an unusual monodentate Zn2+ coordination mode is exploited by sterically bulky HDAC6-selective phenylhydroxamate inhibitors. We additionally report the ultrahigh-resolution structure of the HDAC6–trichostatin A complex, which reveals two Zn2+-binding conformers for the inhibitor: a major conformer (70%) with canonical bidentate hydroxamate-Zn2+ coordination geometry and a minor conformer (30%) with monodentate hydroxamate-Zn2+ coordination geometry, reflecting a free energy difference of only 0.5 kcal/mol. The minor conformer is not visible in lower resolution structure determinations. Structural comparisons of HDAC6-inhibitor complexes with class I HDACs suggest active site features that contribute to the isozyme selectivity observed in biochemical assays.

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Grégoire, Serge, Lin Xiao, Jianyun Nie, Xiaohong Zhang, Minghong Xu, Jiarong Li, Jiemin Wong, Edward Seto, and Xiang-Jiao Yang. "Histone Deacetylase 3 Interacts with and Deacetylates Myocyte Enhancer Factor 2." Molecular and Cellular Biology 27, no. 4 (December 11, 2006): 1280–95. http://dx.doi.org/10.1128/mcb.00882-06.

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ABSTRACT The myocyte enhancer factor 2 (MEF2) family of transcription factors is not only important for controlling gene expression in normal cellular programs, like muscle differentiation, T-cell apoptosis, neuronal survival, and synaptic differentiation, but has also been linked to cardiac hypertrophy and other pathological conditions. Lysine acetylation has been shown to modulate MEF2 function, but it is not so clear which deacetylase(s) is involved. We report here that treatment of HEK293 cells with trichostatin A or nicotinamide upregulated MEF2D acetylation, suggesting that different deacetylases catalyze the deacetylation. Related to the trichostatin A sensitivity, histone deacetylase 4 (HDAC4) and HDAC5, two known partners of MEF2, exhibited little deacetylase activity towards MEF2D. In contrast, HDAC3 efficiently deacetylated MEF2D in vitro and in vivo. This was specific, since HDAC1, HDAC2, and HDAC8 failed to do so. While HDAC4, HDAC5, HDAC7, and HDAC9 are known to recognize primarily the MEF2-specific domain, we found that HDAC3 interacts directly with the MADS box. In addition, HDAC3 associated with the acetyltransferases p300 and p300/CBP-associated factor (PCAF) to reverse autoacetylation. Furthermore, the nuclear receptor corepressor SMRT (silencing mediator of retinoid acid and thyroid hormone receptor) stimulated the deacetylase activity of HDAC3 towards MEF2 and PCAF. Supporting the physical interaction and deacetylase activity, HDAC3 repressed MEF2-dependent transcription and inhibited myogenesis. These results reveal an unexpected role for HDAC3 and suggest a novel pathway through which MEF2 activity is controlled in vivo.

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Imai, Yoichi, Mitsuhito Hirano, Masayuki Kobayashi, Muneyoshi Futami, and Arinobu Tojo. "HDAC Inhibitors Exert Anti-Myeloma Effects through Multiple Modes of Action." Cancers 11, no. 4 (April 4, 2019): 475. http://dx.doi.org/10.3390/cancers11040475.

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HDACs are critical regulators of gene expression that function through histone modification. Non-histone proteins and histones are targeted by these proteins and the inhibition of HDACs results in various biological effects. Moreover, the aberrant expression and function of these proteins is thought to be related to the pathogenesis of multiple myeloma (MM) and several inhibitors have been introduced or clinically tested. Panobinostat, a pan-HDAC inhibitor, in combination with a proteasome inhibitor and dexamethasone has improved survival in relapsing/refractory MM patients. We revealed that panobinostat inhibits MM cell growth by degrading the protein PPP3CA, a catalytic subunit of calcineurin. This degradation was suggested to be mediated by suppression of the chaperone function of HSP90 due to HDAC6 inhibition. Cytotoxicity due to the epigenetic regulation of tumor-associated genes by HDAC inhibitors has also been reported. In addition, HDAC6 inhibition enhances tumor immunity and has been suggested to strengthen the cytotoxic effects of therapeutic antibodies against myeloma. Furthermore, therapeutic strategies to enhance the anti-myeloma effects of HDAC inhibitors through the addition of other agents has been intensely evaluated. Thus, the treatment of patients with MM using HDAC inhibitors is promising as these drugs exert their effects through multiple modes of action.

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Berger, Allison J., Bret Bannerman, Steven N. Quayle, Jie Yu, Khristofer Garcia, Jeffrey Ciavarri, David Tamang, Min Yang, and Simon S. Jones. "Preclinical Combination Of The Oral Investigational Agents ACY-1215, a Selective HDAC6 Inhibitor, and Ixazomib, a Proteasome Inhibitor, Demonstrates Combination Benefit In Multiple Myeloma Cell Lines and Xenograft Models." Blood 122, no. 21 (November 15, 2013): 4437. http://dx.doi.org/10.1182/blood.v122.21.4437.4437.

