Selective Histone Deacetylase Inhibitors Research Articles

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).

Discovery of 1-Benzhydryl-Piperazine-Based HDAC Inhibitors with Anti-Breast Cancer Activity: Synthesis, Molecular Modeling, In Vitro and In Vivo Biological Evaluation.

Isoform-selective histone deacetylase (HDAC) inhibition is promoted as a rational strategy to develop safer anti-cancer drugs compared to non-selective HDAC inhibitors. Despite this presumed benefit, considerably more non-selective HDAC inhibitors have undergone clinical trials. In this report, we detail the design and discovery of potent HDAC inhibitors, with 1-benzhydryl piperazine as a surface recognition group, that differ in hydrocarbon linker. In vitro HDAC screening identified two selective HDAC6 inhibitors with nanomolar IC50 values, as well as two non-selective nanomolar HDAC inhibitors. Structure-based molecular modeling was employed to study the influence of linker chemistry of synthesized inhibitors on HDAC6 potency. The breast cancer cell lines (MDA-MB-231 and MCF-7) were used to evaluate compound-mediated in vitro anti-cancer, anti-migratory, and anti-invasive activities. Experiments on the zebrafish MDA-MB-231 xenograft model revealed that a novel non-selective HDAC inhibitor with a seven-carbon-atom linker exhibits potent anti-tumor, anti-metastatic, and anti-angiogenic effects when tested at low micromolar concentrations.

Potential of histone deacetylase inhibitors for the therapy of ovarian cancer.

Malignant ovarian tumors bear the highest mortality rate among all gynecological cancers. Both late tumor diagnosis and tolerance to available chemotherapy increase patient mortality. Accumulating evidence demonstrates that histone modifications play a key role in cancerization and progression. Histone deacetylases is associated with chromatin condensed structure and transcriptional repression and play a role in chromatin remodeling and epigenetics. Histone deacetylases are promising targets for therapeutic interventions intended to reverse aberrant epigenetic associated with cancer. Therefore, histone deacetylases inhibitors could be used as anti-cancer drugs. Preclinical studies have shown promising outcomes of histone deacetylases inhibitors in ovarian cancer while clinical trials have had mixed results and limited success as monotherapy. Therefore, combination therapy with different anticancer drugs for synergistic effects and newly selective histone deacetylases inhibitors development for lower toxicity are hot issues now. In this review, we summarize the latest studies on the classification and mechanisms of action of histone deacetylase and the clinical application of their inhibitors as monotherapy or combination therapy in ovarian cancer.

Phase 1 Safety and Dose Escalation of Purinostat Mesylate, a Uniquely Potent and Selective Inhibitor of HDAC I and IIb in Relapsed or Refractory Lymphoma and Multiple Myeloma

