HDAC Activity Research Articles

Danggui Buxue decoction alleviates cyclophosphamide-induced myelosuppression by regulating β-hydroxybutyric acid metabolism and suppressing oxidative stress

Context Danggui Buxue Decoction (DBD) is an effective complementary medicine in alleviating myelosuppression after chemotherapy (MAC). However, its mechanism of action is elusive. Objective To illustrate that regulating β-hydroxybutyric acid (β-OHB) metabolism and suppressing oxidative stress could be a potential mechanism of action for DBD in alleviating MAC. Materials and methods After HPLC quantification and dose testing (3, 6 and 10 g/kg, gavage) of DBD, Sprague-Dawley rats were divided into control, cyclophosphamide (CTX) (30 mg/kg CTX for 5 days, intraperitoneal administration) and CTX + DBD groups (6 g/kg DBD for 14 days, gavage). Blood cell counts, thigh bone histological examination, β-OHB levels, oxidative stress indices and HDAC1 activity were tested. The biological function of β-OHB was verified in vitro (hBMSC cells were incubated in culture mediums that contained 40 μM CTX and β-OHB in 0, 1, 2.5, 5, 10 mM) and in vivo (MAC rat model, 3 g/kg β-OHB for 14 days, gavage). Results Rats in the CTX + DBD group showed upregulated blood cell counts (118–243%), β-OHB levels (495 nmol/mL in blood, 122 nmol/mg in marrow supernatant) and downregulated HDAC1 activity (59%), and oxidative stress indices (60–85%). In vitro, 5 mM β-OHB improved hBMSC cell migration (123%) and proliferation (131%). In vivo, rats treated with 3 g/kg β-OHB showed upregulated blood cell counts (121–182%) and downregulated HDAC1 activity (64%) and oxidative stress indices (65–83%). Discussion and conclusions DBD, a traditional Chinese medicine, alleviates MAC by intervening in β-OHB metabolism and oxidative stress.

Abstract 3465: HDAC8 regulates immune escape in melanoma through increased transcription of inhibitory cytokines that increase accumulation of myeloid-derived suppressor cells (MDSCs)

Abstract Immune checkpoint blockade leads to improved and durable responses in 30-40% of melanoma, yet nearly 1/3 of patients relapse from complete remission within 3 years. Recent studies from our group and others demonstrated that upfront use of targeted therapy leads to decreased immune infiltrate and confers resistance to immunotherapy. We have previously showed that HDAC8 reprograming cell transcriptional states leads to reduced recognition by immune system. However, it’s unclear how HDAC8 activity modulates the tumor immune microenvironment (TIME). In this study, we used integrated single cell RNA-seq, ATAC-seq and a novel BRAF-V600E; PTEN+/- melanoma genetically engineered mouse model (GEMM) to explore “cold” TIME modulated by HDAC8. Analysis of human melanoma cells overexpressing HDAC8 by ATAC-seq, ChIP-Seq and RNA-Seq showed that HDAC8 expression significantly increases chromatin accessibility, H3K27 promoter region acetylation and gene expression of IL-11. Increased IL-11 protein expression was confirmed by cytokine array in isogenic SM1 mouse melanoma cells expressing HDAC8.Overexpression of HDAC8 in the GEMM accelerated melanoma development and was associated with worse overall survival rate. Analysis of HDAC8 and control tumors by scRNA-seq and immunohistochemistry (IHC) showed that HDAC8 overexpressing tumors dramatically decreased CD4 and CD8 T cell infiltration and increased MDSCs and IL-11 expression. We next explored whether IL-11 drives the expansion and polarization of MDSCs and subsequent reduction in T cell infiltration and activation. A MDSCs polarization assay demonstrated that addition of IL-11 led to increased expansion and polarization of MDSCs from mouse bone marrow myeloid cells. A MDSCs suppression assay confirmed IL-11 induced MDSCs accumulation significantly reduced T cell proliferation and activation. We show for the first time that HDAC8 expression regulates immune escape by activating IL-11 expression and driving MDSCs expansion. Going forward, inhibition of HDAC8 activity is intriguing as a novel therapeutic strategy to reverse tumor derived immune suppression and to ultimately improve patient responses to immunotherapy. Citation Format: Chao Zhang, Michael Emmons, Richard Bennett, Jonathan Licht, Keiran Smalley. HDAC8 regulates immune escape in melanoma through increased transcription of inhibitory cytokines that increase accumulation of myeloid-derived suppressor cells (MDSCs) [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 3465.