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Introduction The combination of HDAC inhibitors and proteasome inhibitors has demonstrated preclinical benefit in several settings, including multiple myeloma and lymphoma, and is being explored in clinical trials testing various HDAC inhibitors in combination with proteasome inhibitors. ACY-1215 is an investigational, orally available HDAC6-selective inhibitor that has demonstrated preclinical combination benefit with bortezomib in vitro and in vivo (Santos et al, Blood 2012; 119: 2579). These preclinical studies also support the hypothesis that the improved selectivity of ACY-1215 for HDAC6 over class I HDACs (HDAC1,2,3) may provide an improved tolerability profile compared to pan-HDAC inhibitors, while still providing the anti-myeloma effect of other HDACi/proteasome inhibitor combinations. ACY-1215 is currently in a Phase I/II trial in multiple myeloma with bortezomib (VELCADE) and dexamethasone to test this hypothesis (NCT01323751). Ixazomib citrate (MLN9708) is an investigational oral proteasome inhibitor in Phase III clinical trials in multiple myeloma (NCT01850524, NCT01564537). To examine the potential efficacy of the all-oral combination of ixazomib citrate and ACY-1215, we evaluated the combination of these agents in cell lines and xenograft models of multiple myeloma. Results In vitro viability experiments in 2 multiple myeloma cell lines (RPMI-8226 and MM.1S) using a dose matrix format demonstrated a combination benefit of ACY-1215 and ixazomib over a range of concentrations, very similar to the previously reported benefit of ACY-1215 plus bortezomib. Likewise, the combination benefit of the selective HDAC6 inhibitor ACY-1215 with ixazomib was similar to the combination effect observed with the pan-HDAC inhibitor SAHA (vorinostat). Together, these in vitro studies support the hypothesis that the combination of ACY-1215 and ixazomib provides similar levels of benefit as do combinations including other HDACi/proteasome inhibitors. Furthermore, experiments in MM.1S xenograft-bearing mice demonstrated an in vivo combination benefit of ACY-1215 and ixazomib. An all-oral regimen was well tolerated when ACY-1215 was dosed at 100 mg/kg PO twice daily for 5 days per week in combination with ixazomib dosed at 5 mg/kg PO twice weekly, and the combination regimen demonstrated additive antitumor activity (Figure 1). The in vivo combination benefit of ACY-1215 and ixazomib was further demonstrated in MM.1S xenograft-bearing mice using alternate routes of administration (IV dosing of ixazomib and IP dosing of ACY-1215). The combination of ACY-1215 dosed at 30 mg/kg IP once daily for 5 days per week with ixazomib dosed IV at 1.5 mg/kg twice-weekly was also well tolerated and had striking antitumor activity. This combination regimen in fact caused regression of the MM.1S xenograft tumors below the starting volumes, and this level of activity was maintained throughout the entire 17 day dosing period (Figure 2). In an accompanying pharmacodynamic (PD) study of the PO and IP doses of ACY-1215, we confirmed selective HDAC6 inhibition in MM.1S xenograft tumors as evidenced by elevated acetylation levels of the HDAC6 substrate tubulin, with little if any change in the levels of acetylated histone H3, a class I HDAC substrate. In vivo experiments in a second xenograft model, RPMI-8226, also demonstrated a combination benefit of ACY-1215 (30 mg/kg IP for 5 days per week) with ixazomib (0.75 mg/kg IV twice-weekly). Conclusion The combination benefit of ACY-1215 and ixazomib observed here in preclinical experiments utilizing in vitro and in vivo models of multiple myeloma provides rationale for clinical evaluation of this first all-oral combination of a proteasome inhibitor with an HDAC inhibitor. Disclosures: Berger: Takeda Pharmaceutical Company Ltd: Employment. Bannerman:Takeda Pharmaceutical Company Ltd: Employment. Quayle:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yu:Takeda Pharmaceutical Company Ltd: Employment. Garcia:Takeda Pharmaceutical Company Ltd: Employment. Ciavarri:Takeda Pharmaceutical Company Ltd: Employment. Tamang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership.

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Ren, Jingjing, Xiaofeng Liao, Miranda Vieson, Kristin Eden, Miao Chen, Reilly Scott, Jillian Kazmierczak, Xin M. Luo, and Christopher M. Reilly. "Selective HDAC6 inhibitor decreases lupus in mice." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 224.19. http://dx.doi.org/10.4049/jimmunol.198.supp.224.19.

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Abstract Histone deacetylase 6 (HDAC6) is a Class IIb HDACs enzyme primarily residing in the cytoplasm. HDAC6 alters gene transcription by removing acetyl groups from lysine residues on transcription factors. We, and others, have previously demonstrated that HDAC6 expression is increased in systemic lupus erythematosus (SLE) patients and animal models of lupus and that inhibition of HDAC6 decreased disease. In our current studies, we tested if an orally active selective HDAC6 inhibitor would decrease disease pathogenesis in a lupus mouse model with established early disease. Importantly, we sought to delineate the cellular and molecular mechanism(s) of action for the HDAC6 inhibitor. We treated 20-week-old early-diseased NZB/W F1 (lupus) female mice with two different doses of the selective HDAC6 inhibitor (ACY-738) for five weeks. At the termination of the study, our results showed a reduced germinal center B cell response, decreased T follicular helper cells and diminished IFN-g production from T helper cells in splenic tissue. Additionally, we found the IFNa-producing ability of plasmacytoid dendritic cells was decreased along with immunoglobulin isotype switching and the generation of pathogenic autoantibodies. Renal tissue showed decreased immunoglobulin deposition and reduced inflammation as judged by glomerular and interstitial inflammation. Taking together, these studies show selective HDAC inhibition decreased several parameters of disease pathogenesis in lupus prone mice. The decrease was in part due to inhibition of autoreactive B cell differentiation. Studies are currently underway to define further the mechanism of how HDAC6 inhibits auto-reactive B cell differentiation.

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Sharma, Chiranjeev, Yong Jin Oh, Byoungduck Park, Sooyeun Lee, Chul-Ho Jeong, Sangkil Lee, Ji Hae Seo, and Young Ho Seo. "Development of Thiazolidinedione-Based HDAC6 Inhibitors to Overcome Methamphetamine Addiction." International Journal of Molecular Sciences 20, no. 24 (December 9, 2019): 6213. http://dx.doi.org/10.3390/ijms20246213.

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Thiazolidinedione is a five-membered heterocycle that is widely used in drug discovery endeavors. In this study, we report the design, synthesis, and biological evaluation of a series of thiazolidinedione-based HDAC6 inhibitors. In particular, compound 6b exerts an excellent inhibitory activity against HDAC6 with an IC50 value of 21 nM, displaying a good HDAC6 selectivity over HDAC1. Compound 6b dose-dependently induces the acetylation level of α-tubulin via inhibition of HDAC6 in human neuroblastoma SH-SY5Y cell line. Moreover, compound 6b efficiently reverses methamphetamine-induced morphology changes of SH-SY5Y cells via regulating acetylation landscape of α-tubulin. Collectively, compound 6b represents a novel HDAC6-isoform selective inhibitor and demonstrates promising therapeutic potential for the treatment of methamphetamine addiction.

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Lagosz, K. B., A. Bysiek, J. M. Macina, G. P. Bereta, M. Kantorowicz, W. Lipska, M. Sochalska, et al. "HDAC3 Regulates Gingival Fibroblast Inflammatory Responses in Periodontitis." Journal of Dental Research 99, no. 1 (November 6, 2019): 98–106. http://dx.doi.org/10.1177/0022034519885088.

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Histone deacetylases (HDACs) are important regulators of gene expression that are aberrantly regulated in several inflammatory and infectious diseases. HDAC inhibitors (HDACi) suppress inflammatory activation of various cell types through epigenetic and non-epigenetic mechanisms, and ameliorate pathology in a mouse model of periodontitis. Activation of gingival fibroblasts (GFs) significantly contributes to the development of periodontitis and the anaerobic bacterium Porphyromonas gingivalis plays a key role in driving chronic inflammation. Here, we analyzed the role of HDACs in inflammatory responses of GFs. Pan-HDACi suberoylanilide hydroxamic acid (SAHA) and/or ITF2357 (givinostat) significantly reduced TNFα- and P. gingivalis–inducible expression and/or production of a cluster of inflammatory mediators in healthy donor GFs ( IL1B, CCL2, CCL5, CXCL10, COX2, and MMP3) without affecting cell viability. Selective inhibition of HDAC3/6, but not specific HDAC1, HDAC6, or HDAC8 inhibition, reproduced the suppressive effects of pan-HDACi on the inflammatory gene expression profile induced by TNFα and P. gingivalis, suggesting a critical role for HDAC3 in GF inflammatory activation. Consistently, silencing of HDAC3 expression with siRNA largely recapitulated the effects of HDAC3/6i on mRNA levels of inflammatory mediators in P. gingivalis–infected GFs. In contrast, P. gingivalis internalization and intracellular survival in GFs remained unaffected by HDACi. Activation of mitogen-activated protein kinases and NFκB signaling was unaffected by global or HDAC3/6-selective HDACi, and new protein synthesis was not required for gene suppression by HDACi. Finally, pan-HDACi and HDAC3/6i suppressed P. gingivalis–induced expression of IL1B, CCL2, CCL5, CXCL10, MMP1, and MMP3 in GFs from patients with periodontitis. Our results identify HDAC3 as an important regulator of inflammatory gene expression in GFs and suggest that therapeutic targeting of HDAC activity, in particular HDAC3, may be clinically beneficial in suppressing inflammation in periodontal disease.