Introduction Current treatment options for patients with relapsed or refractory (RR) lymphoma and multiple myeloma (MM) are limited, highlighting the unmet need for effective therapies in these disease settings. Purinostat Mesylate (PM) is a small molecule that is designed to selectively inhibit histone deacetylase (HDAC) enzymes class I and IIb, which are members of common oncogenic pathways in hematologic malignancies. This study examined PM monotherapy in patients with RR lymphoma and MM. Methods This open-label, non-randomized, first-in-human, phase 1 two-center trial enrolled adult patients with lymphoma or MM who were refractory to or relapsed after ≥1 prior regimens. PM was administered by 30-minute intravenous infusion in a standard 3+3 dose escalation design. The trial was conducted in three stages sequentially as follows: the first stage as a single dose (Day 1), the second stage as multiple doses (Day 8,11, and 15), and the third stage as extended doses (Day1, 4, 8, 11 in 21-day cycles). The decision to proceed to the third stage is based on patient tolerability in the previous two stages, disease status, and continued benefit determined by investigators. Initially dosing started at 1.2 mg/m2, escalated to 2.4 mg/m2, 4.0 mg/m2, 6.0 mg/m2, 8.4 mg/m2, 11.2 mg/m2 and finally 15.0 mg/m2. Patients continued to receive PM until disease progression or other treatment discontinuation criteria were met. The primary objective was to determine the maximum-tolerated dose and recommended phase 2 dose (RP2D); secondary objectives were to assess the safety and tolerability, pharmacokinetic profiles, and preliminary anti-tumor activity. This ongoing trial is registered at Chinese Clinical Trial Registry, number ChiCTR20191121. Results A total of 11 heavily pretreated MM patients(11/27,41%) and 16 lymphoma patients(16/27,59%) were enrolled to receive PM up to a maximum of 15.0 mg/m2 in the dose escalation. Most adverse events (AEs) were grade 1 to 2 and recovered with observation or symptomatic management. The most common Grade ≥3 AEs were thrombocytopenia (n=48.1%), neutropenia (n=48.1%), and anemia (11.1%), which occurred primarily during the extended dosing stages. AEs led to dose reductions in 4 patients. Dose limiting toxicities (DLTs) have not been observed. Twenty-seven patients were evaluable for responses. Eleven patients with RR MM had a disease control rate (DCR) of 72.7%. Eight patients had stable disease (SD) in cycle 1 and entered the third stage with a maximum of 6 extended cycles, of which 1 patient achieved a minimal remission (MR) in cycle 2. Nine out of 16 patients with RR lymphoma achieved objective responses rates (ORR) of 56.2%. Among them, six of 10 patients with diffuse large B-cell lymphoma (DLBCL) achieved objective responses (3 complete remissions [CR], 2 partial remissions [PR], and 1 MR). CRs occurred in 2 patients with DLBCL in cycle 3 (8.4mg/m2, 11.2 mg/m2) and 1 patient in cycle 2 (11.2 mg/m2). PRs occurred in 4 patient in cycle 1 (2 in 8.4 mg/m2, 2 in 11.2 mg/m2 ) and another patient who relapsed after 2 lines of prior therapy including autologous stem cell transplantation(ASCT) in cycle 3 (11.2 mg/m2). One MR and 1 SD have been observed in patients with double-expression DLBCL in cycle 1(15.0 mg/m2, 8.4 mg/m2) . Thus, the ORR in patients with RR DLBCL was 60.0% (6/10) and DCR was 70.0% (7/10). Two out of 4 follicular lymphoma patients relapsed after 2~4 lines of prior therapy also achieved objective responses (1 MR in cycle 1 and 1 PR in cycle 4). Of note, one patient with peripheral T-cell lymphoma who relapsed after four lines of treatment, including ASCT and Chidamide combination treatment with R-CHOP, obtained PR in cycle 1. Up to date, eight active response patients are still being treated. On the basis of the confirmed objective response and tolerability profile, the RP2D of PM was determined to be 8.4mg/m2 and 11.2mg/m2. Conclusions PM has demonstrated tolerability and good anti-tumor activity across all dosing schedules studied, especially multiple objective responses and disease control in patients with DLBCL. These results support continued development of PM, alone or in combination with other therapies, for the treatment of RR DLBCL. An open-label, non-randomized, phase 2, multicenter trial will be soon conducted in China to evaluate the efficacy of PM monotherapy in RR DLBCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Histone Deacetylase 6 Inhibition Prevents Vaso-Occlusion and Pain in Sickle Cell Mice