Abstract 3606: HDAC8-mediated inhibition of EP300 drives a neural crest-like transcriptional state that increases melanoma brain metastasis

Abstract Melanomas are heterogeneous and adopt multiple transcriptional states that can confer an invasive phenotype and resistance to therapy. Little is known about the epigenetic drivers of these cell states, limiting our ability to regulate melanoma heterogeneity and tumor progression. Here we identify stress-induced HDAC8 activity as the driver of a neural crest stem cell (NCSC)-like transcriptional state that increased the formation of melanoma brain metastases (MBM). Exposure of melanocytes and melanoma cells to multiple different stresses led to HDAC8 activation, a switch to a NCSC gene expression signature and the adoption of an amoeboid, invasive phenotype. This cell state enhanced the survival of melanoma cells under shear stress conditions and increased the formation of metastases in the brain. Single cell RNA-seq analyses showed that HDAC8 expression was correlated with the NCSC cell state in clinical MBM specimens. ATAC-Seq and ChIP-Seq analysis showed HDAC8 to alter chromatin structure by increasing H3K27ac and accessibility at c-Jun binding sites without changing global histone acetylation. The increased accessibility of Jun binding sites was paralleled by decreased H3K27ac and accessibility at MITF binding sites and loss of melanoma-lineage gene expression. Mass spectrometry-based acetylomics demonstrated that HDAC8 deacetylated the histone acetyltransferase (HAT) EP300 leading to its enzymatic inactivation. This, in turn, led to an increased binding of EP300 to Jun-transcriptional sites and decreased binding to MITF-transcriptional sites. Increased expression of EP300 decreased invasion and increased the sensitivity of melanoma cells to multiple stresses while inhibition of EP300 function increased invasion and resistance to stress. We identified HDAC8 as a novel mediator of transcriptional co-factor inactivation and chromatin accessibility that increases MBM development. Citation Format: Michael Emmons, Richard Bennett, Alberto Riva, Chao Zhang, Robert Macaulay, Daphne Dupéré-Richér, Bin Fang, John Koomen, Jiannong Li, Ann Chen, Edward Seto, Jonathan Licht, Keiran Smalley. HDAC8-mediated inhibition of EP300 drives a neural crest-like transcriptional state that increases melanoma brain metastasis. [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 3606.

WNT16b promotes the proliferation and self-renewal of human limbal epithelial stem/progenitor cells via activating the calcium/calcineurin A/NFATC2 pathway.

Our previous finding revealed that WNT16b promoted the proliferation of human limbal epithelial stem cells (hLESCs) through a β-catenin independent pathway. Here, we aimed to explore its underlying molecular mechanism and evaluate its potential in the treatment of limbal stem cell deficiency (LSCD). Based on the findings of mRNA-sequencing, the expression of key molecules in WNT/calcineurin A/NFATC2 signalling pathway was investigated in WNT16b-co-incubated hLESCs and control hLESCs. An epithelial wound healing model was established on Wnt16b-KO mice to confirm the regulatory effect of WNT16b in vivo. The therapeutic potential of WNT16b-co-incubated hLESCs was also evaluated in mice with LSCD. Our findings showed that WNT16b bound with Frizzled7, promoted the release of Ca2+ and activated calcineurin A and NFATC2. With the translocation of NFATC2 into cell nucleus and the activation of HDAC3, WDR5 and GCN5L2, the expression of H3K4me3, H3K14ac and H3K27ac in the promoter regions of FoxM1 and c-MYC increased, which led to hLESC proliferation. The effect of the WNT16b/calcium/calcineurin A/NFATC2 pathway on LESC homeostasis maintenance and corneal epithelial repair was confirmed in Wnt16b-KO mice. Moreover, WNT16b-coincubated hLESCs could reconstruct a stable ocular surface and inhibit corneal neovascularization in mice with LSCD. In conclusion, WNT16b enhances the proliferation and maintains the stemness of hLESCs by activating the non-canonical calcium/calcineurin A/NFATC2 pathway in vitro and in vivo, and accelerates corneal epithelial wound healing.

Endothelial HDAC1-ZEB2-NuRD Complex Drives Aortic Aneurysm and Dissection Through Regulation of Protein S-Sulfhydration.