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Laino, Andressa Sodre, David M. Woods, Fengdong Cheng, Hongwei Wang, and Eduardo M. Sotomayor. "Histone Deacetylase 6 (HDAC6) Influences T-Cell Activation and Survival: Implications For Cancer Immunotherapy." Blood 122, no. 21 (November 15, 2013): 1050. http://dx.doi.org/10.1182/blood.v122.21.1050.1050.

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Abstract The role of histone deacetylases (HDACs) as epigenetic regulators of immune function is becoming increasingly clear. Recently, the role of specific HDACs in orchestrating T-cell maturation, survival and function has begun to emerge, giving rationale to selective therapy to direct immune responses in different disease settings, including cancer. In particular, HDAC6 has recently been characterized as a negative regulator of regulatory T-cell suppressive activity (de Zoeten, Molecular and Cellular Biology, 2011). Here we report an expanded, novel role of HDAC6 in regulating T-cell survival and activation. First, the relative expression of the eleven classic HDACs was evaluated in resting and activated T-cells from mouse and human samples. It was found that the majority of HDACs decrease in expression following activation, including HDAC6. Next, in a HDAC6KO mouse model, it was found that T-cells lacking HDAC6 had skewed survival when compared to wild-type murine T-cells. This difference seems to be the result of an increased CD4+ T-cells population in the lymph nodes, with a concomitant decrease in viable CD8+ T-cells. To determine whether this population skewing was the consequence of defects in HDAC6KO mice T-cell development, wild-type murine T-cells were treated with an isotype-selective HDAC6 inhibitor. The results seen in HDAC6KO T-cells were recapitulated when wild-type T-cells were activated and treated with HDAC6 specific inhibitors, indicating a role of HDAC6 outside of thymic development in promoting CD4+ T-cell survival at the expense of CD8+ T-cells. Interestingly, it was found that activated CD4+ T-cells displayed decreased expression of the apoptosis signaling receptor FAS after HDAC6 inhibition while no differences were observed in CD8+ T-cells under the same conditions. In addition to these results implicating HDAC6 in regulating T-cell survival, expression of surface markers was altered in both CD8+ and CD4+ T-cells, including enhanced expression of the activation molecule CD69 in stimulated T-cells treated with an isotype-selective HDAC6 inhibitor. Finally, in vivo studies in tumor-bearing HDAC6KO mice revealed a significantly delayed in tumor progression. Similar results were observed in lymphoma-bearing mice treated with HDAC6 specific inhibitors. Taken together, this data shows that HDACs are dynamic in expression with regards to T-cell activation state. More specifically, we have unveiled hereto-unexplored roles of HDAC6 in regulating T-cell survival and function, pointing at this specific HDAC as an appealing target to harness T-cell immunity in hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

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Holt, Jason A., Edward P. Garvey, J. David Becherer, William J. Hoekstra, Robert J. Schotzinger, and Christopher M. Yates. "SE-7552, a Highly Selective, Non-Hydroxamate Inhibitor of Histone Deacetylase-6 Blocks Multiple Myeloma Growth In Vivo." Blood 132, Supplement 1 (November 29, 2018): 3215. http://dx.doi.org/10.1182/blood-2018-99-113066.

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Abstract Despite recent advances, disease progression and treatment resistance in multiple myeloma (MM) remains a significant challenge. Current therapies include proteasome inhibitors, immunomodulatory agents, monoclonal antibodies and more recently histone deacetylase (HDAC) inhibitors. Many HDAC inhibitors have progressed into clinical development, however, there has been limited success with the approval of only three pan-HDAC inhibitors (SAHA, belinostat and panobinostat) and the class I selective inhibitor, romidepsin. A challenge to the development of HDAC inhibitors has been the management of toxicities, many of which are dose limiting. Some of these toxicities can be attributed to the use of the hydroxamate, a potent zinc binding group that has been associated with toxicity and poor pharmacokinetic (PK) properties. The very tight binding potency of the hydroxamate to zinc also leads to limited HDAC isoform selectivity and thus further predisposes to toxicity. HDAC6, a member of the class IIb HDAC family, is a target of high interest and has significant therapeutic potential in oncology, particularly MM. It has a unique protein structure and is predominantly located in the cytoplasm where it has multiple non-histone protein substrates including α-tubulin, HSP90, cortactin, and Foxp3. Here, we describe the identification of a highly selective, non-hydroxamate HDAC6 inhibitor with excellent PK properties capable of inhibiting MM growth in vivo. Extensive medicinal chemistry exploration and optimization led to the discovery of a zinc-binding group that was associated with excellent potency and selectivity for HDAC6. Additional optimization of the chemical scaffold led to the identification of SE-7552, a compound with an IC50 of 33nM against HDAC6 and greater than 850-fold selectivity versus all other known HDAC isozymes. SE-7552 demonstrated superior PK compared to hydroxamate-based HDAC inhibitors, with a maximum exposure of 597 ng/ml and a half-life of 7.2 hours after a single oral dose of 5 mg/kg in the mouse. To characterize inhibition of HDAC6 in vivo, acetylated α-tubulin (a biomarker for HDAC6 inhibition) was measured in the spleen of mice treated with SE-7552 or the hydroxamate-based HDAC6 inhibitor, ricolinostat. After a single oral dose of SE-7552 at 30 mg/kg, levels of acetylated α-tubulin were increased for over 24 hours, whereas ricolinostat at a 50 mg/kg IP dose showed increased levels for less than 8 hours. In the same study, SE-7552 had no effect on the acetylation of H3 (a biomarker for inhibition of Class I HDACs), whereas ricolinostat increased the levels of acetylated H3. Based on the positive in vitro and in vivo profile, SE-7552 was progressed into pre-clinical rodent models of MM. Since previously studied HDAC6 inhibitors were not able to significantly inhibit MM growth as monotherapy, SE-7552 was co-administered with either pomalidomide or bortezomib. In a subcutaneous MM model using human H929 MM cells, SE-7552 dosed orally at 10 mg/kg daily in combination with pomalidomide dosed at 1 mg/kg IP daily significantly delayed tumor growth in comparison to pomalidomide alone (p < 0.01), as well as enhanced the survival of the mice. Utilizing human MM.1S cells that can be quantified by luminescence, a disseminated model of MM was conducted in which SE-7552 dosed orally at 10 mg/kg daily in combination with bortezomib dosed at 1.5 mg/kg IP once a week significantly delayed tumor growth compared to bortezomib alone (p < 0.05). In summary, we have identified a non-hydroxamate HDAC6 inhibitor, SE-7552, with superior selectivity and pharmacokinetics compared to current HDAC6 inhibitors. SE-7552 demonstrated high levels of selectivity in an in vivo biomarker study and blocked MM growth in two pre-clinical models when co-administered with current MM therapies. Disclosures Holt: Selenity Therapeutics: Employment, Equity Ownership, Patents & Royalties: METALLOENZYME INHIBITOR COMPOUNDS, U.S. Patent Application No.: 15/917,555 (March 9, 2018). Garvey:Selenity Therapeutics: Employment, Equity Ownership. Becherer:Selenity Therapeutics: Employment, Equity Ownership. Hoekstra:Selenity Therapeutics: Employment, Equity Ownership, Patents & Royalties: METALLOENZYME INHIBITOR COMPOUNDS, U.S. Patent Application No.: 15/917,555 (March 9, 2018). Schotzinger:Mycovia Pharma: Membership on an entity's Board of Directors or advisory committees; Innocrin Pharma: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Selenity Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: METALLOENZYME INHIBITOR COMPOUNDS, U.S. Patent Application No.: 15/917,555 (March 9, 2018). Yates:Selenity Therapeutics: Employment, Equity Ownership, Patents & Royalties: METALLOENZYME INHIBITOR COMPOUNDS, U.S. Patent Application No.: 15/917,555 (March 9, 2018).

