Selective Histone Deacetylase Inhibitors Research Articles

Selective HDAC6 inhibitor TubA offers neuroprotection after intracerebral hemorrhage via inhibiting neuronal apoptosis.

A large body of evidence has demonstrated that neuronal apoptosis is involved in the pathological process of secondary brain injury following intracerebral hemorrhage (ICH). Additionally, our previous studies determined that the inhibition of HDAC6 activity by tubacin or specific shRNA can attenuate neuronal apoptosis in an oxygen-glucose deprivation reperfusion model. However, whether the pharmacological inhibition of HDAC6-attenuated neuronal apoptosis in ICH remains unclear. In this study, we used hemin-induced SH-SY5Y cells to simulate a hemorrhage state in vitro and adopted a collagenase-induced ICH rat model in vivo to assess the effect of the HDAC6 inhibition. We found a significant increase in HDAC6 during the early stages of ICH. As expected, the acetylated α-tubulin significantly decreased in correlation with the expression of HDAC6. Medium and high doses (25, 40 mg/kg) of TubA, a selective inhibitor of HDAC6, both reduced neurological impairments, histological impairments, and ipsilateral brain edema in vivo. TubA or HDAC6 siRNA both alleviated neuronal apoptosis in vivo and in vitro. Finally, HDAC6 inhibition increased the level of acetylated α-tubulin and Bcl-2 and lowered the expression of Bax and cleaved caspase-3 post-ICH. In general, these results suggested that the pharmacological inhibition of HDAC6 may act as a novel and promising therapeutic target for ICH therapy by up-regulating acetylated α-tubulin and reducing neuronal apoptosis.

Dual targeted 2-Benzylideneindanone pendant hydroxamic acid group exhibits selective HDAC6 inhibition along with tubulin stabilization effect

Abnormal epigenetics has been recognised as an early event in tumour progression and aberrant acetylation of lysine in particular has been understood in tumorigenesis. Therefore, it has become an attractive target for anticancer drug development. However, HDAC inhibitors have limited success due to toxicity and drug resistance concerns. Present study deals with design and synthesis of bivalent indanone based HDAC6 and antitubulin ligands as anticancer agents. Two of the analogues 9 and 21 exhibited potent antiproliferative activities (IC50, 0.36–3.27 µM) and high potency against HDAC 6 enzyme. Compound 21 showed high selectivity against HDAC 6 while 9 exhibited low selectivity. Both the compounds also showed microtubule stabilization effects and moderate anti-inflammatory effect. Dual targeted anticancer agents with concomitant anti-inflammatory effects will be more attractive clinical candidates in future.

Origin of the kinetic HDAC2-selectivity of an HDAC inhibitor.

A newly synthesized small molecule, KTT-1, exhibits kinetically selective inhibition of histone deacetylase 2, HDAC2, over its homologous enzyme, HDAC1. KTT-1 is hard to be released from the HDAC2/KTT-1 complex, compared to the HDAC1/KTT-1 complex and the residence time of KTT-1 in HDAC2 is longer than that in HDAC1. To explore the physical origin of this kinetic selectivity, we performed replica-exchange umbrella sampling molecular dynamics simulations for formation of both complexes. The calculated potentials of mean force suggest that KTT-1 is stably bound to HDAC2 and that it is easily disassociated from HDAC1. In the direct vicinity of the KTT-1 binding site in both enzymes, there exists a conserved loop consisting of four consecutive glycine residues (Gly304-307 for HDAC2; Gly299-302 for HDA1). The difference between the two enzymes comes from a single un-conserved residue behind this loop, namely, Ala268 in HDAC2 and Ser263 in HDAC1. The Ala268 contributes to the tight binding of KTT-1 to HDAC2 by the linear orientation of Ala268, Gly306, and one carbon atom in KTT-1. On the other hand, Ser263 cannot stabilize the binding of KTT-1 to HDAC1, because it is relatively further away from the glycine loop and because the directions of the two forces are not in line.