Aortic aneurysm and aortic dissection (AAD) are life-threatening vascular diseases, with endothelium being the primary target for AAD treatment. Protein S-sulfhydration is a newly discovered posttranslational modification whose role in AAD has not yet been defined. This study aims to investigate whether protein S-sulfhydration in the endothelium regulates AAD and its underlying mechanism. Protein S-sulfhydration in endothelial cells (ECs) during AAD was detected and hub genes regulating homeostasis of the endothelium were identified. Clinical data of patients with AAD and healthy controls were collected, and the level of the cystathionine γ lyase (CSE)/hydrogen sulfide (H2S) system in plasma and aortic tissue were determined. Mice with EC-specific CSE deletion or overexpression were generated, and the progression of AAD was determined. Unbiased proteomics and coimmunoprecipitation combined with mass spectrometry analysis were conducted to determine the upstream regulators of the CSE/H2S system and the findings were confirmed in transgenic mice. Higher plasma H2S levels were associated with a lower risk of AAD, after adjustment for common risk factors. CSE was reduced in the endothelium of AAD mouse and aorta of patients with AAD. Protein S-sulfhydration was reduced in the endothelium during AAD and protein disulfide isomerase (PDI) was the main target. S-sulfhydration of PDI at Cys343 and Cys400 enhanced PDI activity and mitigated endoplasmic reticulum stress. EC-specific CSE deletion was exacerbated, and EC-specific overexpression of CSE alleviated the progression of AAD through regulating the S-sulfhydration of PDI. ZEB2 (zinc finger E-box binding homeobox 2) recruited the HDAC1-NuRD complex (histone deacetylase 1-nucleosome remodeling and deacetylase) to repress the transcription of CTH, the gene encoding CSE, and inhibited PDI S-sulfhydration. EC-specific HDAC1 deletion increased PDI S-sulfhydration and alleviated AAD. Increasing PDI S-sulfhydration with the H2S donor GYY4137 or pharmacologically inhibiting HDAC1 activity with entinostat alleviated the progression of AAD. Decreased plasma H2S levels are associated with an increased risk of aortic dissection. The endothelial ZEB2-HDAC1-NuRD complex transcriptionally represses CTH, impairs PDI S-sulfhydration, and drives AAD. The regulation of this pathway effectively prevents AAD progression.

HDAC6-G3BP2 promotes lysosomal-TSC2 and suppresses mTORC1 under ETV4 targeting-induced low-lactate stress in non-small cell lung cancer.

TSC-mTORC1 inhibition-mediated translational reprogramming is a major adaptation mechanism upon many stresses, such as low-oxygen, -ATP, and -amino acids. But how cancer cells hijack the adaptive pathway to survive under low-lactate stress when targeting glycolysis-related signaling remains uncertain. ETV4 is an oncogenic transcription factor frequently dysregulated in human cancer. We previously found that ETV4 is associated with tumor progression and poor prognosis in non-small cell lung cancer (NSCLC). In this study, we report that ETV4 controls HK1 expression and glycolysis-lactate production to activate mTORC1 by relieving TSC2 repression of Rheb in NSCLC cells. Targeting ETV4-induced low-lactate stress is an important input for TSC2 to inhibit mTORC1 and global protein synthesis, while the core stress granule components G3BP2 and HDAC6 are selectively translated. Mechanistically, G3BP2 recruits lysosomal-TSC2 to suppress mTORC1. HDAC6 deacetylates TSC2 to sustain protein stability and associates with G3BP2 to facilitate more recruiting of TSC2 to inactivate mTORC1. In addition, the microtubule retrograde transport activity of HDAC6 drives the aggregate-like perinuclear-mTOR distribution paralleled by lower mTORC1 activity under stress. Thus, HDAC6-G3BP2 is the key complex that promotes lysosomal-TSC2 and suppresses mTORC1 when targeting ETV4, which might represent a critical adaptive mechanism for cell survival under low-lactate challenges.

Phenolic compounds as histone deacetylase inhibitors: binding propensity and interaction insights from molecular docking and dynamics simulations.

Histone deacetylases are well-established target enzymes involved in the pathology of different diseases including cancer and neurodegenerative disorders. The approved HDAC inhibitor drugs are associated with cellular toxicities. Different phenolic compounds have been shown to possess inhibitory activities against HDACs and are, therefore, considered safer alternatives to synthetic compounds. Here, we elucidated the binding mode and calculated the binding propensity of some of the top phenolic compounds against different isoforms representing different classes of Zn2+ ion-containing HDACs using the molecular docking approach. Our data reaffirmed the activity of the studied phenolic compounds against HDACs. Binding interaction analysis suggested that these compounds can block the activity of HDACs with or without binding to the active site zinc metal ion. Furthermore, molecular dynamics (MD) simulations were carried out on the selected crystal and docking complexes of each selected HDAC isoform. Analysis of root-mean-square displacement (RMSD) showed that the phenolic compounds demonstrated a stable binding mode over 50ns in a way that is comparable to the cocrystal ligands. Together, these findings can aid future efforts in the search for natural inhibitors of HDACs.

Introducing HDAC-Targeting Radiopharmaceuticals for Glioblastoma Imaging and Therapy.

Despite recent advances in multimodality therapy for glioblastoma (GB) incorporating surgery, radiotherapy, chemotherapy and targeted therapy, the overall prognosis remains poor. One of the interesting targets for GB therapy is the histone deacetylase family (HDAC). Due to their pleiotropic effects on, e.g., DNA repair, cell proliferation, differentiation, apoptosis and cell cycle, HDAC inhibitors have gained a lot of attention in the last decade as anti-cancer agents. Despite their known underlying mechanism, their therapeutic activity is not well-defined. In this review, an extensive overview is given of the current status of HDAC inhibitors for GB therapy, followed by an overview of current HDAC-targeting radiopharmaceuticals. Imaging HDAC expression or activity could provide key insights regarding the role of HDAC enzymes in gliomagenesis, thus identifying patients likely to benefit from HDACi-targeted therapy.