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Shen, Xiaokun, and Zeng Li. "Abstract P3-05-05: Preclinical evaluation of novel HDAC6 selective inhibitors N008 with potent in vitroandin vivoprofiles in non-solid tumor and solid tumors." Cancer Research 82, no. 4_Supplement (February 15, 2022): P3–05–05—P3–05–05. http://dx.doi.org/10.1158/1538-7445.sabcs21-p3-05-05.

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Abstract HDAC inhibitors are an emerging class of anticancer agents with the approved indications of multiple myeloma, cutaneous and/or peripheral T cell lymphoma. However, non-selective pan-HDAC inhibitors are often reported associated with heart, hematologic and gastrointestinal toxicities. Among HDAC subtypes, inhibiting HDAC6 selectively may not only enhance potency, but may also reduce the toxicity related to off-target effects of pan-HDACis. In previous studies, pioneer HDAC6 selective inhibitors have been demonstrated with the potential to treat both non-solid tumor and solid tumors along or in combination with chemotherapy, proteasome inhibitors or checkpoint inhibitors. Here, we report the design, in vitro and in vivo evaluation of a class of novel HDAC6 selective inhibitors, series N008, with potent antitumor profiles in non-solid tumor such as multiple myeloma, and solid tumors such as breast cancer. Results:1. HDAC enzymes activity, selectivity and cell assaysAccording to the HDAC Caliper assay, N008 series compounds were identified as HDAC6 selective inhibitors bearing 5-20X selectivity over HDAC3, 20-100X selectivity over HDAC1 and HDAC8, with the HDAC6 potency upto <5 nM. The compounds exhibited a priority selectivity than clinical candidates ACY-1215 and ACY-241.Lead compounds inhibited non-solid tumor such as MM.1S, REC-1, and SK-MEL-5 with the IC50 up to 1 micromole, which were equal or better compared with positive control ACY-241 (2-5 micromole). For Breast cancer cell line MCF-7 and MDA-MB-231, exemplified molecules performed higher cytotoxicity (1-5 micromole) over ACY-241 (5-15 micromole). 2. Drug-like profilesExemplified compounds were tested to demonstrate no cytochrome P450 inhibition (IC50 > 25 µm), no hERG inhibition (@ 40 μM). Moreover, these novel molecules showed better metabolic stability in mouse, rat and human liver microsome (MF%>80%) compared with ACY-241 (MF%: 50-70%). Exemplified molecules were identified not P-gp substrates with high permeability in MDRI-MDCKII and potential BBB penetrability. Taken all results together, this series compounds contribute a predominant drug-like advantages than current clinical candidates.3. Xenograft studyIn MM.1s CDX models, lead compounds N008209 and N008219 were more potent than ACY-241 at 30mg/kg in tumor proliferation as monotherapy. N008209 and N008219 in combination with Bortezomib (0.5 mg/kg) significate inhibit tumor growth compared with monotherapy and ACY-241+Bortezomib (p<0.05). N008 monotherapy and in combination with PTX in MCF-7 CDX, and in combination with PD-1 inhibitor in B16F10 CDX models are under investigating. Moreover, the two compounds possessed very promising safety profiles in both monotherapy and combination therapy. Among all groups, no animal was dead and no body weight was declined over 5% in 24-days treatment. Conclusions:Compounds of series N008 exhibited a priority potency than the clinical candidate ACY-241 on HDAC6 inhibition and selectivity with promising drug-like profiles. In CDX models, lead compounds possessed better in vivo efficacy than ACY-241 in both monotherapy and combination therapy with bortezomib, as well as very promising safety profiles. These data reveal optimistic expectation for the clinical development of novel HDAC6 selective inhibitors on anti-cancer indications, especially on both non-solid and solid tumors such as multiple myeloma and breast cancer. Lead compound is expected to enter clinical study in 2022. Citation Format: Xiaokun Shen, Zeng Li. Preclinical evaluation of novel HDAC6 selective inhibitors N008 with potent in vitroandin vivoprofiles in non-solid tumor and solid tumors [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-05-05.

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Bulut, Ipek, Adam Lee, Buse Cevatemre, Dusan Ruzic, Roman Belle, Akane Kawamura, Sheraz Gul, Katarina Nikolic, A. Ganesan, and Ceyda Acilan. "Dual LSD1 and HDAC6 Inhibition Induces Doxorubicin Sensitivity in Acute Myeloid Leukemia Cells." Cancers 14, no. 23 (December 6, 2022): 6014. http://dx.doi.org/10.3390/cancers14236014.

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Defects in epigenetic pathways are key drivers of oncogenic cell proliferation. We developed a LSD1/HDAC6 multitargeting inhibitor (iDual), a hydroxamic acid analogue of the clinical candidate LSD1 inhibitor GSK2879552. iDual inhibits both targets with IC50 values of 540, 110, and 290 nM, respectively, against LSD1, HDAC6, and HDAC8. We compared its activity to structurally similar control probes that act by HDAC or LSD1 inhibition alone, as well as an inactive null compound. iDual inhibited the growth of leukemia cell lines at a higher level than GSK2879552 with micromolar IC50 values. Dual engagement with LSD1 and HDAC6 was supported by dose dependent increases in substrate levels, biomarkers, and cellular thermal shift assay. Both histone methylation and acetylation of tubulin were increased, while acetylated histone levels were only mildly affected, indicating selectivity for HDAC6. Downstream gene expression (CD11b, CD86, p21) was also elevated in response to iDual treatment. Remarkably, iDual synergized with doxorubicin, triggering significant levels of apoptosis with a sublethal concentration of the drug. While mechanistic studies did not reveal changes in DNA repair or drug efflux pathways, the expression of AGPAT9, ALOX5, BTG1, HIPK2, IFI44L, and LRP1, previously implicated in doxorubicin sensitivity, was significantly elevated.

