HDAC Research Articles

Genome Instability and Senescence Are Markers of Cornelia de Lange Syndrome Cells

Cornelia de Lange syndrome (CdLS) is a rare, dominantly inherited multisystem developmental disorder. Pathogenic variants in genes encoding the structural subunits and regulatory proteins of the cohesin complex (NIPBL, SMC1A, SMC3, HDAC8, and RAD21) are the primary contributors to the pathogenesis of CdLS. Pathogenic variations in these genes disrupt normal cohesin function, leading to the syndrome’s diverse and complex clinical presentation. In this study, we discovered that cells harboring variants in the NIPBL, SMC1A and HDAC8 genes exhibit spontaneous genome instability, elevated oxidative stress and premature cellular aging. These findings suggest that cohesin plays a critical role in maintaining proper cellular function and highlight its contribution to the pathophysiology seen in the related diagnoses.

Protective effect of Shenqi Wenfei Formula against lipopolysaccharide/cigarette smoke-induced COPD in Rat based on gut microbiota and network pharmacology analysis.

The incidence of chronic obstructive pulmonary disease (COPD) appears to be increasing and evidence suggests that the intestinal flora may play a causative role in its development. Previous studies found that the Shenqi Wenfei Formula (SQWF) can regulate pyroptosis via the NLRP3/GSDMD pathway, thereby reducing the inflammatory response in the lungs of COPD model rats. However, there is no information on whether the drug's effects are associated with intestinal flora. Therefore, this study investigates whether the effects of SQWF are mediated through the regulation of intestinal flora, aiming to elucidate the underlying mechanisms of its therapeutic impact on COPD. COPD was induced in rats using lipopolysaccharide and cigarette smoke, followed by intragastric administration of SQWF or physiological saline The targets of SQWF, associated signaling pathways, and key bacterial groups were investigated using 16S rRNA sequencing, network pharmacology, and bioinformatics techniques. The prediction results were validated using quantitative reverse transcription PCR, western blotting, and immunofluorescence, among other methods. SQWF treatment was found to alleviate COPD in model rats. Treatment was also observed to restore the balance of the intestinal flora in the rats, especially by reducing the abundance of g_Parabacteroides. Bioinformatics predictions identified g_Parabacteroides metabolites, RelA, HDAC1, and enriched neutrophil extracellular trap formation pathways as core targets of SQWF in COPD. qRT-PCR and Western blotting results showed that SQWF treatment reduced ReLA and HDAC1 mRNA and protein expression, along with decreased myeloperoxidase and neutrophil elastase levels in the nucleus. Treatment with SQWF was found to restore the imbalance of intestinal g_Parabacteroides in COPD and also regulate the expression of the ReLA and HDAC1 genes, thereby reducing pulmonary neutrophil extracellular traps and alleviating lung inflammation.

Recombinant adenoviruses expressing HPV16/18 E7 upregulate the HDAC6 and DNMT3B genes in C33A cells.

High-risk human papillomavirus (HPV) is a carcinogenic virus associated with nearly all cases of cervical cancer, as well as an increasing number of anal and oral cancers. The two carcinogenic proteins of HPV, E6 and E7, can immortalize keratinocytes and are essential for HPV-related cellular transformation. Currently, the global regulatory effects of these oncogenic proteins on the host proteome are not fully understood, and further exploration of the functions and carcinogenic mechanisms of E6 and E7 proteins is needed. We used a previously established platform in our laboratory for constructing recombinant adenoviral plasmids expressing the HPV16 E7 gene to further construct recombinant virus particles expressing HPV16/18 E6, E7, and both E6 and E7 genes. These recombinant viruses were used to infect C33A cells to achieve sustained expression of the HPV16/18 E6/E7 genes. Subsequently, total RNA was extracted and RNA-Seq technology was employed for transcriptome sequencing to identify differentially expressed genes associated with HPV infection in cervical cancer. RNA-Seq analysis revealed that overexpression of the HPV16/18 E6/E7 genes upregulated GP6, CD36, HDAC6, ESPL1, and DNMT3B among the differentially expressed genes (DEGs) associated with cervical cancer. Spearman correlation analysis revealed a statistically significant correlation between the HDAC6 and DNMT3B genes and key pathways, including DNA replication, tumor proliferation signature, G2M checkpoint, p53 pathways, and PI3K/AKT/mTOR signaling pathways. Further, qRT-PCR and Western blot analyses indicated that both HPV16/18 E7 can upregulate the expression of HDAC6 and DNMT3B, genes associated with HPV infection-related cervical cancer. The successful expression of HPV16/18 E6/E7 in cells indicates that the recombinant viruses retain the replication and infection capabilities of Ad4. Furthermore, the recombinant viruses expressing HPV16/18 E7 can upregulate the HDAC6 and DNMT3B genes involved in cervical cancer pathways, thereby influencing the cell cycle. Additionally, HDAC6 and DNMT3B are emerging as important therapeutic targets for cancer. This study lays the foundation for further exploration of the oncogenic mechanisms of HPV E6/E7 and may provide new directions for the treatment of HPV-related cancers.