The HDAC inhibitor domatinostat induces type I interferon α in Merkel cell carcinoma by HES1 repression

BackgroundClass I selective histone deacetylase inhibitors (HDACi) have been previously demonstrated to not only increase major histocompatibility complex class I surface expression in Merkel cell carcinoma (MCC) cells by restoring the antigen processing and presentation machinery, but also exert anti-tumoral effect by inducing apoptosis. Both phenomena could be due to induction of type I interferons (IFN), as has been described for HDACi. However, the mechanism of IFN induction under HDACi is not fully understood because the expression of IFNs is regulated by both activating and inhibitory signaling pathways. Our own preliminary observations suggest that this may be caused by suppression of HES1.MethodsThe effect of the class I selective HDACi domatinostat and IFNα on cell viability and the apoptosis of MCPyV-positive (WaGa, MKL-1) and -negative (UM-MCC 34) MCC cell lines, as well as, primary fibroblasts were assessed by colorimetric methods or measuring mitochondrial membrane potential and intracellular caspase-3/7, respectively. Next, the impact of domatinostat on IFNA and HES1 mRNA expression was measured by RT-qPCR; intracellular IFNα production was detected by flow cytometry. To confirm that the expression of IFNα induced by HDACi was due to the suppression of HES1, it was silenced by RNA interference and then mRNA expression of IFNA and IFN-stimulated genes was assessed.ResultsOur studies show that the previously reported reduction in viability of MCC cell lines after inhibition of HDAC by domatinostat is accompanied by an increase in IFNα expression, both of mRNA and at the protein level. We confirmed that treatment of MCC cells with external IFNα inhibited their proliferation and induced apoptosis. Re-analysis of existing single-cell RNA sequencing data indicated that induction of IFNα by domatinostat occurs through repression of HES1, a transcriptional inhibitor of IFNA; this was confirmed by RT-qPCR. Finally, siRNA-mediated silencing of HES1 in the MCC cell line WaGa not only increased mRNA expression of IFNA and IFN-stimulated genes but also decreased cell viability.ConclusionOur results demonstrate that the direct anti-tumor effect of HDACi domatinostat on MCC cells is at least in part mediated via decreased HES1 expression allowing the induction of IFNα, which in turn causes apoptosis.

Abstract 6245: Selective HDAC6 inhibition by ITF3756 modifies the landscape of gene and protein expression in human monocytes: Possible identification of a specific biomarker signature

Abstract Histone deacetylase 6 (HDAC6) modulates tumor immunogenicity through several pathways, such as the control of PD-L1 expression on tumor and on lymphoid cells. Thus, HDAC6 is a potential pharmacological target for cancer treatment. In humans, HDAC6 inhibition can be monitored by measuring the ratio between tubulin and histone acetylation. However, the identification of biomarkers to be used as a signature for patient stratification that correlates with the treatment efficacy is missing. Since we have previously demonstrated that the activity of the selective HDAC6 inhibitor ITF3756 on myeloid cells plays a role in the antitumor immune response in the TME, we reasoned that an ITF3756-specific signature could be identified in unstimulated monocytes treated with the compound. Monocytes were purified from healthy donors, treated with ITF3756 and RNAseq and proteomic analyses were performed. Gene Ontology analysis shows that ITF3756 has a widespread effect on monocyte transcriptome and proteome. We observed the down-modulation of genes and proteins involved in inflammatory pathways and in transcription regulation. Up-regulation of neutrophil-mediated immunity, metabolic pathways and protein phosphorylation was also identified. Focusing on genes that could be relevant in the tumor context, we selected a panel of genes from the most up- (SCL27A1, ANXA6 and IRF6) and down-modulated (CD84, RANK, CXCL2, CXCL3, STAB-1, CD163, PD-L1, CD206 and ADA). Interestingly, we found among the downmodulated genes markers of M2 macrophages, genes related to the tumor associated macrophages, and STAB-1, whose targeting is currently in clinical trials for cancer immunotherapy. Peripheral Blood Mononuclear Cells (PBMCs) could be a less expensive starting material for biomarker identification to be collected during a clinical trial when compared to purified monocytes. Thus, to ascertain whether the regulation by ITF3756 of our selected genes was detectable also in PBMCs we performed gene expression analysis after ITF3756 treatment and we confirmed that all selected genes were reproducibly modulated. Surface protein expression of M2 macrophages biomarkers in our panel, were determined by flow cytometry and all of them were significantly modulated in PBMCs. These data show how our approach successfully led both to the identification of a biomarker signature that could be used in clinical trials to monitor the pharmacodynamic effect of ITF3756, and to the discovery of previously unknown targets of HDAC6 inhibition. Citation Format: Chiara Ripamonti, Valeria Spadotto, Monica Forino, Christian Steinkuhler, Gianluca Fossati. Selective HDAC6 inhibition by ITF3756 modifies the landscape of gene and protein expression in human monocytes: Possible identification of a specific biomarker signature. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6245.