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Hanemann, Clemens Oliver, and Juri Na. "EXTH-90. HDAC INHIBITOR, CAY10603, IS A POTENT RADIOSENSITIZER IN MENINGIOMAS." Neuro-Oncology 24, Supplement_7 (November 1, 2022): vii230. http://dx.doi.org/10.1093/neuonc/noac209.888.

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Abstract Developing and evaluating potential radiosensitisers to enhance therapeutic efficacy are urgently needed due to the lack of efficient chemotherapy for meningioma. Histone deacetylase (HDACs) expression is generally increased in many cancer types and regulate the expression of numerous proteins involved in tumorigenesis. Targeting HDAC using HDAC inhibitor (HDACi) represent promising radiosensitisers that affect various biological processes, such as cell survival, apoptosis, and DNA repair. Recently, one of the HDAC inhibitor, AR42 monotherapy trials for patients with neurofibromatosis type2 started Nov 2021. However, we were not able to detect the actual HDAC downregulation after applying AR42 (0.5-2 μM) in immortalised meningioma. Hence, we investigated whether pretreatment with the hydroxamate-based HDAC6 inhibitor, Cay10603, impacts HDAC expression, radiation-induced DNA double-strand break (DSB) induction, cell survival, and cell cycle arrest in both immortalised and human meningioma cell lines. Cay10603 at nanomolar level (100 nM) was treated prior to high energy x-ray irradiation (2 Gy) by a medical linear accelerator (LINAC). We found that surviving rate was synergistically decreased after combination treatment of Cay10603 and radiation. Combination therapy induced DNA damage with activation of histone gH2AX and increased G2/M arrest compared to drug or radiation alone. To focus on the mechanisms of action of HDAC6 inhibition followed by radiation, we further investigated nuclear localisation of beta-catenin levels. The results showed the beta-catenin in the nucleus was suppressed after combination therapy. Our findings demonstrate a potential therapeutic strategy of Cay10603 to improve the radiosensitization for meningiomas.

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Harada, Takeshi, Asuka Oda, Hiroto Ohguchi, Yohann Grondin, Hirofumi Tenshin, Masahiro Hiasa, Jumpei Teramachi, et al. "Novel Therapeutic Rationale for Targeting HDAC1 and PIM2 in Multiple Myeloma." Blood 134, Supplement_1 (November 13, 2019): 3111. http://dx.doi.org/10.1182/blood-2019-127679.

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Multiple myeloma (MM) cells preferentially grow and expand in the bone marrow (BM) to elicit the alteration of gene expression thereby acquiring drug resistance. The serine/threonine kinase PIM2 is constitutively overexpressed which is further up-regulated as a critical anti-apoptotic mediator in MM cells by interacting with BM stromal cells (BMSCs) and/or osteoclasts (Leukemia 2011, 2015). Histone deacetylases (HDACs) generally repress gene expression through deacetylation of lysine residues in histone tails. Therefore, HDAC inhibitors are able to restore the expression of tumor suppressor genes, and utilized as anti-cancer agents for various types of malignancies, including MM. Importantly, class-I and a class-IIb (HDAC6) HDACs have been shown as important therapeutic targets in MM (Nat Chem Biol 2010). Among class-I HDAC isoforms, HDAC1 and HDAC3 are highly expressed in MM cells (GSE5900 and GSE2113) and we have already reported that the HDAC3-DNMT1 axis is a critical therapeutic target (Leukemia 2017). However, the significance of HDAC1 expression in MM cell growth and survival is still largely unknown. In the present study, we aimed to clarify the epigenetic regulation of PIM2 and the therapeutic implication of HDAC1 in MM cells. We observed that HDAC1- and HDAC3-selective inhibitor MS-275 (Entinostat) inhibited MM cell growth in a dose-dependent fashion. HDAC1 knockdown using a lentiviral shRNA system induced apoptosis in MM cell lines, indicating a crucial role of HDAC1 in MM cell growth and survival. To identify downstream targets of HDAC1 mediating MM cell survival, we next carried out RNA-Seq using RPMI 8226 cells after HDAC1 knockdown. Expression of a number of genes were altered (adjusted P values < 0.05, log fold change > 0.5). Among these genes, we found that PIM2 and IRF4 were significantly downregulated in HDAC1 knocked down cells. The downregulation of IRF4 and PIM2 was further confirmed at mRNA and protein levels in additional MM cell lines. It has been shown that MS-275 impaired the viability of primary MM cells associated with downregulation of IRF4 and PIM2 expression. However, importantly, HDAC1 knocked down-induced growth inhibition was not observed in RPMI8226 cells with IRF4 overexpression, indicating that IRF4 is a key MM cell survival mediator targeted by HDAC1 inhibition. Previous study shows that HDAC1 is abundantly enriched around at H3K27 acetylation or RNA Pol II- binding sites compared to HDAC2 or HDAC3 (GSE86450), However, our data assessed by ChIP-Seq indicated that HDAC1-occupied genes were not completely upregulated but rather downregulated in HDAC1-knockdown cells. Indeed, MS-275 and a histone acetyltransferase inhibitor C646 downregulated IRF4 and PIM2 expression in MM cells despite upregulation and downregulation of histone H3 acetylation, respectively. The ChIP-Seq data showed HDAC1 binding is enriched around the promotor regions of IRF4 and PIM2 in MM cells; however, MS-275 significantly reduced the HDAC1 enrichment as determined in ChIP-Q-PCR assays, suggesting that IRF4 and PIM2 expression is regulated by the balance between acetylation and deacetylation status of histones in MM cells. In addition, we found that IRF4 binds to the promoter of PIM2 and IRF4 knockdown reduced PIM2 expression, suggesting that IRF4 transcriptionally regulates PIM2. Although PIM2 expression is robustly upregulated in MM cells in an ambient microenvironment with BMSCs and/or osteoclasts, MS-275 and the PIM inhibitor SMI-16a cooperatively induce MM cell death. In conclusion, our data provides a basis of rationale combination strategy targeting of class-I HDAC and PIM2 to improve MM patient outcome. Disclosures Anderson: Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board.