A novel dual-epigenetic inhibitor enhances recombinant monoclonal antibody expression in CHO cells

Epigenetic regulation plays a central role in the regulation of a number of cellular processes such as proliferation, differentiation, cell cycle, and apoptosis. In particular, small molecule epigenetic modulators are key elements that can effectively influence gene expression by precisely regulating the epigenetic state of cells. To identify useful small-molecule regulators that enhance the expression of recombinant proteins in Chinese hamster ovary (CHO) cells, we examined a novel dual-HDAC/LSD1 inhibitor I-4 as a supplement for recombinant CHO cells. Treatment with 2 μM I-4 was most effective in increasing monoclonal antibody production. Despite cell cycle arrest at the G1/G0 phase, which inhibits cell growth, the addition of the inhibitor at 2 µM to monoclonal antibody-expressing CHO cell cultures resulted in a 1.94-fold increase in the maximal monoclonal antibody titer and a 2.43-fold increase in specific monoclonal antibody production. In addition, I-4 significantly increased the messenger RNA levels of the monoclonal antibody and histone H3 acetylation and methylation levels. We also investigated the effect on HDAC-related isoforms and found that interference with the HDAC5 gene increased the monoclonal antibody titer by 1.64-fold. The results of this work provide an effective method of using epigenetic regulatory strategies to enhance the expression of recombinant proteins in CHO cells.Key points• HDAC/LSD1 dual-target small molecule inhibitor can increase the expression level of recombinant monoclonal antibodies in CHO cells.• By affecting the acetylation and methylation levels of histones in CHO cells and downregulating HDAC5, the production of recombinant monoclonal antibodies increased.• It provides an effective pathway for applying epigenetic regulation strategies to enhance the expression of recombinant proteins.

Exogenous miR-29b Reduces DNA Methylation and Apoptosis in Transgenic Cells

Background: MicroRNA-29b (miR-29b) is a 22-base pair long nucleotide, that post-transcriptionally affects the processes like proliferation, differentiation and apoptosis by changes in DNA methyl transferes expressions (DNMTs family gene), which regulate DNA methylation. Methods: This study aimed to investigate the effect of miR-29b mimic and inhibitor on buffalo fibroblast cells containing the human insulin (hINS) gene. Transgenic buffalo cell line containing (hINS) gene was treated with 40 nM miR-29b mimic and inhibitor by lipofectamin-3000. Result: miRNA-29b mimic treated cells showed a significant decrease (P less than 0.05) in expression of DNMT1, DNMT3A, DNMT3B genes but did not find any significant change (P less than 0.05) in HDAC1 expression as compared to control. Whereas, miR-29b inhibitor-treated cells revealed significantly increased (P less than 0.05) the expression of DNMT1, DNMT3A, DNMT3B and HDAC1 genes as compared to control. These miR-29b mimic-treated cells had significantly increased (P less than 0.05) expression of BCL-XL and MCL-1 whereas miR-29b inhibitor-treated cells showed a significantly decreased (P less than 0.05) expression of MCL-1 while BCL-XL showed no significant (P less than 0.05) change in expression. These findings indicate that treatment of miR-29b mimic on transgenic cells reduces the DNA methylation which helps in the reduction of apoptosis levels in cells. miR-29b mimic reduces the methylation status and help in nuclear reprogramming of transgenic cells. Thus in future, these cells can be a better choice as a nuclear donor for efficient animal cloning.

Characterization of a novel HDAC2 pathogenetic variant: a missing puzzle piece for chromatinopathies

Histone deacetylases (HDACs) are enzymes pivotal for histone modification (i.e. acetylation marks removal), chromatin accessibility and gene expression regulation. Class I HDACs (including HDAC1, 2, 3, 8) are ubiquitously expressed and they often participate in multi-molecular protein complexes. To date, three neurodevelopmental disorders caused by mutations in genes encoding for HDACs (HDAC4, HDAC6 and HDAC8) and thus belonging to the group of chromatinopathies, have been described. We performed whole exome sequencing (WES) for a patient (#249) clinically diagnosed with the chromatinopathy Rubinstein-Taybi syndrome (RSTS) but negative for mutations in RSTS genes, identifying a de novo frameshift variant in HDAC2 gene. We then investigated its molecular effects in lymphoblastoid cell lines (LCLs) derived from the patient compared to LCLs from healthy donors (HD). As the variant was predicted to be likely pathogenetic and to affect the sequence of nuclear localization signal, we performed immunocytochemistry and lysates fractionation, observing a nuclear mis-localization of HDAC2 compared to HD LCLs. In addition, HDAC2 total protein abundance resulted altered in patient, and we found that newly identified variant in HDAC2 affects also acetylation levels, with significant difference in acetylation pattern among patient #249, HD and RSTS cells and in expression of a known molecular target. Remarkably, RNA-seq performed on #249, HD and RSTS cells shows differentially expressed genes (DEGs) common to #249 and RSTS. Interestingly, our reported patient was clinically diagnosed with RSTS, a chromatinopathy which known causative genes encode for enzymes antagonizing HDACs. These results support the role of HDAC2 as causative gene for chromatinopathies, strengthening the genotype-phenotype correlations in this relevant group of disorders.