Nuclear SPHK2/S1P induces oxidative stress and NLRP3 inflammasome activation via promoting p53 acetylation in lipopolysaccharide-induced acute lung injury

A bulk of evidence identified that macrophages, including resident alveolar macrophages and recruited macrophages from the blood, played an important role in the pathogenesis of acute respiratory distress syndrome (ARDS). However, the molecular mechanisms of macrophages-induced acute lung injury (ALI) by facilitating oxidative stress and inflammatory responses remain unclear. Herein, we noticed that the levels of mitochondrial reactive oxygen species (mtROS), SPHK2 and activated NLRP3 inflammasome were higher in peripheral blood mononuclear cells (PBMCs) of ARDS patients than that in healthy volunteers. Similar observations were recapitulated in LPS-treated RAW264.7 and THP-1 cells. After exposure to LPS, the SPHK2 enzymatic activity, NLRP3 inflammasome activation and mtROS were significantly upregulated in macrophages. Moreover, knockdown SPHK2 via shRNA or inhibition SPHK2 could prominently decrease LPS-induced M1 macrophage polarization, oxidative stress and NLRP3 inflammasome activation. Further study indicated that upregulated SPHK2 could increase nuclear sphingosine-1-phosphate (S1P) levels and then restrict the enzyme activity of HDACs to facilitate p53 acetylation. Acetylation of p53 reinforced its binding to the specific region of the NLRP3 promoter and drove expression of NLRP3. In the in vivo experiments, it was also observed that treating with Opaganib (ABC294640), a specific SPHK2 inhibitor, could observably alleviate LPS-induced ALI, evidencing by lowered infiltration of inflammatory cells, increased M2 macrophages polarization and reduced oxidative damage in lung tissues. Besides, SPHK2 inhibition can also decrease the accumulation of acetylated p53 protein and the activation of NLRP3 inflammasome. Taken together, our results demonstrated for the first time that nuclear S1P can regulate the acetylation levels of non-histone protein through affecting HDACs enzyme activities, linking them to oxidative stress and inflammation in response to environmental signals. These data provide a theoretical basis that SPHK2 may be an effective therapeutic target of ARDS.

Zingiberene, a non-zinc-binding class I HDAC inhibitor: A novel strategy for the management of neuropathic pain

BackgroundEven though numerous Histone deacetylase inhibitors (HDACi) have been approved for the treatment of different types of cancer, and others are in clinical trials for the treatment of neurodegenerative diseases, the main problem related to the clinical use of available HDACi is their low isoform selectivity which causes undesirable effects and inevitably limits their therapeutic application. Previously, we demonstrated that a standardized Zingiber officinalis Roscoe rhizome extract (ZOE) reduced neuroinflammation through HDAC1 inhibition in a mice model of neuropathy, and this activity was related to terpenes fraction. Hypothesis/PurposeThe aim of this work was to identify the ZOE constituent responsible for the activity on HDAC1 and to study its possible application in trauma-induced neuropathic pain. MethodsThe ability of ZOE and its terpenes fraction (ZTE) to inhibit HDAC and SIRT isoforms activity and protein expression was assessed in vitro. Then, a structure-based virtual screening approach was applied to predict which constituent could be responsible for the activity. In the next step, the activity of selected compound was tested in an in vitro model of neuroinflammation and in an in vivo model of peripheral neuropathy (SNI). ResultsZTE resulted to be more potent than ZOE on HDAC1, 2, and 6 isoforms, while ZOE was more active on HDAC8. Zingiberene (ZNG) was found to be the most promising HDAC1 inhibitor, with an IC50 of 2.3 ± 0.1 µM. A non-zinc-binding mechanism of inhibition was proposed based on molecular docking. Moreover, the oral administration of ZNG reduced thermal hyperalgesia and mechanical allodynia in animals with neuropathy after 60 min from administration, and decreased HDAC-1 levels in the spinal cord microglia. ConclusionWe found a new non-zinc-dependent inhibitor of HDAC class I, with a therapeutic application in trauma-related neuropathic pain forms in which microglia-spinal overexpression of HDAC1 occurs. The non-zinc-binding mechanism has the potential to reduce off target effects, leading to a higher selectivity and better safety profile, compared to other HDAC inhibitors.

Regulation of cellular contractile force, shape and migration of fibroblasts by oncogenes and Histone deacetylase 6.

