HDAC Activity Research Articles

Platycodon grandiflorus polysaccharide inhibits the inflammatory response of 3D4/21 cells infected with PCV2

Porcine circovirus type 2 (PCV2) infection cause multi-systemic inflammation in pigs. Platycodon grandiflorus polysaccharide (PGPSt) has been reported to have the effects of immune regulation and disease resistance. Nevertheless, the role and mechanism of PGPSt in the inflammatory response of 3D4/21 cells induced by PCV2 infection remain unclear. The present study aims to investigate effects of PGPSt on inflammatory response and its possible underlying mechanisms in vitro models. Cells were treated with PCV2 for 36 h to construct a cell inflammation model. The 3D4/21 cell lines were pretreated with or without PGPSt, and the changes of inflammation-related markers and the signaling pathway were detected by CCK-8, ELISA, qPCR and Western blot. The results showed that PGPSt was non-toxic to cells and protected PCV2-infected cells from inflammatory damage. PGPSt could significantly inhibit the high acetylation of histone H3 (AcH3) and histone H4 (AcH4), down-regulate HAT and up-regulate HDAC activity, and reduce the expression of pro-inflammatory enzymes iNOS and COX-2 proteins levels. Then the levels of IL-1β, IL-6 and TNF-α were significantly inhibited, and the level of IL-10 was promoted. We also observed that PGPSt inhibited the phosphorylation of p65, p38 and Erk1/2, which subsequently inhibited nuclear translocation of NF-κB p65 to express pro-inflammatory factors. In conclusion, PGPSt can reduce the inflammatory response by regulating histone acetylation, reducing the release of inflammatory factors, reducing the expression of pro-inflammatory enzymes, and inhibiting the activation of NF-κB and MAPKs signaling pathways. This suggests that PGPSt had an anti-inflammatory effect on the inflammatory response caused by PCV2 infection, which provided theoretical data support for the research.

A novel HDAC6 inhibitor interferes microtubule dynamics and spindle assembly checkpoint and sensitizes cisplatin-induced apoptosis in castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (CRPC), the most refractory prostate cancer, inevitably progresses and becomes unresponsive to hormone therapy, revealing a pressing unmet need for this disease. Novel agents targeting HDAC6 and microtubule dynamics can be a potential anti-CRPC strategy. Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay and anchorage-dependent colony formation assay. Flow cytometric analysis of propidium iodide staining was used to determine cell-cycle progression. Cell-based tubulin polymerization assay and confocal immunofluorescence microscopic examination determine microtubule assembly/disassembly status. Protein expressions were determined using Western blot analysis. A total of 82 novel derivatives targeting HDAC6 were designed and synthesized, and Compound 25202 stood out, showing the highest efficacy in blocking HDAC6 (IC50, 3.5 nM in enzyme assay; IC50, 1.0 μM in antiproliferative assay in CRPC cells), superior to tubastatin A (IC50, 5.4 μM in antiproliferative assay). The selectivity and superiority of 25202 were validated by examining the acetylation of both α-tubulin and histone H3, detecting cell apoptosis and HDACs enzyme activity assessment. Notably, 25202 but not tubastatin A significantly decreased HDAC6 protein expression. 25202 prolonged mitotic arrest through the detection of cyclin B1 upregulation, Cdk1 activation, mitotic phosphoprotein levels, and Bcl-2 phosphorylation. Compound 25202 did not mimic docetaxel in inducing tubulin polymerization but disrupted microtubule organization. Compound 25202 also increased the phosphorylation of CDC20, BUB1, and BUBR1, indicating the activation of the spindle assembly checkpoint (SAC). Moreover, 25202 profoundly sensitized cisplatin-induced cell death through impairment of cisplatin-evoked DNA damage response and DNA repair in both ATR-Chk1 and ATM-Chk2 pathways. The data suggest that 25202 is a novel selective and potent HDAC6 inhibitor. Compound 25202 blocks HDAC6 activity and interferes microtubule dynamics, leading to SAC activation and mitotic arrest prolongation that eventually cause apoptosis of CRPC cells. Furthermore, 25202 sensitizes cisplatin-induced cell apoptosis through impeding DNA damage repair pathways.

Cleavage of HDAC6 to dampen its antiviral activity by nsp5 is a common strategy of swine enteric coronaviruses.