Abstract 2880: Inhibition of HDAC6 and HDAC11 has opposite effects on inflammation and the modulation of the functional phenotype of macrophages in the tumor microenvironment

Abstract Introduction: Pan-histone deacetylase (HDAC) inhibitors have been used as anti-cancer agents due to their cytotoxicity. However, studies from our group have reported that intervention of class-specific HDAC inhibitors can differentially affect immune-related pathways in macrophages and influence their pro- or anti-inflammatory phenotype and function. For example, specific HDAC6 inhibitors demonstrated promising anti-tumor effects by suppressing tumor-promoting M2 macrophages in the tumor microenvironment (TME). In contrast, inhibition of HDAC11 promoted the anti-inflammatory phenotype of macrophages. Besides the divergent roles and distinct signaling pathways, a comprehensive study on the effect of HDCA6 and HDAC11 inhibition on macrophage phenotype and the implications on TME and inflammation is warranted. Objective: To determine the effect of HDAC6 and HDAC11 inhibition on the phenotype and function of macrophages in the context of the TME. Methods: Bone marrow-derived macrophages (BMDMs) isolated from C57BL/6 mice were pre-treated with HDAC6 (NexturastatA) or HDAC11 (FT895) inhibitors prior to polarizing them to M1 and M2 phenotypes. Similarly, BMDMs were isolated from total knockouts (KO) of HDAC6 and HDAC11 mice. The changes in gene expression were identified by RNA-seq analysis. Furthermore, single-cell secretome analysis on Isoplexis platform was performed from human and mice BMDMs on a panel of cytokines and chemokines. Results: Transcriptomic analysis revealed that HDAC6 inhibition in M1 polarized BMDMs sustained inflammatory gene signature while suppressing the tumor-promoting and anti-inflammatory genes in M2 macrophages. This supported our previous reports where HDAC6i increased M1/M2 ratio in murine melanoma models. In contrast, HDAC11 inhibition in M2 macrophages upregulated classical M2 markers, whereas M1-associated gene signature was minimally affected. These results were recapitulated in HDAC6KO and HDAC11KO macrophages, suggesting a high specificity of the inhibitors. Further validation at protein level by single-cell secretome analysis indicated that HDAC11 inhibition in M2 polarized BMDMs increased secretion of growth factors such as Egf and Pdgf and of anti-inflammatory cytokines such as Il10 and Il13. On the contrary, HDAC6 inhibition upregulated inflammatory cytokines such as Ifng and Tnf and T-cell recruiting chemokine Cxcl10 in the M1 phenotype. Conclusions:Transcriptomics and single-cell secretome analyses of macrophages indicate that HDAC6 and HDAC11 affects macrophage function in diametrically opposite directions. Therefore, our study underscores the importance of class-selective inhibition of HDACs over pan-HDAC inhibitors and the potential use of selective HDAC inhibitors as a therapeutic option to control macrophage phenotype in cancer and other conditions. Citation Format: Manasa Suresh, Hawa Coulibaly, Xintang Li, David Quiceno-Torres, Nithya Gajendran, Karen Tan, Matias Hepp, Karthik Musunuri, Satish Noonepalle, Alejandro Villagra. Inhibition of HDAC6 and HDAC11 has opposite effects on inflammation and the modulation of the functional phenotype of macrophages in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2880.

Hydroxamic acid derivatives as selective HDAC3 inhibitors: computer-aided drug design strategies

Histone deacetylases (HDACs) are critical epigenetic drug targets that have gained significant attention in the scientific community for the treatment of cancer. The currently marketed HDAC inhibitors lack selectivity for the various HDAC isoenzymes. Here, we describe our protocol for the discovery of novel potential hydroxamic acid based HDAC3 inhibitors through pharmacophore modeling, virtual screening, docking, molecular dynamics (MD) simulation and toxicity studies. The ten pharmacophore hypotheses were established, and their reliability was validated by different ROC (receiving operator curve) analysis. Among them, the best model (Hypothesis 9 or RRRA) was employed for searching SCHEMBL, ZINC and MolPort database to screen out hit molecules as selective HDAC3 inhibitors, followed by different docking stages. MD simulation (50 ns) and MMGBSA study were performed to study the stability of ligand binding modes and with the help of trajectory analysis, to calculate the ligand-receptor complex RMSD (root-mean-square deviation), RMSF (root-mean-square fluctuation) and H-bond distance, etc. Finally, in-silico toxicity studies were performed on top screened molecules and compared with reference drug SAHA and established structure-activity relationship (SAR). The results indicated that compound 31, with high inhibitory potency and less toxicity (probability value 0.418), is suitable for further experimental analysis. Communicated by Ramaswamy H. Sarma