The capacity of cells to adhere to, exert forces upon and migrate through their surrounding environment governs tissue regeneration and cancer metastasis. The role of the physical contractile forces that cells exert in this process, and the underlying molecular mechanisms are not fully understood. We, therefore, aimed to clarify if the extracellular forces that cells exert on their environment and/or the intracellular forces that deform the cell nucleus, and the link between these forces, are defective in transformed and invasive fibroblasts, and to indicate the underlying molecular mechanism of control. Confocal, Epifluorescence and Traction force microscopy, followed by computational analysis, showed an increased maximum contractile force that cells apply on their environment and a decreased intracellular force on the cell nucleus in the invasive fibroblasts, as compared to normal control cells. Loss of HDAC6 activity by tubacin-treatment and siRNA-mediated HDAC6 knockdown also reversed the reduced size and more circular shape and defective migration of the transformed and invasive cells to normal. However, only tubacin-mediated, and not siRNA knockdown reversed the increased force of the invasive cells on their surrounding environment to normal, with no effects on nuclear forces. We observed that the forces on the environment and the nucleus were weakly positively correlated, with the exception of HDAC6 siRNA-treated cells, in which the correlation was weakly negative. The transformed and invasive fibroblasts showed an increased number and smaller cell-matrix adhesions than control, and neither tubacin-treatment, nor HDAC6 knockdown reversed this phenotype to normal, but instead increased it further. This highlights the possibility that the control of contractile force requires separate functions of HDAC6, than the control of cell adhesions, spreading and shape. These data are consistent with the possibility that defective force-transduction from the extracellular environment to the nucleus contributes to metastasis, via a mechanism that depends upon HDAC6. To our knowledge, our findings present the first correlation between the cellular forces that deforms the surrounding environment and the nucleus in fibroblasts, and it expands our understanding of how cells generate contractile forces that contribute to cell invasion and metastasis.

Network-based assessment of HDAC6 activity predicts preclinical and clinical responses to the HDAC6 inhibitor ricolinostat in breast cancer.

Inhibiting individual histone deacetylase (HDAC) is emerging as well-tolerated anticancer strategy compared with pan-HDAC inhibitors. Through preclinical studies, we demonstrated that the sensitivity to the leading HDAC6 inhibitor (HDAC6i) ricolinstat can be predicted by a computational network-based algorithm (HDAC6 score). Analysis of ~3,000 human breast cancers (BCs) showed that ~30% of them could benefice from HDAC6i therapy. Thus, we designed a phase 1b dose-escalation clinical trial to evaluate the activity of ricolinostat plus nab-paclitaxel in patients with metastatic BC (MBC) (NCT02632071). Study results showed that the two agents can be safely combined, that clinical activity is identified in patients with HR+/HER2- disease and that the HDAC6 score has potential as predictive biomarker. Analysis of other tumor types also identified multiple cohorts with predicted sensitivity to HDAC6i's. Mechanistically, we have linked the anticancer activity of HDAC6i's to their ability to induce c-Myc hyperacetylation (ac-K148) promoting its proteasome-mediated degradation in sensitive cancer cells.

Proteomics-based trapping with single or multiple inactive mutants reproducibly profiles histone deacetylase 1 substrates

Histone deacetylase 1 (HDAC1) plays a key role in diverse cellular processes. With the aberrant expression of HDAC1 linked to many diseases, including cancers, HDAC inhibitors have been used successfully as therapeutics. HDAC1 has been predominantly associated with histone deacetylation and gene expression. Recently, non-histone substrates have revealed diverse roles of HDAC1 beyond epigenetics. To augment discovery of non-histone substrates, we introduced “substrate trapping” to enrich HDAC1 substrates using an inactive mutant. Herein, we performed a series of proteomics studies to test the robustness of HDAC1 substrate trapping. Based on our recent results documenting that different HDAC1 mutants preferentially bound different substrates, which suggested that multiple mutants could be used for efficient trapping, trapping with three single point mutants simultaneously identified several potential substrates uniquely compared to a single mutant alone. However, a greater number of biologically interesting hits were observed using only a single mutant, which suggests that the C151A HDAC1 mutant is the optimal trap. Importantly, comparing independent trials with a single mutant performed by different experimentalists and HEK293 cell populations, trapping was robust and reproducible. Based on the reproducible trapping data, carnosine N-methyltransferase 1 (CARNMT1) was validated as an HDAC1 substrate. The data document that mutant trapping is an effective method for discovery of unanticipated HDAC substrates. SignificanceHistone deacetylase (HDAC) proteins are well established epigenetic transcriptional regulators that deacetylate histone substrates to control gene expression. More recently, deacetylation of non-histone substrates has linked HDAC activity to functions outside of epigenetics. Given the use of HDAC inhibitor drugs as anti-cancer therapeutics, understanding the full functions of HDAC proteins in cell biology is essential to future drug design. To discover unanticipated non-histone substrates and further characterize HDAC functions, inactive mutants have been used to “trap” putative substrates, which were identified with mass spectrometry-based proteomics analysis. Here multiple trapping studies were performed to test the robustness of using inactive mutants and proteomics for HDAC substrate discovery. The data confirm the value of trapping mutants as effective tools to discover HDAC substrates and link HDAC activity to unexpected biological functions.

Enterocyte-specific deletion of metal transporter Zip14 (Slc39a14) alters intestinal homeostasis through epigenetic mechanisms.