HDAC6, a structurally and functionally unique member of the histone deacetylase (HDAC) family, is an important host factor that restricts viral infection. The broad-spectrum antiviral activity of HDAC6 makes it a potent antiviral agent. Previously, we found that HDAC6 functions to antagonize porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus with zoonotic potential. However, the final outcome is typically a productive infection that materializes as cells succumb to viral infection, indicating that the virus has evolved sophisticated mechanisms to combat the antiviral effect of HDAC6. Here, we demonstrate that PDCoV nonstructural protein 5 (nsp5) can cleave HDAC6 at glutamine 519 (Q519), and cleavage of HDAC6 was also detected in the context of PDCoV infection. More importantly, the anti-PDCoV activity of HDAC6 was damaged by nsp5 cleavage. Mechanistically, the cleaved HDAC6 fragments (amino acids 1-519 and 520-1159) lost the ability to degrade PDCoV nsp8 due to their impaired deacetylase activity. Furthermore, nsp5-mediated cleavage impaired the ability of HDAC6 to activate RIG-I-mediated interferon responses. We also tested three other swine enteric coronaviruses (transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and swine acute diarrhea syndrome-coronavirus) and found that all these coronaviruses have adopted similar mechanisms to cleave HDAC6 in both an overexpression system and virus-infected cells, suggesting that cleavage of HDAC6 is a common strategy utilized by swine enteric coronaviruses to antagonize the host's antiviral capacity. Together, these data illustrate how swine enteric coronaviruses antagonize the antiviral function of HDAC6 to maintain their infection, providing new insights to the interaction between virus and host.IMPORTANCEViral infections and host defenses are in constant opposition. Once viruses combat or evade host restriction, productive infection is achieved. HDAC6 is a broad-spectrum antiviral protein that has been demonstrated to inhibit many viruses, including porcine deltacoronavirus (PDCoV). However, whether HDAC6 is reciprocally targeted and disabled by viruses remains unclear. In this study, we used PDCoV as a model and found that HDAC6 is targeted and cleaved by nsp5, a viral 3C-like protease. The cleaved HDAC6 loses its deacetylase activity as well as its ability to degrade viral proteins and activate interferon responses. Furthermore, this cleavage mechanism is shared among other swine enteric coronaviruses. These findings shed light on the intricate interplay between viruses and HDAC6, highlighting the strategies employed by viruses to evade host antiviral defenses.

CYLD induces high oxidative stress and DNA damage through class I HDACs to promote radiosensitivity in nasopharyngeal carcinoma

Abnormal expression of Cylindromatosis (CYLD), a tumor suppressor molecule, plays an important role in tumor development and treatment. In this work, we found that CYLD binds to class I histone deacetylases (HDAC1 and HDAC2) through its N-terminal domain and inhibits HDAC1 activity. RNA sequencing showed that CYLD-HDAC axis regulates cellular antioxidant response via Nrf2 and its target genes. Then we revealed a mechanism that class I HDACs mediate redox abnormalities in CYLD low-expressing tumors. HDACs are central players in the DNA damage signaling. We further confirmed that CYLD regulates radiation-induced DNA damage and repair response through inhibiting class I HDACs. Furthermore, CYLD mediates nasopharyngeal carcinoma cell radiosensitivity through class I HDACs. Thus, we identified the function of the CYLD-HDAC axis in radiotherapy and blocking HDACs by Chidamide can increase the sensitivity of cancer cells and tumors to radiation therapy both in vitro and in vivo. In addition, ChIP and luciferase reporter assays revealed that CYLD could be transcriptionally regulated by zinc finger protein 202 (ZNF202). Our findings offer novel insight into the function of CYLD in tumor and uncover important roles for CYLD-HDAC axis in radiosensitivity, which provide new molecular target and therapeutic strategy for tumor radiotherapy.

Ribosomal frameshifting at normal codon repeats recodes functional chimeric proteins in human.

Ribosomal frameshifting refers to the process that ribosomes slip into +1 or -1 reading frame, thus produce chimeric trans-frame proteins. In viruses and bacteria, programmed ribosomal frameshifting can produce essential trans-frame proteins for viral replication or regulation of other biological processes. In humans, however, functional trans-frame protein derived from ribosomal frameshifting is scarcely documented. Combining multiple assays, we show that short codon repeats could act as cis-acting elements that stimulate ribosomal frameshifting in humans, abbreviated as CRFS hereafter. Using proteomic analyses, we identified many putative CRFS events from 32 normal human tissues supported by trans-frame peptides positioned at codon repeats. Finally, we show a CRFS-derived trans-frame protein (HDAC1-FS) functions by antagonizing the activities of HDAC1, thus affecting cell migration and apoptosis. These data suggest a novel type of translational recoding associated with codon repeats, which may expand the coding capacity of mRNA and diversify the regulation in human.

A GSH-resistant FK228 analogue containing a stable disulfide bond

FK228 is a potent natural pan HDAC inhibitor approved by the FDA for the treatment of cutaneous T-cell lymphoma as well as peripheral T-cell lymphoma. It is generally believed that the mechanism of FK228 acting on HDACs is by reducing its disulfide bond after entering the cell, and the dithiol group may chelate with Zn2+ and form a weak reversible covalent bond with cysteine in the catalytic pocket of HDACs, therefore inhibiting the activity of HDACs. However, due to the weak stability of the disulfide bond in FK228, it has been difficult to obtain direct evidence for the above conjecture. Thus, improving the stability of the FK228 disulfide bond will help to explore the exact mechanism of FK228. In this study, based on the stability and target-induced covalent properties of the Cysteine-Penicillamine (Cys-Pen) disulfide bond reported previously, the Pen was introduced into the modification of FK228. Specifically, the d-Cys in FK228 was replaced by d-Pen, the total synthetic pathway was optimized, and the novel synthetic FK228 analogue (FK-P) stability was verified. FK-P can also be used as a new drug molecule in the future to participate in the research of related biological mechanisms or the treatment of diseases.