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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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Sixto-López, Yudibeth, Martha Cecilia Rosales-Hernández, Arturo Contis-Montes de Oca, Leticia Guadalupe Fragoso-Morales, Jessica Elena Mendieta-Wejebe, Ana María Correa-Basurto, Edgar Abarca-Rojano, and José Correa-Basurto. "N-(2′-Hydroxyphenyl)-2-Propylpentanamide (HO-AAVPA) Inhibits HDAC1 and Increases the Translocation of HMGB1 Levels in Human Cervical Cancer Cells." International Journal of Molecular Sciences 21, no. 16 (August 16, 2020): 5873. http://dx.doi.org/10.3390/ijms21165873.

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N-(2′-hydroxyphenyl)-2-propylpentanamide (HO-AAVPA) is a VPA derivative designed to be a histone deacetylase (HDAC) inhibitor. HO-AAVPA has better antiproliferative effect than VPA in cancer cell lines. Therefore, in this work, the inhibitory effect of HO-AAVPA on HDAC1, HDAC6, and HDAC8 was determined by in silico and in vitro enzymatic assay. Furthermore, its antiproliferative effect on the cervical cancer cell line (SiHa) and the translocation of HMGB1 and ROS production were evaluated. The results showed that HO-AAVPA inhibits HDAC1, which could be related with HMGB1 translocation from the nucleus to the cytoplasm due to HDAC1 being involved in the deacetylation of HMGB1. Furthermore, an increase in ROS production was observed after the treatment with HO-AAVPA, which also could contribute to HMGB1 translocation. Therefore, the results suggest that one of the possible antiproliferative mechanisms of HO-AAVPA is by HDAC1 inhibition which entails HMGB1 translocation and ROS increased levels that could trigger the cell apoptosis.

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Hess, Lena, Verena Moos, Arnel A. Lauber, Wolfgang Reiter, Michael Schuster, Natascha Hartl, Daniel Lackner, et al. "A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation." PLOS Genetics 18, no. 8 (August 22, 2022): e1010376. http://dx.doi.org/10.1371/journal.pgen.1010376.

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The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimicks class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.

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Masselli, Elena, Lilian Varricchio, Barbara Ghinassi, Carolyn Whitsett, Patricia A. Shi, and Anna Rita F. Migliaccio. "Class IIa HDAC Inhibitors Reduce HDAC1 Activity by off-Target Effects Which Reduce GATA1 Expression In Human Erythroblasts Expanded Ex-Vivo." Blood 116, no. 21 (November 19, 2010): 4780. http://dx.doi.org/10.1182/blood.v116.21.4780.4780.

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Abstract Abstract 4780 Histone deacetylation maintains chromatin in a condensed configuration preventing gene expression in eukaryotic cells. The deacetylation reaction is catalyzed by enzymes of the histone deacetylase (HDAC) superfamily, which perform their functions as multiprotein complexes including at least 2 HDAC isoforms, DNA docking factors (transcription factors and methyl-binding proteins) and protein kinases (PKC and Erk). The well established role of HDACs in gene silencing has suggested studies to identify HDAC inhibitors (HDACi) that, by re-activating γ-globin expression, might treat the anemia due to insufficient β-globin expression (Cao et al Blood 103:701, 2004). Over the years several HDACi have been documented to induce γ-globin expression in human erythroid cultures, adult baboons, and β-thalassemia and sickle cell patients. Among those, Class I HDACi, and in particular those that inhibit HDAC3, appear to be more potent as γ-globin gene activators (Mankidy et al, Blood 108:3179, 2006). We have recently identified two new HDACi (compound 9 and 24) which both improved maturation and reactivated γ-globin expression in β°-thalassemic erythroblasts in vitro (Mai et al Mol Pharmacol 72:111, 2007). Compound 24 inhibits both class I (HDAC1 ID50 =0.2 μ M) and class IIa (HDAC4 ID50=0.6 μ M) HDAC. Compound 9 is a class IIa specific inhibitor (HDAC4 ID50=20 μ M) and does not affect HDAC1 activity but is a more potent γ-globin inducer than compound 24. This observation suggests that HDACi may also affect HDAC activity through indirect effects which alter overall complex activity. To clarify possible off-target effects of Class II and Class I/IIa inhibitors and their consequences for erythroid maturation, we analysed expression and activity of different HDAC isoforms during maturation of normal human erythroblasts in vitro at baseline and with treatment with compounds 9 and 24. The proteins studied included GATA1 (the major transcription regulator of erythroid maturation), p21/p27kip1, two cyclin D dependent kinase inhibitors which favor maturation, Caspase 3 (the protease which specifically cleaves GATA1) and Erk (a component of the HDAC complex). During normal erythroid maturation (without HDACi), all the HDAC isoforms were expressed at the mRNA and protein levels. Immunoprecipitation studies followed by determination of HDAC activity indicated that the activities which changed most during maturation are those of HDAC1 (class I), increased by 2-fold, and HDAC5 (class IIa), decreased by 2-fold. In addition, co-immunoprecipitation studies revealed an increase in the association between HDAC1 and GATA1 with erythroid maturation. Changes in the expression of key regulatory proteins were observed with normal erythroid maturation: activation of Caspase 3 decreased with resultant increase in GATA-1, and phosphorylation of pErk decreased while expression of p21 and p27 increased. With exposure to increasing HDACi concentrations (0.2, 2 and 6 μ M), there were class-specific, concentration-dependent alterations in protein expression: compound 9 (Class IIa inhibitor) induced Caspase 3 activation and reduced GATA1 content, while compound 24 decreased Caspase3 activation and greatly increased GATA1 content. In addition, compound 9 did not induce Erk phosphorylation and decreased p21 expression, while compound 24 did induce Erk phosphorylation and inhibited p27 expression (see figure). These results confirm the hypothesis that, in addition to class I inhibitors that directly inhibit class I HDAC, class II HDACi can also affect class I HDAC activity, through indirect effects that involve other components of the complex (repression of GATA1 expression and decrease of Erk phosphorylation). Disclosures: No relevant conflicts of interest to declare.

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Kikuchi, Jiro, Taeko Wada, Rumi Shimizu, Tohru Izumi, Miyuki Akutsu, Kanae Mitsunaga, Kaoru Noborio-Hatano, et al. "Histone deacetylases are critical targets of bortezomib-induced cytotoxicity in multiple myeloma." Blood 116, no. 3 (July 22, 2010): 406–17. http://dx.doi.org/10.1182/blood-2009-07-235663.

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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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Leucker, Thorsten M., Yohei Nomura, Jae Hyung Kim, Anil Bhatta, Victor Wang, Andrea Wecker, Sandeep Jandu, et al. "Cystathionine γ-lyase protects vascular endothelium: a role for inhibition of histone deacetylase 6." American Journal of Physiology-Heart and Circulatory Physiology 312, no. 4 (April 1, 2017): H711—H720. http://dx.doi.org/10.1152/ajpheart.00724.2016.