To Investigate the Influence of Smoking Cessation Intention and Common Downstream Variants of HDAC9 Gene on Large Artery Atherosclerotic Cerebral Infarction.

To investigate the association of smoking cessation intention and single nucleotide polymorphism of HDAC9 gene with LAA-S in Han people in Hainan province. A case-control study was conducted. Six single nucleotide polymorphisms (SNPS) of HDAC9 gene were genotyped by SNPscan genotyping technique in 248 patients with LAA-S and 237 controls in Hainan Han population. SNP loci (rs10227612, rs12669496, rs1548577, rs2074633, rs2526626, and rs2717344) were genotyped, and the genotype and allele frequencies were compared between the case and control group. At the same time, the distribution of smoking between the case and control group was compared, and the 3-year and 7-year follow-up smoking cessation between the case and control group was compared, so as to find out the effects of smoking cessation intention and HDAC9 SNP on LAA-S. (1) The GT genotype at rs10227612, GG genotype at rs2717344, and GA genotype at rs1548577 in the case group were significantly higher than those in the control group, and the differences were statistically significant. (2) There were significant differences in the distribution of smoking between the case and control group (P < 0.05), and there were significant differences in the smoking cessation after 3 years and 7 years of follow-up between the case and control group (P < 0.05). The intention to quit smoking was positively correlated with the incidence of LAA-S. (1) The rs10227612, rs1548577, rs2074633, rs2717344 of HDAC9 gene may be significantly related to atherosclerotic cerebral infarction of great arteries in Hainan Han population, while rs12669496 and rs2526626 may not be related. (2) According to the statistics of smoking in the case and control group, smoking was related to large artery atherosclerotic cerebral infarction, and the intention to quit smoking was a very important factor affecting the success of smoking cessation.

HDAC4 Genomic Methylation and Subsequent DNA Binding Following Joint Injury Confers a Sustained Epigenetic Memory of Disrupted Muscle Plasticity

Joint injury is associated with protracted atrophy and weakness that poorly recovers; our objective was to define molecular effectors of lost muscle size, strength, and plasticity following joint injury. We hypothesized that epigenetic imprinting of joint injury in muscle impairs muscle plasticity and contributes to poor functional recovery following surgical repair. We recruited a total of sixteen ACL-injured participants from whom we obtained vastus lateralis muscle biopsies Pre-, 7-days post-, and 4-months post- reconstruction surgery (Sx) and strength measures up to 6-months post-Sx. We performed immunohistochemistry, RNA-seq, reduced representation bisulfite sequencing (RRBS), and HDAC4 chromatin immunoprecipitation sequencing (HDAC4 ChIP-seq) to determine the impact of epigenetics and gene expression on histological profiles, muscle size and muscle strength following injury. Binding and Expression Target Analysis (BETA) of DNA methylation revealed a conserved 985bp regulatory genomic region that was differentially methylated upstream of the HDAC4 promoter. We observed 108 (Pre-Sx), 111 (7d post-Sx), and 110 (4-months post-Sx) differentially methylated promoter sites of which 95 sites were common across all timepoints in the injured limb. Additionally, we observed 1293 differentially methylated intragenic sites within the HDAC4 locus that were also common across all timepoints following injury. Altered HDAC4 methylation was associated with a 2.2 and 4.3 fold change in HDAC4 transcript abundance Pre-Sx and 7d post-Sx, respectively (FDR &lt; 0.05). Notably, HDAC4 binding to critical contractile genes coincided with their downregulation 7d post-Sx (Log2 fold change: ACTA1: -2.02; CKM: -0.97; ATP2A1: -2.34; HOMER2: -1.09; all FDR&lt;0.05). Altered transcript abundance of these genes coincided with lower mean muscle fiber cross-sectional area in the injured limb compared to Healthy (Healthy: 4845 ± 213.4μm2; pre-Sx: 4313 ± 190.6μm2, p&lt;0.05; 7d post-Sx: 3685 ± 228.1μm2, p&lt;0.05; 4-months post-Sx: 3422 ± 142.8μm2, p&lt;0.05) and lower peak torque compared to Healthy (Healthy: 174.8 ± 10.72nm; pre-Sx: 135.2 ± 10.19nm, p&lt;0.05; 4-months post-Sx: 82.64 ± 6.80nm, p&lt;0.05; 6-months post-Sx: 105.9 ± 8.26nm, p&lt;0.05) that was sustained throughout follow-up. Our results suggest that joint injury confers HDAC4 promoter-proximal and intragenic epigenetic imprinting by DNA methylation that increases HDAC4 transcript abundance and HDAC4-DNA binding to represses expression of genes regulating muscle contractile function. This HDAC4 gene regulatory program likely contributes to prolonged deficits in muscle size and strength following joint injury. This work was supported by NIH-NIAMS R01 AR072061. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Collagen sponge scaffolds loaded with Trichostatin A pretreated BMSCs-derived exosomes regulate macrophage polarization to promote skin wound healing