Propionate functions as a feeding state-dependent regulatory metabolite to counter proinflammatory signaling linked to nutrient load and obesity.

Generally, fasting and refeeding confer anti- and proinflammatory effects, respectively. In humans, these caloric-load interventions function, in part, via regulation of CD4+ T cell biology. However, mechanisms orchestrating this regulation remain incomplete. We employed integrative bioinformatics of RNA sequencing and high-performance liquid chromatography-mass spectrometry data to measure serum metabolites and gene expression of peripheral blood mononuclear cells isolated from fasting and refeeding in volunteers to identify nutrient-load metabolite-driven immunoregulation. Propionate, a short chain fatty acid (SCFA), and the SCFA-sensing G protein-coupled receptor 43 (ffar2) were coordinately and inversely regulated by fasting and refeeding. Propionate and free fatty acid receptor agonists decreased interferon-γ and interleukin-17 and significantly blunted histone deacetylase activity in CD4+ T cells. Furthermore, propionate blunted nuclear factor κB activity and diminished interleukin-6 release. In parallel, propionate reduced phosphorylation of canonical T helper 1 (TH1) and TH17 regulators, STAT1 and STAT3, respectively. Conversely, knockdown of free fatty acid receptors significantly attenuated the anti-inflammatory role of propionate. Interestingly, propionate recapitulated the blunting of CD4+ TH cell activation in primary cells from obese individuals, extending the role of this metabolite to a disease associated with low-grade inflammation. Together, these data identify a nutrient-load responsive SCFA-G protein-coupled receptor linked pathway to regulate CD4+ TH cell immune responsiveness.

HRpn13 shapes the proteome and transcriptome through epigenetic factors HDAC8, PADI4, and transcription factor NF-κB p50

The anti-cancer target hRpn13 is a proteasome substrate receptor. However, hRpn13-targeting molecules do not impair its interaction with proteasomes or ubiquitin, suggesting other critical cellular activities. We find that hRpn13 depletion causes correlated proteomic and transcriptomic changes, with pronounced effects in myeloma cells for cytoskeletal and immune response proteins and bone-marrow-specific arginine deiminase PADI4. Moreover, a PROTAC against hRpn13 co-depletes PADI4, histone deacetylase HDAC8, and DNA methyltransferase MGMT. PADI4 binds and citrullinates hRpn13 and proteasomes, and proteasomes from PADI4-inhibited myeloma cells exhibit reduced peptidase activity. When off proteasomes, hRpn13 can bind HDAC8, and this interaction inhibits HDAC8 activity. Further linking hRpn13 to transcription, its loss reduces nuclear factor κB (NF-κB) transcription factor p50, which proteasomes generate by cleaving its precursor protein. NF-κB inhibition depletes hRpn13 interactors PADI4 and HDAC8. Altogether, we find that hRpn13 acts dually in protein degradation and expression and that proteasome constituency and, in turn, regulation varies by cell type.

Epigenetic and Metabolic Reprogramming of Fibroblasts in Crohn's Disease Strictures Reveals Histone Deacetylases as Therapeutic Targets.

No effective therapeutic intervention exists for intestinal fibrosis in Crohn's disease [CD]. We characterized fibroblast subtypes, epigenetic and metabolic changes, and signalling pathways in CD fibrosis to inform future therapeutic strategies. We undertook immunohistochemistry, metabolic, signalling pathway and epigenetic [Transposase-Accessible Chromatin using sequencing] analyses associated with collagen production in CCD-18Co intestinal fibroblasts and primary fibroblasts isolated from stricturing [SCD] and non-stricturing [NSCD] CD small intestine. SCD/NSCD fibroblasts were cultured with TGFβ and valproic acid [VPA]. Stricturing CD was characterized by distinct histone deacetylase [HDAC] expression profiles, particularly HDAC1, HDAC2, and HDAC7. As a proxy for HDAC activity, reduced numbers of H3K27ac+ cells were found in SCD compared to NSCD sections. Primary fibroblasts had increased extracellular lactate [increased glycolytic activity] and intracellular hydroxyproline [increased collagen production] in SCD compared to NSCD cultures. The metabolic effect of TGFβ stimulation was reversed by the HDAC inhibitor VPA. SCD fibroblasts appeared 'metabolically primed' and responded more strongly to both TGFβ and VPA. Treatment with VPA revealed TGFβ-dependent and TGFβ-independent Collagen-I production in CCD-18Co cells and primary fibroblasts. VPA altered the epigenetic landscape with reduced chromatin accessibility at the COL1A1 and COL1A2 promoters. Increased HDAC expression profiles, H3K27ac hypoacetylation, a significant glycolytic phenotype and metabolic priming characterize SCD-derived as compared to NSCD fibroblasts. Our results reveal a novel epigenetic component to Collagen-I regulation and TGFβ-mediated CD fibrosis. HDAC inhibitor therapy may 'reset' the epigenetic changes associated with fibrosis.