Ellagitannin Punicalagin Disrupts the Pathways Related to Bacterial Growth and Affects Multiple Pattern Recognition Receptor Signaling by Acting as a Selective Histone Deacetylase Inhibitor

Punicalagin (PA) is a key ellagitannin abundant in pomegranate with wide-ranging biological activities. In this study, we examined the biological processes by which PA regulates bacterial growth and inflammation in human cells using multiomics and molecular docking approaches. PA promoted macrophage-mediated bacterial killing and inhibited the growth of Citrobacter rodentium by inducing a distinct metabolome pattern. PA acted as a selective regulator of histone deacetylases (HDACs) and affected 37 pathways in macrophages, including signaling mediated by pattern recognition receptors, such as Toll-like and NOD-like receptors. In silico simulation showed that PA can bind with high affinity to HDAC7. PA downregulated HDAC7 at both mRNA and protein levels and resulted in a decrease in the level of histone 3 lysine 27 acetylation. Our findings provide evidence that PA exerts its biological effects via multiple pathways, which can be exploited in the development of this bioactive food ingredient for disease management.

Selectivity of Hydroxamate- and Difluoromethyloxadiazole-Based Inhibitors of Histone Deacetylase 6 In Vitro and in Cells.

Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family of enzymes due to its complex domain organization and cytosolic localization. Experimental data point toward the therapeutic use of HDAC6-selective inhibitors (HDAC6is) for use in both neurological and psychiatric disorders. In this article, we provide side-by-side comparisons of hydroxamate-based HDAC6is frequently used in the field and a novel HDAC6 inhibitor containing the difluoromethyl-1,3,4-oxadiazole function as an alternative zinc-binding group (compound 7). In vitro isotype selectivity screening uncovered HDAC10 as a primary off-target for the hydroxamate-based HDAC6is, while compound 7 features exquisite 10,000-fold selectivity over all other HDAC isoforms. Complementary cell-based assays using tubulin acetylation as a surrogate readout revealed approximately 100-fold lower apparent potency for all compounds. Finally, the limited selectivity of a number of these HDAC6is is shown to be linked to cytotoxicity in RPMI-8226 cells. Our results clearly show that off-target effects of HDAC6is must be considered before attributing observed physiological readouts solely to HDAC6 inhibition. Moreover, given their unparalleled specificity, the oxadiazole-based inhibitors would best be employed either as research tools in further probing HDAC6 biology or as leads in the development of truly HDAC6-specific compounds in the treatment of human disease states.

Discovery of Indole-Piperazine Hybrid Structures as Potent Selective Class I Histone Deacetylases Inhibitors.

Histone deacetylases (HDACs) are important targets in cancer treatment, and the development of selective and broad-spectrum HDACs inhibitors (HDACis) is urgent. In this research, a series of aroylpiperazine hybrid derivatives were designed and synthesized. Among these, indole-piperazine hybrids 6a (IC50 = 205 nM) and 6b (IC50 = 280 nM) showed submicromolar activity against HDAC1. Moreover, 6a showed a preferable affinity toward class I HDACs, especially for HDAC1-3. In vitro, 6a exhibited better antiproliferative activities against K562 and HCT116 cell lines than chidamide.

Selective inhibition of HDAC6 promotes bladder cancer radiosensitization and mitigates the radiation-induced CXCL1 signalling