The process of wound healing includes the inflammatory stage, which plays an important role. Macrophages can promote inflammatory response and also promote angiogenesis, wound contraction and tissue remodeling required for wound healing. It is crucial to promote macrophages to polarize from M1 pro-inflammatory phenotype to M2 anti-inflammatory phenotype at a critical time for the quality of wound healing. Because mesenchymal stem cell-derived exosomes have broad therapeutic prospects in the field of tissue repair and regeneration, in this study, we explored whether trichostatin A pretreated bone marrow mesenchymal stem cells (BMSCs)-derived exosomes (T-Exo) could promote wound healing by binding to biomaterial scaffolds through certain anti-inflammatory effects. In the cell experiment, we established macrophage inflammation model and then treated with T-Exo, and finally detected the expression levels of macrophage polarization proteins CD206, CD86 and TNF-α, iNOS, and Arg-1 by Western Blot and immunofluorescence staining; detected the expression levels of inflammation-related genes TNF-α, iNOS, IL-1β, IL-10 and anti-inflammatory genes CD206 and Arg-1 by qRT-PCR; explored the promoting ability of T-Exo to promote cell migration and tube formation by cell scratch experiment and angiogenesis experiment. The results showed that T-Exo could promote the polarization of M1 macrophages to M2 macrophages, and promote the migration and angiogenesis of HUVECs. Because TSA pretreatment may bring about changes in the content and function of BMSCs-derived exosomes, proteomic analysis was performed on T-Exo and unpretreated BMSCs-derived exosomes (Exo). The results showed that the differentially expressed proteins in T-Exo were related to some pathways that promote angiogenesis, cell migration, proliferation, and re-epithelialization. Then, exosome/collagen sponge (T-Exo/Col) biological scaffolds were prepared, and the physicochemical properties and biocompatibility of the scaffolds were investigated. Animal skin wound models were established, and the therapeutic effect and anti-inflammatory effect of T-Exo/Col in wound repair were evaluated by small animal in vivo imaging, H&E staining, Masson trichrome staining, immunohistochemical staining, Western Blot, and qRT-PCR. The results showed that T-Exo significantly promoted wound healing by inhibiting inflammation, thereby further promoting angiogenesis and collagen formation in vivo. Moreover, the existence of Col scaffold in T-Exo/Col enabled T-Exo to achieve a certain sustained release effect. Finally, we further explored whether TSA exerts beneficial effects by inhibiting HDAC6 gene of BMSCs, but the results showed that knockdown of HDAC6 gene would cause oxidative stress damage to BMSCs, which means that TSA does not produce these beneficial effects by inhibiting HDAC6 gene. What molecular mechanisms TSA exerts beneficial effects through needs to be further elucidated in the future.

Ginger Polyphenols Reverse Molecular Signature of Amygdala Neuroimmune Signaling and Modulate Microbiome in Male Rats with Neuropathic Pain: Evidence for Microbiota-Gut-Brain Axis.

Emerging evidence shows that the gut microbiota plays an important role in neuropathic pain (NP) via the gut-brain axis. Male rats were divided into sham, spinal nerve ligation (SNL), SNL + 200 mg GEG/kg BW (GEG200), and SNL + 600 mg GEG/kg BW (GEG600) for 5 weeks. The dosages of 200 and 600 mg GEG/kg BW for rats correspond to 45 g and 135 g raw ginger for human daily consumption, respectively. Both GEG groups mitigated SNL-induced NP behavior. GEG-supplemented animals had a decreased abundance of Rikenella, Muribaculaceae, Clostridia UCG-014, Mucispirillum schaedleri, RF39, Acetatifactor, and Clostridia UCG-009, while they had an increased abundance of Flavonifactor, Hungatella, Anaerofustis stercorihominis, and Clostridium innocuum group. Relative to sham rats, Fos and Gadd45g genes were upregulated, while Igf1, Ccl2, Hadc2, Rtn4rl1, Nfkb2, Gpr84, Pik3cg, and Abcc8 genes were downregulated in SNL rats. Compared to the SNL group, the GEG200 group and GEG600 group had increases/decreases in 16 (10/6) genes and 11 (1/10) genes, respectively. GEG downregulated Fos and Gadd45g genes and upregulated Hdac2 genes in the amygdala. In summary, GEG alleviates NP by modulating the gut microbiome and reversing a molecular neuroimmune signature.