Exploration of 5-Azacytidine and Trichostatin A in the modulation of postharvest quality of 'Shine Muscat' grape berries

Grapes have excellent commercial characteristics and high nutritional value, but postharvest storage inevitably reduces their quality. Previously, we studied the regulatory mechanisms of methylation for grape ripening. To further investigate the effects of epigenetic modifications on postharvest quality of grape berries, Transcriptome deep sequencing (RNA-seq) and metabolome sequencing were performed on 5-Azacytidine (5azaC) or Trichostatin A (TSA) treated grape berries. TSA delayed the depletion of soluble sugar and organic acid content in postharvest grapes compared to controls, while 5azaC had the opposite effect. In addition, TSA and 5azaC affected the content and composition of volatile organic compounds (VOCs) in the berries, of which the decrease in linalool content may be directly responsible for the reduction of muscat flavor in the berries. RNA-Seq analysis showed that 626 and 2253 differentially expressed genes (DEGs) were generated by 5azaC and TSA treatments, respectively. GO and KEGG enrichment analyses revealed significant differences in the biological pathways involved in the two collections of DEGs. Metabolomic analyses further clarified the marker metabolites induced by TSA and 5azaC related to fruit quality. In addition, the results of HDAC activity and transcript levels of acetylation-related genes suggest that VvHDA15 may acetylation modification affects the key regulators of postharvest fruit quality. This study not only provides abundant data information, but also offers new insights into the regulation of fruit quality by epimodification.

Exploration of quinazoline-tethered hydroxamic acid derivatives as HDAC inhibitors for anticancer activity: Design, synthesis, molecular docking, and biological evaluation

In the past two decades, targeted cancer therapy has emerged as a novel class of anticancer therapeutics, besides traditional chemotherapy, surgery, and radiotherapy. Inhibition of HDACs represents a promising avenue for cancer therapeutics and initial success in treating select T-cell lymphomas has led to the FDA approval of four HDAC-targeted agents. The majority of currently available HDAC inhibitors have weak anticancer activity, with significant side effects. To address these drawbacks, in the present work we have designed and synthesized a novel series of histone deacetylase inhibitors combining 2-(4-substituted phenyl) quinazoline bioactive fragment as cap group and N-hydroxycinnamamide/ N-hydroxybenzamide as linker group, and evaluated for their cytotoxic activity against the three cancer cell lines (HeLa, MCF-7 and HCT-116) and HDAC inhibitory activity. All three cancer cell lines were most sensitive to the N-hydroxyacrylamide derivatives (5a-f) as compared to N-hydroxybenzamides (7a-f) derivatives. In particular, compound 5a (0.39, 0.26, 0.41 µM respectively) and 5b (0.27, 0.57, 0.32 µM respectively) were found to be the most potent derivatives among all tested cell lines. For HDAC inhibitory activity, it was found that compounds 5a, 5b, and 5d possessed comparable, even better HDAC inhibitory activity as compared to SAHA. These compounds also tested in vitro heamolytic activity, most of the compounds found biocompatible with human erythrocytes thereby evidencing the lack of haemolytic toxicity of anticancer drugs. In silico ADMET and molecular docking studies further corroborated with the experimental results. Overall, the new synthesized compounds exhibited significant HDAC and cytotoxic activity. Also, the different substituents and unsaturated bond influenced both HDAC inhibitory and cytotoxic activity. The outcome of the current work is that several potential HDAC inhibitors have emerged for further design of more potent HDAC inhibitors with better cytotoxicity.

A histone deacetylase network regulates epigenetic reprogramming and viral silencing in HIV-infected cells

A long-lived latent reservoir of HIV-1-infected CD4 Tcells persists with antiretroviral therapy and prevents cure. We report that the emergence of latently infected primary CD4 Tcells requires the activity of histone deacetylase enzymes HDAC1/2 and HDAC3. Data from targeted HDAC molecules, an HDAC3-directed PROTAC, and CRISPR-Cas9 knockout experiments converge on a model where either HDAC1/2 or HDAC3 targeting can prevent latency, whereas all three enzymes must be targeted to achieve latency reversal. Furthermore, HDACi treatment targets features of memory Tcells that are linked to proviral latency and persistence. Latency prevention is associated with increased H3K9ac at the proviral LTR promoter region and decreased H3K9me3, suggesting that this epigenetic switch is a key proviral silencing mechanism that depends on HDAC activity. These findings support further mechanistic work on latency initiation and eventual clinical studies of HDAC inhibitors to interfere with latency initiation.