BackgroundAlthough trimodality therapy resecting tumours followed by chemoradiotherapy is emerged for muscle-invasive bladder cancer (MIBC), chemotherapy produces toxicities. Histone deacetylase inhibitors have been identified as an effective strategy to enhance cancer radiotherapy (RT).MethodsWe examined the role of HDAC6 and specific inhibition of HDAC6 on BC radiosensitivity by performing transcriptomic analysis and mechanism study.ResultsHDAC6 knockdown or HDAC6 inhibitor (HDAC6i) tubacin exerted a radiosensitizing effect, including decreased clonogenic survival, increased H3K9ac and α-tubulin acetylation, and accumulated γH2AX, which are similar to the effect of panobinostat, a pan-HDACi, on irradiated BC cells. Transcriptomics of shHDAC6-transduced T24 under irradiation showed that shHDAC6 counteracted RT-induced mRNA expression of CXCL1, SERPINE1, SDC1 and SDC2, which are linked to cell migration, angiogenesis and metastasis. Moreover, tubacin significantly suppressed RT-induced CXCL1 and radiation-enhanced invasion/migration, whereas panobinostat elevated RT-induced CXCL1 expression and invasion/migration abilities. This phenotype was significantly abrogated by anti-CXCL1 antibody, indicating the key regulator of CXCL1 contributing to BC malignancy. Immunohistochemical evaluation of tumours from urothelial carcinoma patients supported the correlation between high CXCL1 expression and reduced survival.ConclusionUnlike pan-HDACi, the selective HDAC6i can enhance BC radiosensitization and effectively inhibit RT-induced oncogenic CXCL1-Snail-signalling, thus further advancing its therapeutic potential with RT.

Chidamide suppresses adipogenic differentiation of bone marrow derived mesenchymal stem cells via increasing REEP2 expression

Increased propensity of bone marrow-derived mesenchymal stem cells (BM-MSCs) toward adipogenic differentiation at the expense of osteogenesis has been implicated in obesity, diabetes, and age-related osteoporosis as well as various hematopoietic disorders. Defining small molecules with role in rectifying the adipo-osteogenic differentiation imbalance is of great significance. Here, we unexpectedly found that Chidamide, a selective histone deacetylases inhibitor, exhibited remarkably suppressive effect on the invitro induced adipogenic differentiation of BM-MSCs. Multifaceted alterations in the spectrum of gene expression were observed in Chidamide-managed BM-MSCs during adipogenic induction. Finally, we focused on REEP2, which presented decreased expression in BM-MSCs-mediated adipogenesis and was restored by Chidamide treatment. REEP2 was subsequently demonstrated as a negative regulator of adipogenic differentiation of BM-MSCs and mediated the suppressive effect of Chidamide on adipocyte development. Our findings provide the theoretical and experimental foundation for the clinical application of Chidamide for disorders associated with excessive marrow adipocytes.

Cytotoxic evaluation of YSL-109 in a triple negative breast cancer cell line and toxicological evaluations.

Breast cancer (BC) is the leading cause of cancer-related death in women worldwide. Triple negative breast cancer (TNBC) is the most aggressive form of BC being with the worst prognosis and the worst survival rates. There is no specific pharmacological target for the treatment of TNBC; conventional therapy includes the use of non-specific chemotherapy that generally has a poor prognosis. Therefore, the search of effective therapies against to TNBC continues at both preclinical and clinical level. In this sense, the exploration of different pharmacological targets is a continue task that pave the way to epigenetic modulation using novel small molecules. Lately, the inhibition of histone deacetylases (HDACs) has been explored to treat different BC, including TNBC. HDACs remove the acetyl groups from the ɛ-amino lysine resides on histone and non-histone proteins. In particular, the inhibition of HDAC6 has been suggested to be useful for the treatment of TNBC due to it is overexpressed in TNBC. Therefore, in this work, an HDAC6 selective inhibitor, the (S)-4-butyl-N-(1-(hydroxyamino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl) benzamide (YSL-109), was assayed on TNBC cell line (MDA-MB231) showing an antiproliferative activity (IC50 = 50.34 ± 1.11µM), whereas on fibroblast, it was lesser toxic. After corroborating the in vitro antiproliferative activity of YSL-109 in TNBC, the toxicological profile was explored using combined approach with in silico tools and experimental assays. YSL-109 shows moderate mutagenic activity on TA-98 strain at 30 and 100µM in the Ames test, whereas YSL-109 did not show in vivo genotoxicity and its oral acute toxicity (LD50) in CD-1 female mice was higher than 2000mg/kg, which is in agreement with our in silico predictions. According to these results, YSL-109 represents an interesting compound to be explored for the treatment of TNBC under preclinical in vivo models.