Zinc has anti-inflammatory properties using mechanisms that are unclear. Zip14 (Slc39a14) is a zinc transporter induced by proinflammatory stimuli and is highly expressed at the basolateral membrane of intestinal epithelial cells (IECs). Enterocyte-specific Zip14 ablation (Zip14ΔIEC) in mice was developed to study the functions of this transporter in enterocytes. This gene deletion led to increased intestinal permeability, increased IL-6 and IFNγ expression, mild endotoxemia, and intestinal dysbiosis. RNA sequencing was used for transcriptome profiling. These analyses revealed differential expression of specific intestinal proinflammatory and tight junction (TJ) genes. Binding of transcription factors, including NF-κβ, STAT3, and CDX2, to appropriate promoter sites of these genes supports the differential expression shown with chromatin immunoprecipitation assays. Total histone deacetylase (HDAC), and specifically HDAC3, activities were markedly reduced with Zip14 ablation. Intestinal organoids derived from ΔIEC mice display TJ and cytokine gene dysregulation compared with control mice. Differential expression of specific genes was reversed with zinc supplementation of the organoids. We conclude that zinc-dependent HDAC enzymes acquire zinc ions via Zip14-mediated transport and that intestinal integrity is controlled in part through epigenetic modifications.NEW & NOTEWORTHY We show that enterocyte-specific ablation of zinc transporter Zip14 (Slc39a14) results in selective dysbiosis and differential expression of tight junction proteins, claudin 1 and 2, and specific cytokines associated with intestinal inflammation. HDAC activity and zinc uptake are reduced with Zip14 ablation. Using intestinal organoids, the expression defects of claudin 1 and 2 are resolved through zinc supplementation. These novel results suggest that zinc, an essential micronutrient, influences gene expression through epigenetic mechanisms.

Effects of Suberoylanilide Hydroxamic Acid (SAHA) on the Inflammatory Response in Lipopolysaccharide-Induced N9 Microglial Cells.

Epigenetics has shown promising results for understanding the different behaviors of microglia under the context of neuroinflammation. However, to our knowledge, the results of this complex mechanism with novel pharmacological agents such as histone deacetylase inhibitors (HDACis) are still missing. In this study, we aimed to investigate the effects of suberoylanilide hydroxamic acid (SAHA), a pan-HDACi, on the lipopolysaccharide (LPS)-induced neuroinflammation model in the N9 microglial cells. Microglial cells were treated with SAHA (0.25, 0.5, 1.0, 1.25, 1.5 µM) and LPS (100 ng/mL) for 24 hours. Then, levels of the pro/anti-inflammatory cytokines interleukin-1 beta (IL-1β), IL-6, tumor necrosis factor alpha (TNF-α), and IL-10were determined by the enzyme-linked immunosorbent assay. The total cellular HDACactivity was determined by colorimetric analysis. Additionally, the expression levels of nuclear factor kappa-B (NF-κB) were quantified via western blotting. SAHA (1.0 and 1.25 µM) attenuated theLPS-induced inflammatory response of microglial cells via decreasing NF-κB expression and pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) in the N9 microglial cells. Moreover, SAHA treatment improved IL-10 levels and prevented the LPS-induced increase in the HDACactivity in the microglial cells. Our results suggest SAHA attenuates the LPS-induced inflammatory response in the N9 microglial cells, and regulation of histone acetylation withHDACis might be a rational approach for the treatment of neuroinflammation.

Histone acetylation functions in the wound-induced spore formation in nori.

The red macroalgae Pyropia yezoensis is one of the most economically important marine crops. In the asexual reproduction process, released archeospores could provide secondary seedling resources in nori farming and be used to establish asexual seeding strategies. We previously found that wounds could induce the somatic cells in sectioned Pyropia thalli to develop into large number of asexual wound-induced spores (WIS) in a short time. Many genes involved in signaling pathways, cell division, cell wall remodeling, etc. exhibited transcriptional variation in this cell fate transition process. However, the regulatory mechanisms controlling gene transcription remain elusive. In this study, we found that suberoylanilide hydroxamic acid (SAHA), the inhibitor of histone deacetylase, strongly repressed WIS formation after wounding. The lack of a sharp increase in HDAC activity after wounding, as well as the hyperacetylated status of histone H3 and H4, were observed in SAHA-treated thalli fragments, thus confirming a histone deacetylation-related epigenetic mechanism of wound-induced cell fate reprogramming. Moreover, histone deacetylation is required in the whole process of WIS formation and release. We further compared the genome-wide transcriptional variations after SAHA treatment. SAHA-responsive genes were identified, including some transcriptional factors, chromatin remodeling complex proteins, protein kinases, etc. Transcription of RBOH genes was also altered by SAHA, and moreover, ROS signals in cut fragments were attenuated, both indicating that the ROS systematic signaling pathway is closely associated with histone deacetylation. Our findings provide insights into the biological significance of dynamic histone acetylation states in WIS formation in P. yezoensis.