HDAC8-mediated inhibition of EP300 drives a transcriptional state that increases melanoma brain metastasis

Melanomas can adopt multiple transcriptional states. Little is known about the epigenetic drivers of these cell states, limiting our ability to regulate melanoma heterogeneity. Here, we identify stress-induced HDAC8 activity as driving melanoma brain metastasis development. Exposure of melanocytes and melanoma cells to multiple stresses increases HDAC8 activation leading to a neural crest-stem cell transcriptional state and an amoeboid, invasive phenotype that increases seeding to the brain. Using ATAC-Seq and ChIP-Seq we show that increased HDAC8 activity alters chromatin structure by increasing H3K27ac and enhancing accessibility at c-Jun binding sites. Functionally, HDAC8 deacetylates the histone acetyltransferase EP300, causing its enzymatic inactivation. This, in turn, increases binding of EP300 to Jun-transcriptional sites and decreases binding to MITF-transcriptional sites. Inhibition of EP300 increases melanoma cell invasion, resistance to stress and increases melanoma brain metastasis development. HDAC8 is identified as a mediator of transcriptional co-factor inactivation and chromatin accessibility that drives brain metastasis.

Dysregulated Alternative Splicing in Inv(16) Acute Myeloid Leukemia Via U2AF1 Deacetylation during DNA Damage Response

Inversion of chromosome 16, inv(16) [inv(16)(p13q22) or t(16;16)(p13.1;q22)], is a recurrent chromosomal translocation observed in 5-8% of acute myeloid leukemia (AML) cases. Inv(16) creates a fusion gene CBFb-MYH11 (CM) which impairs hematopoietic differentiation and creates abnormal progenitor populations prone to leukemic transformation. We reported that CM interacts with HDAC8 and enhances its activity, and high HDAC8 activity impaired DNA damage response (DDR) in CM knock-in (KI) hematopoietic stem and progenitor cells (HSPCs) (Zhang, L et al, ASH meeting 2022, https://doi.org/10.1182/blood-2022-160280). The recruitment of BRCA1-mRNA splicing machinery is critical for efficient DDR and the U2 small nuclear RNA auxiliary factor 1 (U2AF1) is required for accurate 3'-splice site selection. Here, we show how U2AF1 is post-translationally regulated following DNA damage and its impact on alternative splicing in CM KI HSPCs upon DNA damage. First, we identified TIP60 as the histone acetyltransferase (HAT) responsible for the increased U2AF1 acetylation induced by ionizing radiation (IR). We detected enhanced interaction between U2AF1 and TIP60 but not with other HATs (GCN5, PCAF, CBP) along with the increased U2AF1 acetylation in response to IR. In addition, TIP60 knockdown (KD) reduced U2AF1 acetylation, while TIP60 expression increased U2AF1 acetylation, indicating that TIP60 mediates the acetylation of U2AF1. To pinpoint the specific lysine residue subjected to TIP60 catalysis, we introduced lysine site-specific mutants U2AF1-HA-K15R, K23R, K39R, K175R, or 4 lysine sites mutant (U2AF1-HA-4mut) with TIP60. Co-immunoprecipitation (anti-HA) followed by western blotting (anti-acetyl-lysine) showed undetectable acetylation of U2AF1-HA-K23R and K175R mutants (similar to U2AF1-HA-4mut), suggesting that K23 and K175 residues are essential for TIP60-mediated acetylation. To investigate the impact of K23 or K175 acetylation on the assembly of the BRCA1-mRNA splicing complex and DDR, we subjected U2AF1-K23R or K175R expressing 32D cells to IR (3.5 Gy). Both U2AF1-K23R and K175R mutants exhibited markedly reduced acetylation levels after IR, confirming that K23 and K175 sites are the acetylation sites responding to DNA damage. Furthermore, the interaction of U2AF1-K23R and K175R with BRCA1 and BCLAF1 was impaired. We observed increased apoptosis in U2AF1-K23R and K175R cells compared to wild-type (WT) U2AF1 cells (K23R vs WT, 51.975±7.867% vs 6.055±0.634%, P=0.0011; K175R vs WT,22.645±3.037% vs 6.055±0.634%, P=0.0017) after KD of endogenous U2AF1 and subjected to IR (3.5 Gy). These results indicate that U2AF1 is acetylated by TIP60 upon DNA damage on K23 and K175 which is critical for cell survival and assembly of the BRCA1-mRNA splicing complex in DDR. Our studies revealed that HDAC8 interacts with U2AF1 and modulates U2AF1 acetylation following DNA damage and acetylation on U2AF1-K23 is important for HDAC8 binding. Consistent with the enhanced HDAC8 activity induced by CM, we show that CM also diminished U2AF1 acetylation and impaired the assembly of the BRCA1-mRNA splicing complex after IR. To examine the consequences in alternative splicing events upon IR, we sorted CM KI (n=5) or control (n=3) Lin-Sca1+Kit+ (LSK) cells and exposed them to IR (2.0 Gy) followed by RNA-seq. We identified a total of 829 splicing events in WT-IR vs WT-NIR (NIR: no IR) and 643 splicing events in CM-IR vs CM-NIR. Specifically, we found that skipped exon (SE) and retained intron (RI) events are selectively reduced in CM vs WT (SE events: 496 vs 653; RI events: 2 vs 26). Gene set enrichment analysis revealed striking differential enrichment in 38 BRCA1-related pathways, including regulation of response to DNA damage stimulus (NES: -1.0816 vs 0.8707), regulation of DNA repair (NES: -1.1557 vs 0.7729), cell cycle checkpoint signaling (NES: -0.9719 vs 0.7895), and recombinational repair (NES: -0.9215 vs 0.7299). Altogether, these studies reveal mechanistic insights into DNA damage induced regulation of post-translational acetylation of U2AF1 splicing factor and provide a mechanistic link to the dysregulated alternative splicing and impaired DNA damage response in inv(16) AML.