Abstract 6413: Biomarkers of response and progression to Pembrolizumab and Vorinostat combination in late-stage squamous cell carcinoma patients of different locations included in the PEVOsq basket trial

Abstract Background: Epigenetic modulation plays a major role in escaping tumor immunosurveillance. PEVOsq (NCT04357873) was an open-label, non-randomized, multi-center, basket phase II trial evaluating the efficacy of pembrolizumab (P) and the vorinostat (V) HDAC inhibitor in 112 patients (pts) with recurrent and/or metastatic squamous cell carcinoma (SCC) of the cervix, head and neck, anus, vulva/vagina, penis, and lung. Results showed an objective response rate (ORR) of 25%, the highest being in anal (31%) and cervical (39%) (ASCO #397612). We report here early biomarkers of response and progression to P+V. Patients and Methods: Pts had to undergo a tumor biopsy at baseline, and optional biopsies on-treatment (after cycle 2), and at progression for translational analyses. PD-L1 expression (n=102), tumor-infiltrating lymphocytes quantification (n=39), HPV typing (n=111), WES (n=83), RNAseq (n=82) and liquid chromatography-mass spectrometry (LC-MS) quantitative analysis of histone post-translational modifications (PTMs) (n=44) were performed on baseline samples.SNVs, CNVs, TMB, MSI status (by MSI sensor), aneuploidy score (AS), mutational signatures, immune signatures, and histone modifications were correlated with response and progression. Results: Sixty-six pts were HPV+ (59%). Most frequently altered genes were PIK3CA (30%), TP53 (30%), KMT2D/C (18 and 17%), and FGF3/4 (17%). MSI was detected in 4 pts (5%), and TMB-high (&amp;gt;10mut/Mb) in 13 pts (16%). High AS (&amp;gt;10) was detected in 34 pts (40%), and was significantly associated with HPV- status (p=0.03). TMB high and MSI were associated with MMR mutational signatures (p&amp;lt;0.01). TMB high was also associated with APOBEC (p=0.02) and DNA polymerase (POL) signatures (p &amp;lt;0.01). Biomarkers associated with improved overall survival (OS) included HPV positivity (7.8 months versus 17.4 months, p&amp;lt;0.01), high TMB (not reached versus 10.3 months, p=0.01), and high PD-L1 CPS score (13.2 months versus 7.0 months, p=0.06).Deconvolution of immune cell infiltration showed an expected trend for increased CD8 and decreased neutrophil infiltration in non progressor pts.Interestingly, LC-MS histone PTMs profiling showed a significant decrease in H4K5K8K12K16-4ac (p=0.05) and increase in H3K27me2K36me1 in non progressor pts (p=0.03). At the transcriptomic level, although not statistically significant, we observed modulation in the expression of genes associated with H3K36me1 (KDM2A, SETD3, SMYD2), and H3K27me3 (KDM6A) regulation, as well as HDAC genes. Conclusions: HPV+ status was associated with improved OS, together with a high TMB and PD-L1 expression. CD8, macrophages and neutrophil signatures at baseline were associated with response. Promising epigenetic biomarkers were identified using LC/MS and will be correlated to transcriptomic and genomic data. Citation Format: Christophe Le Tourneau, Luca Mazzarella, Bastien Cabarrou, Miguel Francisco, Célia Dupain, Maral Halladjian, Gianmaria Frige, Bruno Duso, Yinxiu Zhan, Elena Guerini-Rocco, Roberta Noberini, Tiziana Bonaldi, Maria Manuela Tonini, Grégoire Marret, Clélia Coutzac, Elodie Coquan, Mathilde Saint-Ghislain, Olivia Le Saux, Laetitia Chanas, François Legrand, Daria Maria Filippini, Emmanuelle Jeannot, Stephan Bernhart, Gabor Balogh, Marta Jimenez, Thomas Filleron, Nicolas Servant, Maud Kamal. Biomarkers of response and progression to Pembrolizumab and Vorinostat combination in late-stage squamous cell carcinoma patients of different locations included in the PEVOsq basket trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6413.