Introduction: In sickle cell disease (SCD) mouse models, heme activates toll-like receptor 4 (TLR4) MyD88-dependent signaling and activates NOD-like receptor family pyrin domain containing (NLRP) 3 inflammasome assembly, leading to vascular endothelial activation and vaso-occlusion (VO). In SCD patients, cycles of hemolysis-driven VO contribute to acute and chronic pain/hyperalgesia. Several studies have evaluated non-selective histone deacetylase (HDAC) inhibitors for SCD; however, side-effects have limited clinical advancement. HDAC6 is an attractive target as it exhibits cytoplasmic AND nuclear activity and knockout mice lack hematologic consequences. In the cytoplasm, HDAC6 helps assemble NLRP3 inflammasomes and functions downstream of TLR4 to inhibit signaling. In SCD mouse models, TLR4 signaling is also critical for mechanical hyperalgesia. Intriguingly, in non-SCD hyperalgesia models, HDAC6 inhibition reduces TLR4-mediated mechanical hypersensitivity. We hypothesized that by preventing TLR4/NLRP3 activation in SCD mice, HDAC6 inhibition will prevent VO and pain. Methods: For in vitro analysis, TLR-4 signaling was stimulated in human umbilical vein endothelial cells (HUVEC) or mouse microglial cells (SIM-A9, ATTC) using 10-20 µM hemin or 10 ng/mL lipopolysaccharide (LPS) ± HDAC6 inhibitor BML-281 (2 µM, Enzo). To assess for pro-adhesive (VCAM-1, P-selectin, VWF) and pro-inflammatory (IL-6, NF-κB, IL-1) gene and protein expression, cell lysates or conditioned media were collected for analysis using qRT-PCR, immunoblotting, and enzyme-linked immunosorbent assay. Immunofluorescence (IF) for P-selectin and VWF was performed on fresh frozen organ sections or cells fixed with 4% paraformaldehyde. To assess for reactive oxygen species generation (ROS), the TMRE assay was used (Abcam). For dorsal skin fold chamber (DSFC) assessment of VO, HbSS Townes mice (n=7 per condition) were subcutaneously injected with either vehicle (DMSO) or BML-281 (1 mg/kg) 24h and 1h prior to DSFC implantation. After DSFC implantation, baseline venules were assessed. To initiate VO, panhematin (3.2 µmole/kg) was infused via tail vein, and at 1-4 hours post-infusion venules were reexamined for stasis. Mice were then sacrificed and organs stored for pathology assessment. For pain studies, HbSS Townes mice (n=6) were assessed for mechanical hyperalgesia for 4 days prior to injections. Only non-hyperalgesic mice were injected subcutaneously with either vehicle (DMSO, n=3) or BML-281 (1 mg/kg, n=3) 24h and 1h prior to panhematin infusion. To induce acute pain, panhematin (3.2 µmole/kg) was infused via tail vein, and 1h, 4h, 24h and 48h post-infusion mechanical hyperalgesia measurements were performed. Results: Compared to LPS or hemin treated ECs, addition of 2 µM BML-281 prevented upregulation of , VCAM-1, P-selectin and E-selection mRNA, this was inhibited by the addition of 2 µM BML-281. Likewise, compared to hemin-treated ECs, BML-281+hemin-treated ECs had reduced pro-adhesive VCAM-1 and E-selectin protein expression. In IF studies, ECs treated with BML-281+hemin had reduced VWF expression and P-selectin release. Hemin-treated HUVEC exhibit increased NLRP3-mediated ROS production, which BML-281 treatments was significantly decreased. In DSFC studies, compared to vehicle-treated mice, BML-281-treated SS Townes mice had significantly fewer static vessels after panhematin infusion (Fig 1). In pathology assessment, BML-281 treated mice, showed reduced lung VWF and P-selectin vessel deposition. To assess neuroinflammation, mouse microglial cells were treated with LPS. Compared to LPS alone, BML-281 treatment significantly reduced IL-6 secretion and NLRP3 mRNA upregulation. Importantly, in in vivo pain assessments, compared to vehicle-treated SS-Townes mice at 1 h and 4 h post-hemin infusion, BML-281-treated SS-Townes mice exhibited significantly less mechanical hyperalgesia. Conclusion: Our studies show that HDAC6 inhibition prevented hemin-mediated VO, potentially through reduction in hemin-mediated ROS and TLR4 driven pro-adhesive endothelial activation. Furthermore, HDAC6 inhibition prevented hemin-mediated pro-inflammatory microglial activation and reduced hemin-mediated acute pain stimulation. Collectively, these studies suggest that selective HDAC6 inhibition may be a strategy to reduce acute inflammation and pain in SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Synergistic Sensitivity of Selinexor with Chidamide By Targeting NF-Κb /c-FLIP Signaling Via Suppressing HDAC1/XPO1 Activity in T-ALL

Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a poor prognosis. Exportin-1 (XPO1) overexpression is observed in ALL and is associated with a poor prognosis for the patients. Recently, XPO1 inhibitor is reported to show anti-tumor effects in T-ALL. HDAC inhibitors (HDACi) are also reported to inhibit T-cell leukemia, and Chidamide is a new selective HDACi. Here, we explored the effect of Chidamide combined with the XPO1 inhibitor Selinexor in T-ALL and the underlying mechanisms. Methods Cell Counting Kit-8 (CCK-8) was used for cell viability assay, Annexin-V/PI staining following flow cytometry analysis for apoptosis, and qPCR and Western Blot for gene expression in CEM and Molt4 T-ALL cell lines. CalcuSyn analysis was performed for a synergistic effect. Cellular fractionation combined with western blot was used to detect the distribution of observed proteins. The co-immunoprecipitation(Co-IP) assay was utilized for the protein-protein interaction. Results We first observed that both Chidamide and Selinexor induce cell proliferation arrest of T-ALL cells in a dose-dependent manner. Then we found that Selinexor significantly increased the Chidamide-mediated cell proliferation arrest in both CEM and Molt4 cells and CalcuSyn analysis showed they have a synergistic effect (Fig. 1A). Moreover, the combination significantly increased the apoptosis (Fig.1B) and cell cycle arrest at G0/G1 phase in CEM cells (Fig. IC) and Molt-4 cells compared to the single drug controls. Consistently, the combination significantly up-regulated the expression of pro-apoptotic factors Cleaved caspase-3 and BAX, but down-regulated that of anti-apoptotic effectors BcL2 and MCL1, and the cell cycle regulators CDK2 and CyclinE2 compared to that of the single drug controls in the cells (Fig.1D). These data together indicate the combination of Chidamide with Selinexor has synergistic anti-leukemia effects in T-ALL. To understand the underlying mechanism of the synergy, we observed the apoptosis regulatory proteins Cellular FLICE inhibitory protein(c-FLIP) is significantly down-regulated upon the combination treatment compared to the single drug controls (Fig. 1E). c-FLIP is reported to be transcriptionally regulated by NF-kB. Interestingly, we observed that Chidamide significantly down-regulates the expression of HDAC1 and NF-κB p50 in T-ALL cells (Fig. 1F). Moreover, IκB-α and NF-κB p65 are XPO1 cargoes, and the interaction of IκB-α with NF-κB p65 prevents NF-κB binding to target DNA. We also observed the accumulation of IκB-α, and NF-κB p65 in the nuclear fraction in CEM (Fig. 1G) and Molt4 cells upon the Selinexor treatment. The co-IP assay showed the IκB-α bound to NF-κB p65 subunit in the cells and this interaction is enhanced in nuclear faction upon Selinexor treatment (Fig.1H), which might prevent NF-κB dependent transcription of target genes such as c-FLIP. Furthermore, the combination of Selinexor with Chidamide showed significant effects on cell growth arrest, the expression of CDK2, CyclinE2, Cleaved caspase3, and BCL2 in primary cells from T-ALL patients (Fig.1I). Finally, qPCR results revealed c-FLIP was overexpressed in T-ALL patients compared to the normal controls in our T-ALL patient cohort (Fig.1J). These data together indicated that the combination exerts its anti-tumor effect by targeting XPO1/HDAC1/NF-kB/c-FLIP signaling. The mechanism model is shown in Fig.1K. Conclusions Our results indicated that the combination of Selinexor with Chidamide has synergistic sensitivity on cell proliferation arrest, apoptosis, and cell cycle arrest in T-ALL by targeting NF-kB/c-FLIP oncogenic signaling through suppressing XPO1/HDAC1 activity. Our data also provide experimental evidence for the further clinical trial of the new potential combination in therapy of T-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Optimized dose selective HDAC inhibitor tucidinostat overcomes anti-PD-L1 antibody resistance in experimental solid tumors

BackgroundAlthough immune checkpoint inhibitors (ICIs) have influenced the treatment paradigm for multiple solid tumors, increasing evidence suggests that primary and adaptive resistance may limit the long-term efficacy of ICIs. New therapeutic strategies with other drug combinations are hence warranted to enhance the antitumor efficacy of ICIs. As a novel tumor suppressor, histone deacetylase (HDAC) inhibitor tucidinostat has been successfully confirmed to act against hematological malignancies. However, the underlying mechanisms of action for tucidinostat and whether it can manipulate the tumor microenvironment (TME) in solid tumors remain unclear.MethodsThree murine tumor models (4T1, LLC, and CT26) were developed to define the significant role of different doses of tucidinostat in TME. The immunotherapeutic effect of tucidinostat combined with anti-programmed cell death ligand 1 antibody (aPD-L1) was demonstrated. Furthermore, the effect of tucidinostat on phenotypic characteristics of peripheral blood mononuclear cells (PBMCs) from lung cancer patients was investigated.ResultsWith an optimized dose, tucidinostat could alter TME and promote the migration and infiltration of CD8+ T cells into tumors, partially by increasing the activity of C-C motif chemokine ligand 5 (CCL5) via NF-κB signaling. Moreover, tucidinostat significantly promoted M1 polarization of macrophages and increased the in vivo antitumor efficacy of aPD-L1. Tucidinostat also enhanced the expression of the costimulatory molecules on human monocytes, suggesting a novel and improved antigen-presenting function.ConclusionsA combination regimen of tucidinostat and aPD-L1 may work synergistically to reduce tumor burden in patients with cancer by enhancing the immune function and provided a promising treatment strategy to overcome ICI treatment resistance.