Butyrate functions as a histone deacetylase inhibitor to protect pancreatic beta cells from IL-1β-induced dysfunction.

Butyrate, a gut microbial metabolite, has beneficial effects on glucose homeostasis and has become an attractive drug candidate for type 2 diabetes (T2D). Recently, we showed that butyrate protects pancreatic beta cells against cytokine-induced dysfunction. In this study, we explored the underlying mechanisms of butyrate action. Pancreatic mouse islets were exposed to a non-cytotoxic concentration of interleukin-1β (IL-1β) for 10 days to mimic low-grade inflammation in T2D. Similar to the effect of butyrate, an isoform-selective histone deacetylase 3 (HDAC3) inhibitor normalized IL-1β-reduced glucose-stimulated insulin secretion and insulin content. In contrast, free fatty acid receptor 2 and 3 (FFAR2/3) agonists failed to normalize IL-1β-induced beta cell dysfunction. Furthermore, butyrate inhibited HDAC activity and increased the acetylation of histone H3 and H4 by 3- and 10-fold, respectively. Genome-wide analysis of histone H3 lysine 27 acetylation (H3K27ac) revealed that butyrate mainly increased H3K27ac at promoter regions (74%), while H3K27ac peaks regulated by IL-1β were more equally distributed at promoters (38%), introns (23%) and intergenic regions (23%). Gene ontology analysis showed that butyrate increased IL-1β-reduced H3K27ac levels near several genes related to hormone secretion and reduced IL-1β-increased H3K27ac levels near genes associated with inflammatory responses. Butyrate alone increased H3K27ac near many genes related to MAPK signaling, hormone secretion, and differentiation, and decreased H3K27ac at genes involved in cell replication. Together, these results suggest that butyrate prevents IL-1β-induced pancreatic islet dysfunction by inhibition of HDACs resulting in changes in H3K27ac levels at genes relevant for beta cell function and inflammatory responses.

HDAC3 Is Required for Pathognomonic Features of Langerhans Cell Histiocytes

Langerhans cell histiocytosis (LCH) is a pediatric inflammatory myeloid neoplasm that develops due to dysregulated myeloid cell development. BRAFV600E is the most common disease-causing mutation and constitutively activates the mitogen-activated protein kinase (MAPK) pathway in myeloid lineage precursors leading to the key pathognomonic features of LCH cells. Enhanced myelopoiesis and reduced CCR7 expression promote accumulation of LCH cells in tissues by simultaneously increasing the production of pathological DCs and preventing tissue egress. Furthermore, LCH cells acquire an oncogene induced senescence-associated secretory phenotype (SASP) that depends on mammalian target of rapamycin (mTOR), which is hallmarked by increased expression of anti-apoptotic proteins, inflammatory cytokines, and matrix metalloproteinases enhancing survival of LCH cells and promoting recruitment of inflammatory immune cells forming characteristic granulomatous lesions. These pathognomonic features result in the accumulation of LCH cells in any organ causing a wide range of clinical symptoms. Frontline therapy for LCH involves combination chemotherapy and steroidal anti-inflammatories, or MAPK inhibitors, which have significant toxicity and fail to eliminate disease causing precursors. New therapeutic approaches are urgently needed. Here, using multiple genetic mouse models, we show that normal epidermal Langerhans cells (LCs) depend on HDAC3 for their development, differentiation, and survival. Integrative RNA and chromatin-immunoprecipitation-sequencing show loss of HDAC3 abrogates the expression of master regulators of myeloid development and function including Csf1r, Spi1, Id2 and Runx3. LCH cells are also known to rely on Csf1r and Pu.1 for their development and homeostasis, thus we hypothesized that LCH cells similarly rely on HDAC3 for their development and survival. CD11c Cre LSL-BRAFV600E ( BRAFV600E CD11c) mice develop severe multifocal LCH with pronounced lesion development in their livers and lungs, hepatosplenomegaly, and a reduced lifespan due to the accumulation of pathological dendritic cells (DCs). We generated BRAFV600E CD11c HDAC3 fl/fl ( BRAFV600E HD3KO) mice, which produce pathological DCs that simultaneously express BRAFV600E and harbor a conditional deletion in the deacetylase domain of HDAC3. Compared to BRAFV600E CD11c mice, BRAFV600E HD3KO mice exhibited significantly less hepatosplenomegaly, reduced lesional burden (Panel A), attenuated disease progression, and improved survival indicating reduced LCH disease burden. Compared to BRAFV600E CD11c flow cytometry showed BRAFV600E HD3KO had reduced numbers of LCH cells in lungs and livers linking improved disease outcomes to abrogation of pathological DCs. Flow cytometric analysis of circulating myeloid cells further found reduced frequency of circulating DCs and DC progenitors, indicating that a lack of HDAC3 activity prevents the development of pathological DCs (Panel B). LCH-like cells can be generated in vitro by culturing BRAFV600E CD11c bone marrow with granulocyte-monocyte colony-stimulating factor (GM-CSF), providing a valuable drug screening tool. Treating LCH-like cells with RGFP966, an HDAC3-specific inhibitor, increased apoptosis indicated by annexin-V and DAPI staining, reduced expression of Bcl-2, increased CCR7 expression, and decreased S6 phosphorylation (an indication of decreased mTOR activity), showing that pharmacological inhibition of HDAC3 may prove therapeutically efficacious by abrogating pathognomonic features of LCH cells. Together, our findings identify HDAC3 as a critical epigenetic regulator for both healthy and pathological LCs. We further show, HDAC3 is required for multiple pathognomonic features of LCH cells and could be a promising drug target. Furthermore, if HDAC3 is required for the development of pathological DC and DC progenitors, HDAC3 blockade would address a great need in treatment of patients with LCH.