Abstract 6595: HDAC8 targeting impairs Foxp3+Treg cell function and promotes anti-tumor immunity

Abstract HDAC8 is an evolutionarily distinct, X-linked, zinc-dependent class I histone/protein deacetylase implicated in developmental disorders, parasitic infections, myopathy, and cancers such as neuroblastoma, gastric cancer, hepatocellular carcinoma, oral squamous cell carcinoma, and childhood acute lymphoblastic leukemia. Here, we highlight a hitherto unknown role of HDAC8 in immune cells by presenting data on conditional targeting of HDAC8 in T cells and the effects of selective HDAC8 inhibitors (HDAC8i) on T cell-dependent immune responses. First, as a genetic approach, we produced C57BL/6 mice with floxed exon 3 of the HDAC8 gene suitable for targeted deletion of HDAC8 and, after crossing with syngeneic CD4cre mice, demonstrated conditional deletion of HDAC8 and enhanced acetylation of SMC3, a classic HDAC8 substrate, in their T-cells. Next, in a pharmacologic approach, we tested isoform-selective inhibitors of HDAC8 (OJ-1, PCI-34051) and assessed effects on Foxp3+ T-regulatory (Treg) cell suppressive function. Finally, we performed flow cytometry, RNAseq, CHIP-seq and mass spectrometry to investigate the underlying mechanisms by which HDAC8 knockout affected Treg function. Our data demonstrate that knocking down or inhibiting HDAC8: (i) impaired murine Treg suppressive function in vitro and in vivo, including inhibiting the ability of Tregs to promote mouse cardiac allograft survival (p&amp;lt;0.01); (ii) decreased Treg function in homeostatic proliferation assays (p&amp;lt;0.01), e.g., involving adoptive transfer of WT or HDAC8-/- Tregs plus WT conventional T cells into immunodeficient hosts; (iii) decreased intratumoral Tregs, increased CD4 and CD8 T cells, increased production of effectors such as IFN-g and granzyme-B, and limited growth of syngeneic mouse non-small cell lung cancer cell lines (p&amp;lt;0.01) (whereas no effect of Hdac8i on tumor growth was seen in immunodeficient hosts, indicating the key action was via immune cell targeting). Mechanistically, HDAC8 co-precipitated with Foxp3 and NFAT5 and promoted the deacetylation of Foxp3 and NFAT5, and knockout of HDAC8 enhanced the acetylation level of NFAT5 and promoted cytokine expression. Hence, our combined genetic and pharmacologic studies establish the importance of HDAC8 in Foxp3+ Treg cells and show that therapy with selective HDAC8 inhibitors can be a novel therapeutic approach in immuno-oncology. Citation Format: Fanhua Kong, Liqing Wang, Aaron Beeler, Wayne W. Hancock. HDAC8 targeting impairs Foxp3+Treg cell function and promotes anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6595.

Targeting HDAC6 to treat heart failure with preserved ejection fraction in mice

Heart failure with preserved ejection fraction (HFpEF) poses therapeutic challenges due to the limited treatment options. Building upon our previous research that demonstrates the efficacy of histone deacetylase 6 (HDAC6) inhibition in a genetic cardiomyopathy model, we investigate HDAC6’s role in HFpEF due to their shared mechanisms of inflammation and metabolism. Here, we show that inhibiting HDAC6 with TYA-018 effectively reverses established heart failure and its associated symptoms in male HFpEF mouse models. Additionally, in male mice lacking Hdac6 gene, HFpEF progression is delayed and they are resistant to TYA-018’s effects. The efficacy of TYA-018 is comparable to a sodium-glucose cotransporter 2 (SGLT2) inhibitor, and the combination shows enhanced effects. Mechanistically, TYA-018 restores gene expression related to hypertrophy, fibrosis, and mitochondrial energy production in HFpEF heart tissues. Furthermore, TYA-018 also inhibits activation of human cardiac fibroblasts and enhances mitochondrial respiratory capacity in cardiomyocytes. In this work, our findings show that HDAC6 impacts on heart pathophysiology and is a promising target for HFpEF treatment.

Chidamide and orelabrutinib synergistically induce cell cycle arrest and apoptosis in diffuse large B-cell lymphoma by regulating the PI3K/AKT/mTOR pathway

ObjectiveThe initial therapeutic approach for diffuse large B-cell lymphoma (DLBCL) entails a rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) regimen. However, 40% of patients exhibit suboptimal responses, with some experiencing relapse and refractory conditions. This study aimed to explore novel therapeutic strategies and elucidate their underlying mechanisms in DLBCL.MethodsBioinformatics techniques were employed to scrutinize correlations between the HDAC1, HDAC2, HDAC3, HDAC10, BTK, MYC, TP53, and BCL2 genes in DLBCL. In vitro experiments were conducted using DB and SU-DHL-4 cells treated with chidamide, orelabrutinib, and a combination of both. Cell viability was assessed by cell counting kit-8. Cell apoptosis and the cell cycle were determined using flow cytometry. Reactive oxygen species (ROS) production and mitochondrial function were assessed through ROS and JC-1 staining. RNA sequencing and western blot analyses were conducted to elucidate the molecular mechanisms underlying the combined action of chidamide and orelabrutinib in DLBCL cells.ResultsThis investigation revealed markedly enhanced antiproliferative effects when chidamide was combined with orelabrutinib. Compusyn software analysis indicated a synergistic effect of chidamide and orelabrutinib in inhibiting DLBCL cell proliferation, with a combination index (CI) < 1. This synergy further manifested as augmented cell cycle arrest, apoptosis induction, the downregulation of cell cycle-associated and antiapoptotic proteins, and the upregulation of proapoptotic proteins. Furthermore, the western blot and RNA-Seq findings suggested that combining chidamide and orelabrutinib modulated the PI3K/AKT/mTOR signaling pathway, thereby promoting DLBCL cell cycle arrest and apoptosis.ConclusionThe findings of this study provide a compelling justification for the clinical utilization of chidamide and orelabrutinib to treat relapsed/refractory DLBCL.