Synthesis and biological evaluation of novel N-benzyltriazolyl-hydroxamate derivatives as selective histone deacetylase 6 inhibitors

Histone deacetylases (HDAC) regulate post-translational acetylation and the inhibition of these enzymes has emerged as an intriguing disease therapeutic. Among them, class IIb HDAC6 has the unique characteristic of mainly deacetylating cytoplasmic proteins, suggesting clinical applications for neurodegenerative diseases, inflammation, and cancer. In this study, we designed a novel N-benzyltriazolyl-hydroxamate scaffold based on the known HDAC6 inhibitors nexturastat A and tubastatin A. Among the 27 derivatives, 3-fluoro-4-((3-(2-fluorophenyl)-1H-1,2,4-triazol-1-yl)methyl)-N-hydroxybenzamide 4u (HDAC6 IC50 = 7.08 nM) showed nanomolar HDAC6 inhibitory activity with 42-fold selectivity over HDAC1. Structure-activity relationship (SAR) and computational docking studies were conducted to optimize the triazole capping group. Docking analysis revealed that the capping group aligned with the conserved L1 pocket of HDAC6 and was associated with subtype selectivity. Overall, our study explored the triazole-based biaryl capping group and its substitution and orientation, suggesting a rationale for the design of HDAC6-selective inhibitors.

Tacedinaline (CI-994), a class I HDAC inhibitor, targets intrinsic tumor growth and leptomeningeal dissemination in MYC-driven medulloblastoma while making them susceptible to anti-CD47-induced macrophage phagocytosis via NF-kB-TGM2 driven tumor inflammation

BackgroundWhile major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant...

HDAC6 regulates human erythroid differentiation through modulation of JAK2 signalling

Among histone deacetylases, HDAC6 is unusual in its cytoplasmic localization. Its inhibition leads to hyperacetylation of non-histone proteins, inhibiting cell cycle, proliferation and apoptosis. Ricolinostat (ACY-1215) is a selective inhibitor of the histone deacetylase HDAC6 with proven efficacy in the treatment of malignant diseases, but anaemia is one of the most frequent side effects. We investigated here the underlying mechanisms of this erythroid toxicity. We first confirmed that HDAC6 was strongly expressed at both RNA and protein levels in CD34+ -cells-derived erythroid progenitors. ACY-1215 exposure on CD34+ -cells driven in vitro towards the erythroid lineage led to a decreased cell count, an increased apoptotic rate and a delayed erythroid differentiation with accumulation of weakly hemoglobinized immature erythroblasts. This was accompanied by drastic changes in the transcriptomic profile of primary cells as shown by RNAseq. In erythroid cells, ACY-1215 and shRNA-mediated HDAC6 knockdown inhibited the EPO-dependent JAK2 phosphorylation. Using acetylome, we identified 14-3-3ζ, known to interact directly with the JAK2 negative regulator LNK, as a potential HDAC6 target in erythroid cells. We confirmed that 14-3-3ζ was hyperacetylated after ACY-1215 exposure, which decreased the 14-3-3ζ/LNK interaction while increased LNK ability to interact with JAK2. Thus, in addition to its previously described role in the enucleation of mouse fetal liver erythroblasts, we identified here a new mechanism of HDAC6-dependent control of erythropoiesis through 14-3-3ζ acetylation level, LNK availability and finally JAK2 activation in response to EPO, which is crucial downstream of EPO-R activation for human erythroid cell survival, proliferation and differentiation.

Increased recombinant adeno-associated virus production by HEK293 cells using small molecule chemical additives.

Recombinant adeno-associated virus (rAAV) has established itself as a highly efficacious gene delivery vector with a well characterised safety profile allowing broad clinical application. Recent successes in rAAV-mediated gene therapy clinical trials will continue to drive demand for improved rAAV production processes to reduce costs. Here, we demonstrate that small molecule bioactive chemical additives can significantly increase recombinant AAV vector production by human embryonic kidney (HEK) cells up to three-fold. Nocodazole (an anti-mitotic agent) and M344 (a selective histone deacetylase inhibitor) were identified as positive regulators of rAAV8 genome titre in a microplate screening assay. Addition of nocodazole to triple-transfected HEK293 suspension cells producing rAAV arrested cells in G2/M phase, increased average cell volume and reduced viable cell density relative to untreated rAAV producing cells at harvest. Final crude genome vector titre from nocodazole treated cultures was >2-fold higher compared to non-treated cultures. Further investigation showed nocodazole addition to cultures to be time critical. Genome titre improvement was found to be scalable and serotype independent across two distinct rAAV serotypes, rAAV8 and rAAV9. Furthermore, a combination of M344 and nocodazole produced a positive additive effect on rAAV8 genome titre, resulting in a three-fold increase in genome titre compared to untreated cells.