Histone deacetylation and cytosine methylation compartmentalize heterochromatic regions in the genome organization of Neurospora crassa.

Chromosomes must correctly fold in eukaryotic nuclei for proper genome function. Eukaryotic organisms hierarchically organize their genomes, including in the fungus Neurospora crassa, where chromatin fiber loops compact into Topologically Associated Domain-like structures formed by heterochromatic region aggregation. However, insufficient data exist on how histone posttranslational modifications (PTMs), including acetylation, affect genome organization. In Neurospora, the HCHC complex [composed of the proteins HDA-1, CDP-2 (Chromodomain Protein-2), Heterochromatin Protein-1, and CHAP (CDP-2 and HDA-1 Associated Protein)] deacetylates heterochromatic nucleosomes, as loss of individual HCHC members increases centromeric acetylation, and alters the methylation of cytosines in DNA. Here, we assess whether the HCHC complex affects genome organization by performing Hi-C in strains deleted of the cdp-2 or chap genes. CDP-2 loss increases intra- and interchromosomal heterochromatic region interactions, while loss of CHAP decreases heterochromatic region compaction. Individual HCHC mutants exhibit different patterns of histone PTMs genome-wide, as CDP-2 deletion increases heterochromatic H4K16 acetylation, yet smaller heterochromatic regions lose H3K9 trimethylation and gain interheterochromatic region interactions; CHAP loss produces minimal acetylation changes but increases heterochromatic H3K9me3 enrichment. Loss of both CDP-2 and the DIM-2 DNA methyltransferase causes extensive genome disorder as heterochromatic-euchromatic contacts increase despite additional H3K9me3 enrichment. Our results highlight how the increased cytosine methylation in HCHC mutants ensures genome compartmentalization when heterochromatic regions become hyperacetylated without HDAC activity.

Mettl3-Mediated m6A Methylation Controls Pancreatic Bipotent Progenitor Fate and Islet Formation.

The important role of m6A RNA modification on β cell function has been established, yet how it regulates pancreas development and endocrine differentiation remains unknown. Here, we generated transgenic mice lacking RNA methyltransferase-like 3 (Mettl3) specifically in Pdx1+ pancreatic progenitor cells and found the mutant mice developed hyperglycemia and hypo-insulinemia at 2 weeks of age, with atrophic pancreas, reduced islet mass and abnormal increase in duct formation. At E15.5, Mettl3 deletion caused a significant loss of Ngn3+ endocrine progenitor cells, which was accompanied by increased Sox9+ duct precursor cells. We identified histone deacetylase 1(Hdac1) as the critical direct m6A target in bipotent progenitor, whose degeneration caused abnormal activation of Wnt/Notch signaling pathway and blocked endocrine differentiation. This transformation could be manipulated in embryonic pancreas culture in vitro through the regulation of the axis of Mettl3-Hdac1-Wnt/Notch signaling. Our finding that Mettl3 determines endocrine lineage through modulating Hdac1 activity during the transition of bipotent progenitor might help in the development of targeted endocrine cell protocols for diabetes treatment.

Complexation of histone deacetylase inhibitor belinostat to Cu(II) prevents premature metabolic inactivation in vitro and demonstrates potent anti-cancer activity in vitro and ex vivo in colon cancer.