Development of Fluorinated Peptoid-Based Histone Deacetylase (HDAC) Inhibitors for Therapy-Resistant Acute Leukemia.

Using a microwave-assisted protocol, we synthesized 16 peptoid-capped HDAC inhibitors (HDACi) with fluorinated linkers and identified two hit compounds. In biochemical and cellular assays, 10h stood out as a potent unselective HDACi with remarkable cytotoxic potential against different therapy-resistant leukemia cell lines. 10h demonstrated prominent antileukemic activity with low cytotoxic activity toward healthy cells. Moreover, 10h exhibited synergistic interactions with the DNA methyltransferase inhibitor decitabine in AML cell lines. The comparison of crystal structures of HDAC6 complexes with 10h and its nonfluorinated counterpart revealed a similar occupation of the L1 loop pocket but slight differences in zinc coordination. The substitution pattern of the acyl residue turned out to be crucial in terms of isoform selectivity. The introduction of an isopropyl group onto the phenyl ring provided the highly HDAC6-selective inhibitor 10p, which demonstrated moderate synergy with decitabine and exceeded the HDAC6 selectivity of tubastatin A.

Abstract 15701: Histone Deacetylase 6Inhibition Demonstrates Comparable Efficacy as Empagliflozin in a Mouse Model of Heart Failure With Preserved Ejection Fraction

Background: Heart failure with preserved ejection fraction (HFpEF) is associated with high morbidity and mortality, yet there are few effective therapies for HFpEF. Previously, we have demonstrated that TN-301, a highly selective small molecule inhibitor of histone deacetylase 6 (HDAC6), works directly on the heart and on systemic metabolism and inflammation in mouse models of HFpEF. Recently, empagliflozin, a selective inhibitor of sodium-glucose transport protein (SGLT2) inhibitor, was approved in the U.S. for patients with HFpEF. Purpose: We aimed to compare the efficacy of HDAC6i and SGLT2i in a mouse model of established diastolic dysfunction with preserved ejection fraction. Methods and Results: To recapitulate systemic and cardiovascular features of HFpEF in humans, we induced diastolic dysfunction with a combination of high fat diet and inhibition of constitutive nitric oxide synthase using N ω -nitrol-arginine methyl ester (L-NAME). After the HFpEF phenotypes were established, mice received TYA-11018 (TN-301-like HDAC6i) or empagliflozin, or vehicle orally once daily for 9 weeks. We found that TYA-11018 reduced left ventricular hypertrophy and restored diastolic function with comparable efficacy to empagliflozin. In addition, TYA-11018 reduced fasting glucose and improved glucose tolerance in HFpEF mice, to levels similar to empagliflozin. Preliminary data suggested TYA-11018 corrects hyperglycemia independently of glucose urine excretion, which is distinct from SGLT2i. Using whole transriptome RNA-seq, TYA-11018 showed greater effects on correcting expression of gene sets associated with inflammation, fibrosis and mitochondrial energy production compared to empagliflozin in HFpEF hearts. Conclusion: Our results show that with its distinct HDAC6 inhibition mechanism, TYA-11018 reverses preexisting hypertrophy, diastolic dysfunction and glucose tolerance, demonstrated similar efficacy to that with a SGLT2 inhibitor in HFpEF model. The comparable efficacy seen in this HFpEF model with TYA-11018 and empagliflozin provides early but encouraging evidence of the potential translatability of these findings to clinical development. We are developing TN-301 for treating HFpEF patients.