Association of changes in expression of HDAC and SIRT genes after drug treatment with cancer cell line sensitivity to kinase inhibitors

ABSTRACT Histone deacetylases (HDACs) and sirtuins (SIRTs) are important epigenetic regulators of cancer pathways. There is a limited understanding of how transcriptional regulation of their genes is affected by chemotherapeutic agents, and how such transcriptional changes affect tumour sensitivity to drug treatment. We investigated the concerted transcriptional response of HDAC and SIRT genes to 15 approved antitumor agents in the NCI-60 cancer cell line panel. Antitumor agents with diverse mechanisms of action induced upregulation or downregulation of multiple HDAC and SIRT genes. HDAC5 was upregulated by dasatinib and erlotinib in the majority of the cell lines. Tumour cell line sensitivity to kinase inhibitors was associated with upregulation of HDAC5, HDAC1, and several SIRT genes. We confirmed changes in HDAC and SIRT expression in independent datasets. We also experimentally validated the upregulation of HDAC5 mRNA and protein expression by dasatinib in the highly sensitive IGROV1 cell line. HDAC5 was not upregulated in the UACC-257 cell line resistant to dasatinib. The effects of cancer drug treatment on expression of HDAC and SIRT genes may influence chemosensitivity and may need to be considered during chemotherapy.

Epigenome-wide DNA methylation analysis of late-stage mild cognitive impairment.

Background: Patients with late-stage mild cognitive impairment (LMCI) have a higher risk of progression to Alzheimer's disease (AD) than those with early-stage mild cognitive impairment (EMCI). However, previous studies have often pooled EMCI and LMCI patients into a single MCI group, with limited independent investigation into the pathogenesis of LMCI. Methods: In this study, we employed whole-genome methylation association analysis to determine the differences in peripheral blood methylation profiles between 663 cognitive aging (CN) and 554 LMCI patients. Results: Our results revealed 2,333 differentially methylated probes (DMPs) and 85 differentially methylated regions (DMRs) specific to LMCI. The top hit methylation sites or regions were associated with genes such as SNED1, histone deacetylases coding gene HDACs, and HOX and ZNF gene family. The DNA methylations upregulated the expression of HDAC4, HDAC8, and HOX family genes HOXC5 and HOXC9, but they downregulated the expression of SNED1, ADCYAP1, and ZNF family genes ZNF415 and ZNF502. Gene Ontology (GO) and KEGG analysis showed that the genes associated with these methylation sites were predominantly related to the processes of addiction disorders, neurotransmission, and neurogenesis. Out of the 554 LMCI patients included in this study, 358 subjects (65%) had progressed to AD. Further association analysis between the LMCI subjects with a stable course (sLMCI) and those who progressed to AD (pLMCI) indicated that the methylation signal intensities of HDAC6, ZNF502, HOXC5, HOXC6, and HOXD8 were associated with increased susceptibility to AD. Protective effects against progression to AD were noticed when the methylation of SNED1 and ZNF727 appeared in LMCI patients. Conclusion: Our findings highlight a substantial number of LMCI-specific methylated biomarkers that differ from those identified in previous MCI case-control studies. These biomarkers have the potential to contribute to a better understanding of the pathogenesis of LMCI.

Identification of neutrophil extracellular traps and crosstalk genes linking inflammatory bowel disease and osteoporosis by integrated bioinformatics analysis and machine learning

Musculoskeletal deficits are among the most common extra-intestinal manifestations and complications of inflammatory bowel disease (IBD). This study aimed to identify crosstalk genes between IBD and osteoporosis (OP) and potential relationships between crosstalk and neutrophil extracellular traps (NETs)-related genes. Three common hub genes from different compared groups are actually the same, namely HDAC6, IL-8, and PPIF. ROC showed that the combined diagnostic value of HDAC6, IL-8, and PPIF was higher than each of the three key hub genes. Immune infiltration results showed that HDAC6 and IL-8 key genes negatively correlated with CD65 bright natural killer cells. USF1 was the common upstream TFs between HDAC6 and PPIF, and MYC was the common upstream TFs between IL-8 and PPIF in RegNetwork. Taken together, this study shows a linked mechanism between IBD and OP via NETs and crosstalk genes. These findings may show light on better diagnosis and treatment of IBD complicated with OP.