Solid-Phase Synthesis of Cereblon-Recruiting Selective Histone Deacetylase 6 Degraders (HDAC6 PROTACs) with Antileukemic Activity.

In this work, we utilized the proteolysis targeting chimera (PROTAC) technology to achieve the chemical knock-down of histone deacetylase 6 (HDAC6). Two series of cereblon-recruiting PROTACs were synthesized via a solid-phase parallel synthesis approach, which allowed the rapid preparation of two HDAC6 degrader mini libraries. The PROTACs were either based on an unselective vorinostat-like HDAC ligand or derived from a selective HDAC6 inhibitor. Notably, both PROTAC series demonstrated selective degradation of HDAC6 in leukemia cell lines. The best degraders from each series (denoted A6 and B4) were capable of degrading HDAC6 via ternary complex formation and the ubiquitin-proteasome pathway, with DC50 values of 3.5 and 19.4 nM, respectively. PROTAC A6 demonstrated promising antiproliferative activity via inducing apoptosis in myeloid leukemia cell lines. These findings highlight the potential of this series of degraders as effective pharmacological tools for the targeted degradation of HDAC6.

Effects of Histone Deacetylase Inhibitors on Different Memory Domains in an Animal Model of Aging and Alzheimer’s Disease

AbstractBackgroundHistone deacetylases (HDACs) have been involved in regulation of memory and previous work from our group and others have shown promising beneficial effects of HDAC inhibitors (HDACis) in aging and neurodegenerative disorders, including Alzheimer’s disease. However, currently, there are no reports on cross comparison of none‐selective and selective HDACis for their beneficial effects on memory and AD‐like pathologies in both aging and AD models.MethodIn this study, 3‐, 12‐, and 18‐months of age WT and APP/PS1 mice were administered a broad acting HDACi valproic acid (VPA), and two selective Class 1 HDACis entinostat (MS‐275, targeting HDAC 1 and 3) and tacedinaline (CI‐994, targeting HDAC 1, 2 and 3) for 30 days. After HDACi administration, mice underwent three memory tests: the novel object recognition (NOR) for executive memory, Y‐maze for short‐term working memory, and Morris water maze (MWM) for long‐term spatial reference memory (n = 8‐10). After memory assessment, mouse prefrontal cortex and hippocampus were collected for biochemical studies.ResultWe found a decrease in executive memory, and long‐term spatial reference memory in WT mice at 18 months of age and, severe memory deficits of three memory domains in APP/PS1 mice at 12‐ and 18‐months of age, and most importantly, we found an age and genotype interaction in the severity of executive and long‐term spatial reference memory deficits in APP/PS1 mice. Both MS‐275 and CI‐994 rescued executive memory, and short‐term spatial reference memory, however, only CI‐994 was sufficient to rescue long‐term spatial reference memory in APP/PS1 mice. We also found a decrease in H3K9ac in APP/PS1 mice with aging at the gene promoters related to memory and synaptic plasticity, however, only CI‐994 rescued H3K9ac levels in APP/PS1 mice.ConclusionOur results suggest that selective HDACis could rescue memory function in APP/PS1 mice, and HDAC2 may be an important modulator for hippocampal dependent memory. We are continuing our investigation of changes of specific HDAC and histone acetylation marks on genes related to memory and synaptic plasticity in the hippocampus, but also in the PFC to identify regional specific of HDAC modulation across aging and AD.

156P A phase Ib/II study of nanatinostat (Nstat) plus valganciclovir (VGCV) in advanced Epstein-Barr virus positive (EBV+) solid tumors and with pembrolizumab (PEM) in recurrent/metastatic nasopharyngeal carcinoma (RM-NPC)

EBV is associated with NPC, with the virus in a latent state. Nstat is a Class-I selective oral HDAC inhibitor that induces expression of the lytic BGLF4 EBV protein kinase in EBV+ tumor cells, activating ganciclovir (GCV) via phosphorylation. This results in GCV-mediated inhibition of viral and cellular DNA synthesis and apoptosis. Nstat plus VGCV (oral prodrug of GCV) showed a favorable safety profile with anti-tumor activity in a phase 1b/2 study in R/R EBV+ lymphoma (recommended phase 2 dose [RP2D]: Nstat 20 mg daily [QD], 4 d/wk + VGCV 900 mg QD).