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Endothelial cystathionine γ-lyase (CSEγ) contributes to cardiovascular homeostasis, mainly through production of H2S. However, the molecular mechanisms that control CSEγ gene expression in the endothelium during cardiovascular diseases are unclear. The aim of the current study is to determine the role of specific histone deacetylases (HDACs) in the regulation of endothelial CSEγ. Reduced CSEγ mRNA expression and protein abundance were observed in human aortic endothelial cells (HAEC) exposed to oxidized LDL (OxLDL) and in aortas from atherogenic apolipoprotein E knockout (ApoE−/−) mice fed a high-fat diet compared with controls. Intact murine aortic rings exposed to OxLDL (50 μg/ml) for 24 h exhibited impaired endothelium-dependent vasorelaxation that was blocked by CSEγ overexpression or the H2S donor NaHS. CSEγ expression was upregulated by pan-HDAC inhibitors and by class II-specific HDAC inhibitors, but not by other class-specific inhibitors. The HDAC6 selective inhibitor tubacin and HDAC6-specific siRNA increased CSEγ expression and blocked OxLDL-mediated reductions in endothelial CSEγ expression and CSEγ promoter activity, indicating that HDAC6 is a specific regulator of CSEγ expression. Consistent with this finding, HDAC6 mRNA, protein expression, and activity were upregulated in OxLDL-exposed HAEC, but not in human aortic smooth muscle cells. HDAC6 protein levels in aortas from high-fat diet-fed ApoE−/− mice were comparable to those in controls, whereas HDAC6 activity was robustly upregulated. Together, our findings indicate that HDAC6 is upregulated by atherogenic stimuli via posttranslational modifications and is a critical regulator of CSEγ expression in vascular endothelium. Inhibition of HDAC6 activity may improve endothelial function and prevent or reverse the development of atherosclerosis. NEW & NOTEWORTHY Oxidative injury to endothelial cells by oxidized LDL reduced cystathionine γ-lyase (CSEγ) expression and H2S production, leading to endothelial dysfunction, which was prevented by histone deacetylase 6 (HDAC6) inhibition. Our data suggest HDAC6 as a novel therapeutic target to prevent the development of atherosclerosis.

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Osko, Jeremy D., and David W. Christianson. "Binding of inhibitors to active-site mutants of CD1, the enigmatic catalytic domain of histone deacetylase 6." Acta Crystallographica Section F Structural Biology Communications 76, no. 9 (August 19, 2020): 428–37. http://dx.doi.org/10.1107/s2053230x20010250.

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The zinc hydrolase histone deacetylase 6 (HDAC6) is unique among vertebrate deacetylases in that it contains two catalytic domains, designated CD1 and CD2. Both domains are fully functional as lysine deacetylases in vitro. However, the in vivo function of only the CD2 domain is well defined, whereas that of the CD1 domain is more enigmatic. Three X-ray crystal structures of HDAC6 CD1–inhibitor complexes are now reported to broaden the understanding of affinity determinants in the active site. Notably, cocrystallization with inhibitors was facilitated by using active-site mutants of zebrafish HDAC6 CD1. The first mutant studied, H82F/F202Y HDAC6 CD1, was designed to mimic the active site of human HDAC6 CD1. The structure of its complex with trichostatin A was generally identical to that with the wild-type zebrafish enzyme. The second mutant studied, K330L HDAC6 CD1, was prepared to mimic the active site of HDAC6 CD2. It has previously been demonstrated that this substitution does not perturb inhibitor binding conformations in HDAC6 CD1; here, this mutant facilitated cocrystallization with derivatives of the cancer chemotherapy drug suberoylanilide hydroxamic acid (SAHA). These crystal structures allow the mapping of inhibitor-binding regions in the outer active-site cleft, where one HDAC isozyme typically differs from another. It is expected that these structures will help to guide the structure-based design of inhibitors with selectivity against HDAC6 CD1, which in turn will enable new chemical biology approaches to probe its cellular function.

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Cao, Biyin, Mingyun Shen, Depei Wu, Jianhong Du, Jingyu Zhu, Suning Chen, Aining Sun, et al. "The Proteasomal Inhibitor Clioquinol Induces Apoptosis in Leukemia and Myeloma Cells by Inhibiting Histone Deacetylase Activity." Blood 120, no. 21 (November 16, 2012): 2449. http://dx.doi.org/10.1182/blood.v120.21.2449.2449.

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Abstract Abstract 2449 Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline, CQ) is an anti-fungal and anti-parasitic drug and is also a strong chelator of divalent metal ions such as zinc, and copper. Recent studies suggested by formation a metal complex, CQ becomes an inhibitor of proteasomes and displays anti-cancer activity in several types of solid cancers including prostate cancers and breast cancers. Although addition of copper and other divalent mental ions increases the activity of CQ in terms of proteasomal inhibition and cell death, our recent studies found that CQ and its analog 5-amino-8-hydroquinoline display potent anti-leukemia and anti-myeloma activity without addition of such metal ions. Because CQ is a potent chelator of zinc that is indispensable for many biological enzymes, such as histone deacetylases (HDACs). HDACs are a class of zinc-dependent enzymes regulating gene expression, cell survival and cell death. We questioned that whether CQ induces apoptosis by inhibiting HDAC activity via interfering with zinc in the active sites of these enzymes. To answer this question, we first analyzed the effects of clioquinol on transcription of HDAC-regulated genes including p21, p27 and p53. After 24 hr treatment, expression of these genes was significantly increased by CQ in a concentration-dependent manner. Consistent with these findings, CQ also induced cell cycle arrest and cell apoptosis, a sign of HDAC inhibition. We then examined HDAC activity by evaluating the expression level of acetylated histone 3 (Ac-H3). As expected, Ac-H3 was increased by CQ in all examined cell lines and bone marrow cells from primary leukemia and myeloma patients. CQ also induced accumulation of acetylated p53 and acetylated HSP90. In the mechanistic study, we further surveyed the effects of CQ on a panel of HDACs, including HDAC-1, −2, −3, −5 and −8, and found that most enzymes but HDAC2 were decreased by CQ at concentrations of 20 mM or higher in both myeloma and leukemia cells. Since CQ increased Ac-H3 at a concentration as low as 5 mM, we wondered whether CQ binds to HDACs thus directly interfering with their activity. To this end, we next screened the effects of CQ on all 11 zinc-dependent HDACs, including Class I (HDAC1, 2, 3, 8), Class 2A (HDAC4, -5, -7, -9) and Class 2B (HDAC6, -10) and Class IV (HDAC11) and measured the values of IC50 to each enzyme. The results showed that CQ had no effects on activities of Class 2B (HDAC6-, -10) and Class IV (HDAC11). Compared with trichostatin, the classic HDAC inhibitor, CQ displayed similar inhibition to Class 2A HDACs, but the IC50 values to Class I HDACs were 1000 fold higher than trichostatin. Thus, CQ probably mainly targeted Class 2A HDACs. To demonstrate this hypothesis, we analyzed the interaction between CQ and HDAC by computer modeling. The result indicated that CQ was well docked into the active pocket of the enzyme, where the oxygen and nitrogen atoms in CQ formed stable coordinate bonds with the zinc ion, and the hydroxyl group from CQ formed an effective hydrogen bond with Asp267. Moreover, CQ formed extensive van der Waals interactions with hydrophobic residues Trp141, Phe152 and Try306. To further verify this prediction, we co-treated cells with CQ and zinc chloride, and found that CQ-induced accumulation of Ac-H3 was attenuated by zinc. Thus, the proteasomal inhibitor CQ can induce apoptosis in leukemia and myeloma by inhibiting the HDAC activity, especially Class 2A enzymes. This study proposed a new line of mechanism for understanding CQ in the treatment of leukemia and myeloma. Disclosures: No relevant conflicts of interest to declare.