The histone deacetylase inhibitor (HDACi), belinostat, has had limited therapeutic impact in solid tumors, such as colon cancer, due to its poor metabolic stability. Here we evaluated a novel belinostat prodrug, copper-bis-belinostat (Cubisbel), in vitro and ex vivo, designed to overcome the pharmacokinetic challenges of belinostat. The in vitro metabolism of each HDACi was evaluated in human liver microsomes (HLMs) using mass spectrometry. Next, the effect of belinostat and Cubisbel on cell growth, HDAC activity, apoptosis and cell cycle was assessed in three colon cancer cell lines. Gene expression alterations induced by both HDACis were determined using RNA-Seq, followed by in silico analysis to identify master regulators (MRs) of differentially expressed genes (DEGs). The effect of both HDACis on the viability of colon cancer patient-derived tumor organoids (PDTOs) was also examined. Belinostat and Cubisbel significantly reduced colon cancer cell growth mediated through HDAC inhibition and apoptosis induction. Interestingly, the in vitro half-life of Cubisbel was significantly longer than belinostat. Belinostat and its Cu derivative commonly dysregulated numerous signalling and metabolic pathways while genes downregulated by Cubisbel were potentially controlled by VEGFA, ERBB2 and DUSP2 MRs. Treatment of colon cancer PDTOs with the HDACis resulted in a significant reduction in cell viability and downregulation of stem cell and proliferation markers. Complexation of belinostat to Cu(II) does not alter the HDAC activity of belinostat, but instead significantly enhances its metabolic stability in vitro and targets anti-cancer pathways by perturbing key MRs in colon cancer. Complexation of HDACis to a metal ion might improve the efficacy of clinically used HDACis in patients with colon cancer.

Epigenetic Reprogramming By Histone Deacetylase Inhibitors Improves CAR-T Cell Functions

Background: The exhaustion and diminished persistence of CAR-T cells primarily affect clinical efficacy and long-term outcomes. Epigenetic is an innate mechanism in regulating the biological function CAR-T cells. We previously dissected comprehensive dynamic changes of chromatin accessibility and transcription factors during CAR-T cell exhaustion (Jiang P, et al. Leukemia. 2022 Nov;36(11):2656-2668). Therefore, combined application with epigenetic drugs have much therapeutic potential. Methods: We initially performed an extensive screening among 370 epigenetic drugs to select candidates that could resist exhaustion and maintain phenotype of memory subsets, and thoroughly verify their effects on CAR-T cells in vitro. Next, we conducted multi-omics analysis comprising RNA-seq, ATAC-seq, and CUT&Tag-seq to explore underlying regulative mechanisms of epigenetic manipulation by epi-drugs. In the end, we used preclinical B-ALL NSG mouse model to verify anti-tumor activities of drug-treated CAR-T cells in vivo. Results: Among those screened epigenetic drugs, we identified two histone deacetylase inhibitors (HDACi), Chidamide and M344, could best promote maintenance of central memory subsets and resistance to exhaustion of CAR-T cells, with enhanced anti-leukemia activities in vitro (Figure 1a-c). Chidamide and M344 inhibited enzymatic activity and expression of HDAC1, which elevated the acetylation level of histone H3, particularly at H3K27 in CD19 CAR-T cells. Genetic knock-down of HDAC1 could partially reflect the phenotypic mimicry by Chidamide and M344 on CAR-T cells. By combining multi-omics data from bulk RNA-seq, ATAC-seq, and H3K27ac-targeted CUT&Tag-seq in Chidamide-or M344-treated CAR-T cells, we identified distinct patterns of epigenetic reprogramming, and enrichment of genes associated with exhaustion, as well as regulation of cellular differentiation and formation of memory subsets. In addition, analysis of motif and interaction network among transcription factors revealed altered regulation of gene transcription (data not shown). Finally, either Chidamide-pretreated CAR-T cells or in vivo administration of Chidamide after CAR-T cells infusion had exhibited enhanced and persistent tumor clearance in preclinical B-ALL NSG mouse model (Figure 1d). Conclusion: we conducted comprehensive screening of 370 epigenetic drugs and identified that administration of HDACi, particularly Chidamide and M344, could induce significantly enhanced anti-tumor function with maintenance of memory phenotype and reduced exhaustion in CAR-T cells through epigenetic reprogramming and altered regulation of gene transcription, which provided with basis and therapeutic targets for the rational combination of CAR-T cell therapy and epigenetic drugs in patients with hematological malignancies. Figure legends: Figure1. Screening of 370 epigenetic drugs identified M344 and Chidamide that enhanced maintenance of memory phenotype, resistance to exhaustion, and ability of tumor clearance in CAR-T cells. (a) The scatterplot of screening 370 compounds based on relative CD45RO +CD62L + proportion of central memory in treated CAR-T cell, showing of compared with DMSO control group by flow cytometry. Red indicates an increase in the proportion of memory CAR-T cells, while blue represents a decrease. (b) Frequency of co-expression of exhaustion-related inhibitory receptors (LAG-3, TIM-3, and PD-1) in DMSO or HDACi-treated groups. Data presented as mean ± SEM of independent donors (n=3 or more). (c) Flow cytometric analysis of central memory and effector memory subsets in CAR-T cells in each group. Data presented as mean ± SEM of independent donors (n=3 or more). (d) Bioluminescent imaging of tumor clearance in B-ALL NSG mouse. ‘X’ represents dead subject (end point). Survival of B-ALL NSG mice i.v. infused with T cells or treated CAR-T cells. Statistical analysis by Mantel-Cox test. Data presented as pooled from independent experiments (n=2).