MDMX elevation by a novel Mdmx–p53 interaction inhibitor mitigates neuronal damage after ischemic stroke

Mdmx and Mdm2 are two major suppressor factors for the tumor suppressor gene p53. In central nervous system, Mdmx suppresses the transcriptional activity of p53 and enhances the binding of Mdm2 to p53 for degradation. But Mdmx dynamics in cerebral infarction remained obscure. Here we investigated the role of Mdmx under ischemic conditions and evaluated the effects of our developed small-molecule Protein–Protein Interaction (PPI) inhibitors, K-181, on Mdmx–p53 interactions in vivo and in vitro. We found ischemic stroke decreased Mdmx expression with increased phosphorylation of Mdmx Serine 367, while Mdmx overexpression by AAV-Mdmx showed a neuroprotective effect on neurons. The PPI inhibitor, K-181 attenuated the neurological deficits by increasing Mdmx expression in post-stroke mice brain. Additionally, K-181 selectively inhibited HDAC6 activity and enhanced tubulin acetylation. Our findings clarified the dynamics of Mdmx in cerebral ischemia and provide a clue for the future pharmaceutic development of ischemic stroke.

A neuronal cell-based reporter system for monitoring the activity of HDAC2.

Given that histone acetylation via histone acetyltransferases (HATs) and histone deacetylases (HDACs) is significant in memory formation, HDAC2 has been thoroughly investigated as a potential therapeutic target for the treatment of cognitive dysfunction. Although HDAC inhibitors have been discovered through in vitro enzyme assay, off-target effects on other HDACs are common due to their conserved catalytic domains. Each HDAC could be regulated by specific intracellular molecular mechanisms, raising the possibility that a cell-based assay could identify selective inhibitors targeting specific HDACs through their regulatory mechanisms. Here, we propose a versatile, cell-based reporter system for screening HDAC2 inhibitors. Through RNA-sequencing from human cultured neuronal cells, we determined that expression of a transcriptional repressor, inhibitor of DNA binding 1 (ID1), is increased by knockdown of HDAC2. We also established the knock-in neuronal cell lines of a bioluminescence reporter gene to ID1. The knock-in cell lines showed significant reporter activity by known HDAC inhibitors and by HDAC2-knockdown but not by HDAC1-knockdown. Thus, our neuronal cell-based reporter system is a promising method for screening the specific inhibitors of HDAC2 but not HDAC1, by potentially targeting not only HDAC2, but also the regulatory mechanisms of HDAC2 in neurons.

Abstract PR001: Mutant NPM1 hijacks transcriptional hub to maintain pathogenic gene programs and block differentiation in acute myeloid leukemia

Abstract Nucleophosmin (NPM1) is a ubiquitously expressed nucleolar protein with a wide range of functions including ribosome biogenesis, mRNA processing, and maintenance of genomic stability. In acute myeloid leukemia (AML), the terminal exon of NPM1 is often mutated (~30% of adult AMLs), changing the nucleolar localization signal into a nuclear export signal, which results in a shift of the protein to the cytoplasm (NPM1c). AMLs carrying this mutation have an aberrant expression of the HOXA genes, whose overexpression leads to leukemogenic transformation. The gene regulatory role of NPM1c has long been an enigma. Recently, it was shown that depletion or re-localization of the NPM1c protein into the nucleus [PG1] causes downregulation of the HOXA genes, leading to the speculation that NPM1c modulates transcription of HOXA genes. Here, we found NPM1c binds to chromatin and is located genome-wide at gene promoters and colocalized at active regions marked by H3K27ac in NPM1c leukemia cell lines and primary leukemia blasts. NPM1c clearly binds to known leukemia-driving genes - posterior HOXA, HOXB, and MEIS1/PBX3 genes as well as novel targets -IRX5 and NKX2-3. After PROTAC degradation of NPM1c, we observed a significant RNA polymerase II elongation defect genome-wide. The dramatic elongation defect is caused by the loss of the Super Elongation Complex (SEC) chromatin binding. Further tests suggest that loss of NPM1c leads to disruption of RNA polymerase II condensates as well as other direct transcriptional regulators of HOX genes - Menin and AFF4 through IDR interactions. We found that pulse PROTAC degradation of NPM1c leads to sustained differentiation with the loss of histone acetylation at NPM1c target loci. Further, NPM1c maintains the active chromatin state of target genes by antagonizing HDAC activity. Additionally, we found that NPM1c’s activation of the leukemia driving genes depends on the pre-established active chromatin state in the transformed hematopoietic stem and progenitor cells. NPM1c alone is not sufficient to activate these leukemia driving genes with heterochromatin marks accumulated. We also found that NPM1c binds to genomic regions that have been pre-bound by its export partner, XPO1. The XPO1 inhibitors Selinexor and Eltanexor can displace NPM1c from chromatin. The combination of clinical-grade XPO1 inhibitor eltanexor and Menin inhibitor MI-3454 displayed a strong synergy in inducing differentiation and reducing leukemia driving gene expression in the NPM1 mutated leukemia cell lines and PDX model, which promises to be an effective combination therapy for NPM1c leukemia patients. Citation Format: Xue Qing Wang, Dandan Fan, Yiman Liu, Qinyu Han, Jolanta Grembecka, Tomasz Cierpicki, Jianzhong Su, Shasha Chong, Liling Wan, Xiaotian Zhang. Mutant NPM1 hijacks transcriptional hub to maintain pathogenic gene programs and block differentiation in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR001.