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Iaconelli, Jonathan, Lucius Xuan, and Rakesh Karmacharya. "HDAC6 Modulates Signaling Pathways Relevant to Synaptic Biology and Neuronal Differentiation in Human Stem-Cell-Derived Neurons." International Journal of Molecular Sciences 20, no. 7 (March 31, 2019): 1605. http://dx.doi.org/10.3390/ijms20071605.

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Recent studies show that histone deacetylase 6 (HDAC6) has important roles in the human brain, especially in the context of a number of nervous system disorders. Animal models of neurodevelopmental, neurodegenerative, and neuropsychiatric disorders show that HDAC6 modulates important biological processes relevant to disease biology. Pan-selective histone deacetylase (HDAC) inhibitors had been studied in animal behavioral assays and shown to induce synaptogenesis in rodent neuronal cultures. While most studies of HDACs in the nervous system have focused on class I HDACs located in the nucleus (e.g., HDACs 1,2,3), recent findings in rodent models suggest that the cytoplasmic class IIb HDAC, HDAC6, plays an important role in regulating mood-related behaviors. Human studies suggest a significant role for synaptic dysfunction in the prefrontal cortex (PFC) and hippocampus in depression. Studies of HDAC inhibitors (HDACi) in human neuronal cells show that HDAC6 inhibitors (HDAC6i) increase the acetylation of specific lysine residues in proteins involved in synaptogenesis. This has led to the hypothesis that HDAC6i may modulate synaptic biology not through effects on the acetylation of histones, but by regulating acetylation of non-histone proteins.

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Quayle, Steven N., David Tamang, Min Yang, and Simon S. Jones. "Inhibition Of HDAC6 In Combination With Targeted Agents Results In Broad Synergistic Decreases In Viability In Non-Hodgkin’s Lymphoma (NHL) Cells." Blood 122, no. 21 (November 15, 2013): 3071. http://dx.doi.org/10.1182/blood.v122.21.3071.3071.

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Abstract Histone Deacetylase (HDAC) inhibitors have demonstrated significant clinical benefit as single agents in cutaneous and peripheral T cell lymphomas, and have received FDA approval for these indications. Numerous clinical studies are also ongoing to investigate the potential benefit of combining HDAC inhibitors with other standard of care and investigational agents in various subtypes of lymphoma. ACY-1215 is a first-in-class, orally available selective inhibitor of HDAC6 (approximately 11-fold selective over class I HDAC’s) that is currently in two Phase Ib clinical trials (NCT01323751 and NCT01583283) in combination with dexamethasone and either bortezomib or lenalidomide in multiple myeloma (MM) patients. Beyond MM, preclinical studies in NHL cell lines also demonstrated increased benefit from treatment with ACY-1215 in combination with the proteasome inhibitors bortezomib or carfilzomib (Amengual, et al, ASH, 2012; Dasmahapatra, et al, ASH, 2012). In addition to standard of care therapies, a number of novel targeted agents have recently demonstrated potential clinical benefit in subtypes of NHL, including agents targeting Bruton’s tyrosine kinase (eg. ibrutinib) and the phosphatidyl inositol-3’ kinase family (eg. GS-1101, IPI-145, and GDC-0941). We describe here the therapeutic potential of ACY-1215 in a collection of NHL cell lines both as a single agent and in combination with these novel targeted agents. NHL cell lines derived from diffuse large B cell lymphomas (DLBCL; both germinal center and activated B cell type) and mantle cell lymphomas (MCL) exhibit significantly decreased viability in response to treatment with selective inhibitors of HDAC6, including both ACY-1215 and ACY-775 (approximately 300-fold selective for HDAC6 over HDAC1/2, and 1500-fold selective over HDAC3). Furthermore, combination treatment of NHL lines with ACY-1215 and either ibrutinib, GDC-0941, or GS-1101, in a dose-matrix format resulted in synergistic decreases in cell viability. The relevance of HDAC6 inhibition to this synergistic response was confirmed through the observation of similarly decreased viability when treating cells in combination with the highly selective HDAC6 inhibitor ACY-775. Detailed molecular mechanism of action studies are ongoing and will be presented. Additional studies are also underway to assess the tolerability and efficacy of combinations of these targeted agents with ACY-1215 in xenograft models of lymphoma. Overall, these data support the continued investigation of the activity of ACY-1215 in lymphoma both as a single agent and in combination with the next generation of targeted therapies for B cell lymphomas. Disclosures: Quayle: Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Tamang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership.

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Wen, Zhi-Hong, Jhy-Shrian Huang, Yen-You Lin, Zhi-Kang Yao, Yu-Cheng Lai, Wu-Fu Chen, Hsin-Tzu Liu, et al. "Chondroprotective Effects of a Histone Deacetylase Inhibitor, Panobinostat, on Pain Behavior and Cartilage Degradation in Anterior Cruciate Ligament Transection-Induced Experimental Osteoarthritic Rats." International Journal of Molecular Sciences 22, no. 14 (July 7, 2021): 7290. http://dx.doi.org/10.3390/ijms22147290.

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Osteoarthritis (OA) is the most common articular degenerative disease characterized by chronic pain, joint inflammation, and movement limitations, which are significantly influenced by aberrant epigenetic modifications of numerous OA-susceptible genes. Recent studies revealed that both the abnormal activation and differential expression of histone deacetylases (HDACs) might contribute to OA pathogenesis. In this study, we investigated the chondroprotective effects of a marine-derived HDAC inhibitor, panobinostat, on anterior cruciate ligament transection (ACLT)-induced experimental OA rats. The intra-articular administration of 2 or 10 µg of panobinostat (each group, n = 7) per week from the 6th to 17th week attenuates ACLT-induced nociceptive behaviors, including secondary mechanical allodynia and weight-bearing distribution. Histopathological and microcomputed tomography analysis showed that panobinostat significantly prevents cartilage degeneration after ACLT. Moreover, intra-articular panobinostat exerts hypertrophic effects in the chondrocytes of articular cartilage by regulating the protein expressions of HDAC4, HDAC6, HDAC7, runt-domain transcription factor-2, and matrix metalloproteinase-13. The study indicated that HDACs might have different modulations on the chondrocyte phenotype in the early stages of OA development. These results provide new evidence that panobinostat may be a potential therapeutic drug for OA.

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Journal articles on the topic 'Inhibitor of HDAC6'

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