Alterations in DNA methylation associate with reduced migraine and headache days after medication withdrawal treatment in chronic migraine patients: a longitudinal study

BackgroundChronic migraine, a highly disabling migraine subtype, affects nearly 2% of the general population. Understanding migraine chronification is vital for developing better treatment and prevention strategies. An important factor in the chronification of migraine is the overuse of acute headache medication. However, the mechanisms behind the transformation of episodic migraine to chronic migraine and vice versa have not yet been elucidated. We performed a longitudinal epigenome-wide association study to identify DNA methylation (DNAm) changes associated with treatment response in patients with chronic migraine and medication overuse as part of the Chronification and Reversibility of Migraine clinical trial. Blood was taken from patients with chronic migraine (n = 98) at baseline and after a 12-week medication withdrawal period. Treatment responders, patients with ≥ 50% reduction in monthly headache days (MHD), were compared with non-responders to identify DNAm changes associated with treatment response. Similarly, patients with ≥ 50% versus < 50% reduction in monthly migraine days (MMD) were compared.ResultsAt the epigenome-wide significant level (p < 9.42 × 10–8), a longitudinal reduction in DNAm at an intronic CpG site (cg14377273) within the HDAC4 gene was associated with MHD response following the withdrawal of acute medication. HDAC4 is highly expressed in the brain, plays a major role in synaptic plasticity, and modulates the expression and release of several neuroinflammation markers which have been implicated in migraine pathophysiology. Investigating whether baseline DNAm associated with treatment response, we identified lower baseline DNAm at a CpG site (cg15205829) within MARK3 that was significantly associated with MMD response at 12 weeks.ConclusionsOur findings of a longitudinal reduction in HDAC4 DNAm status associated with treatment response and baseline MARK3 DNAm status as an early biomarker for treatment response, provide support for a role of pathways related to chromatin structure and synaptic plasticity in headache chronification and introduce HDAC4 and MARK3 as novel therapeutic targets.

The Role of Histone H2B Acetylation Modification in Aluminum-Induced Cognitive Dysfunction.

Aluminum (Al) is a low toxic trace element that can accumulate in the nervous system and induce cognitive disorders characterized by reduced learning and memory ability. Neuroepigenetic effects are structural changes in cellular function by the brain in response to environmental stimuli by altering the expression of specific genes and repressing normal cellular transcription, leading to abnormalities in a variety of biological processes within the nervous system and affecting neurobehavioral responses. One of the most important mechanisms of epigenetic control on chromatin shape is histone modification. In the present study, we established an offspring rat model of Al intoxication to investigate the changes in spatial learning and memory retention abilities and the relationship with histone H2B acetylation modification in rats exposed to different doses of Al over a long period of time. The results demonstrated that long-term AlCl3 staining resulted in decreased CBP gene and protein expression, increased HDAC3 gene and protein levels, as well as decreased histone H2B and acH2BK20 protein expression levels in the hippocampus of rats. In conclusion, long-term exposure to Al may vary the expression of histone H2B and acH2BK20 through the regulation of enzymes that specifically regulate histone acetylation, hence hastening the deterioration of the nervous system that impairs cognitive function.

Upregulation of HDAC9 in hippocampal neurons mediates depression-like behaviours by inhibiting ANXA2 degradation

Major depressive disorder (MDD) is a pervasive and devastating mental disease. Broad spectrum histone deacetylase (HDAC) inhibitors are considered to have potential for the treatment of depressive phenotype in mice. However, due to its non-specific inhibition, it has extensive side effects and can not be used in clinical treatment of MDD. Therefore, finding specific HDAC subtypes that play a major role in the etiology of MDD is the key to develop corresponding specific inhibitors as antidepressants in the future. Copy number variation in HDAC9 gene is thought to be associated with the etiology of some psychiatric disorders. Herein, we found that HDAC9 was highly expressed in the hippocampus of chronic restraint stress (CRS) mouse model of depression. Upregulation of HDAC9 expression in hippocampal neurons of mice induced depression-like phenotypes, including anhedonia, helplessness, decreased dendritic spine density, and neuronal hypoexcitability. Moreover, knockdown or knockout of HDAC9 in hippocampal neurons alleviated depression-like phenotypes caused by chronic restraint stress (CRS) in WT mice. Importantly, using immunoprecipitation–mass spectrometry (IP–MS), we further found that Annexin A2 (ANXA2) was coupled to and deacetylated by HDAC9. This coupling resulted in the inhibition of ubiquitinated ANXA2 degradation and then mediates depression-like behavior. Overall, we discovered a previously unrecognized role for HDAC9 in hippocampal neurons in the pathogenesis of depression, indicating that inhibition of HDAC9 might be a promising clinical strategy for the treatment of depressive disorders.