A Preliminary Analysis of Chidamide Combined with R-GemOx (rituximab, gemcitabine and oxaliplatin) As Salvage Treatment for Transplant-Ineligible Patients with Relapsed/Refractory Diffuse Large B-Cell Lymphoma: A Prospective, Multicenter, Single-Arm, Phase II Study

Background: The clinical outcomes remain significantly poor for patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL), especially transplantation-ineligible R/R DLBCL. R-GemOx (rituximab, gemcitabine and oxaliplatin) is considered as one of the effective and acceptable side-effect therapeutic regimens for those patients. Chidamide, a histone deacetylase (HDAC) inhibitor, plays an anti-tumor effect by selectively inhibiting activity of HDAC1, 2, 3 and 10. Our preclinical finding indicated that chidamide combined with R-GemOx may have potential synergistic anti-tumor effect in DLBCL (unpublished data). Therefore, a prospective, multicenter, single-arm, phase II study (ClinicalTrials.gov: NCT04022005) was initiated to evaluate the efficacy and toxicity of chidamide in combination with R-GemOx (CR-GemOx) regimen for transplant-ineligible R/R DLBCL. Here, we reported the preliminary results of this ongoing study. Methods: Patients aged 18-75 years with transplantation-ineligible R/R DLBCL who failed anthracycline-based chemotherapy were enrolled. The CR-GemOx regimen was administered as follows: chidamide, 20 mg, P.O., twice per week; rituximab 375mg/m2, d1, intravenous drip; gemcitabine 1000mg/m2, d2, intravenous drip; oxaliplatin 100mg/m2, d2, intravenous drip. Repeat cycle every 21 days (up to 6 cycles). Patients achieved complete response (CR) or partial response (PR) were administered chidamide as maintenance therapy. The primary endpoint was overall response rate (ORR). Secondary endpoints included duration of response, progression-free survival (PFS), overall survival (OS) and toxicities. The response was evaluated by using Lugano 2014 criteria. The safety was assessed according to Common Terminology Criteria for Adverse Events version 5.0. Results: A total of 41 patients were enrolled between August 2019 and June 2022, including 26 patients (63.4%) with relapsed diseases and 15 patients (36.6%) with refractory diseases. The median age was 60 years (range: 26-74 years). Twenty patients were male (48.8%), and the median number of prior lines of therapy was 1 (range: 1-3). Baseline characteristics were listed in Table 1. All patients received response assessments, the ORR and CR rate were 61.0% and 31.7%, respectively. Notably, among 14 patients with bcl-2/c-myc double-expression (DE), 3 patients achieved CR (21.4%) and 5 patients had PR (35.7%). Sixteen patients received chidamide maintenance therapy. At the cut-off date of July 21st, 2022, the median PFS was 7.6 months (95% confidence interval: 2.7 - 12.5 months) with a median follow-up of 10.1 months. There were not significant differences in ORR (57.1% versus 64.0%, p=0.673) and median PFS (4.6 versus 12.4 months, p=0.079) between DE and non-DE patients. In the safety analysis population, the most common (≥20%) treatment-related adverse events (AEs) at any grade were neutropenia (65.9%, 27/41), thrombocytopenia (63.4%, 26/41), anemia (56.1%, 23/41), fatigue (43.9%, 18/41) (Table 2). Grade 3/4 AEs were neutropenia (41.5%, 17/41), thrombocytopenia (34.1%, 14/41), anemia (4.9%, 2/41), febrile neutropenia (2.4%, 1/41), diarrhea (2.4%, 1/41), and elevated triglyceride (2.4%, 1/41) (Table 2). Lung infection of grade ≥3 was observed in one patient only (2.4%, 1/41). Dose reductions of chidamide occurred in 13 patients due to AEs (thrombocytopenia, n=5; neutropenia, n=5; febrile neutropenia, n=1; diarrhea, n=2). Conclusion: The CR-GemOx regimen demonstrates encouraging efficacy in transplant-ineligible R/R DLBCL patients. The common AEs at any grade and grade 3/4 were neutropenia, and thrombocytopenia. Furthermore, lone-term efficacy and safety evaluation in larger cohort is ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal