HDAC2 Expression Research Articles

Ethanolic extract of Arctium lappa leaves alleviates cerebral ischemia reperfusion-induced inflammatory injury via HDAC9-mediated NF-κB pathway

BackgroundIschemic stroke (IS) is a major cause of mortality and disability worldwide. Inflammatory response is crucial in the pathogenesis of tissue injury in cerebral infarction. Arctium lappa leaves are traditionally used to treat IS. PurposesTo investigate the neuroprotective effects and molecular mechanisms of the ethanolic extract of A. lappa leaves (ALLEE) on cerebral ischemia-reperfusion (CIR). MethodsMiddle cerebral artery obstruction reperfusion (MCAO/R) rats and an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model were used to evaluate ALLEE pharmacodynamics. Various methods, including neurological function, 2,3,5-triphenyltetrazolium chloride, hematoxylin and eosin, and Nissl, enzyme-linked immunosorbent, and TdT-mediated dUTP nick-end labeling assays, were used to analyze the neuroprotective effects of ALLEE in vitro and in vivo. The major chemical components and potential target genes of ALLEE were screened using network pharmacology. Molecular docking, western blotting, and immunofluorescence analyses were performed to confirm the effectiveness of the targets in related pathways. ResultsALLEE exerted potent effects on the MCAO/R model by decreasing the neurological scores, infarct volumes, and pathological features (p < 0.01). Furthermore, network pharmacology results revealed that the treatment of IS with ALLEE involved the regulation of various inflammatory pathways, such as the tumor necrosis factor (TNF) and chemokine signaling pathways. ALLEE also played key roles in targeting key molecules, including nuclear factor (NF)-κBIA, NF-κB1, interleukin (IL)-6, TNF-α and IL1β, and regulating the histone deacetylase (HDAC)-9-mediated signaling pathway. In vivo and in vitro analyses revealed that ALLEE significantly regulated the NF-κB pathway, promoted the phosphorylation activation of NF-κB P65, IκB and IKK (p < 0.01 or p < 0.05), and decreased the expression levels of the inflammatory factors, IL-1β, IL-6 and TNF-α (p < 0.01). Moreover, ALLEE significantly decreased the expression of HDAC9 (p < 0.01) that is associated with inflammatory responses. However, HDAC9 overexpression partially reversed the neuroprotective effects of ALLEE and its suppressive effects on inflammation and phosphorylation of NF-κB (p < 0.01). ConclusionsIn conclusion, our results revealed that ALLEE ameliorates MCAO/R-induced experimental CIR by modulating inflammatory responses via the inhibition of HDAC9-mediated NF-κB pathway.

Abstract LB210: Identification of histone deacetylase 10 as a repressor of cancer cell migration

Abstract Histone deacetylases (HDACs) are a family of 18 enzymes with the potential to be epigenetic modifiers. Of the 4 classes of HDACs, HDAC6 and HDAC10 belong to the class IIb subgroup. Unlike HDAC6, HDAC10 contains two deacetylase-like domains, with one active deacetylase domain and one inactive leucine-rich domain, and a preference for a specific acetylated polyamine over other substrates. Aberrant expression of HDAC10 has been implicated in cancer and other diseases. However, the deacetylation targets and biological functions of HDAC10 have not been well characterized due to a lack of specific and potent HDAC10 inhibitors with cellular activity. We previously established CRISPR/Cas9-mediated HDAC10 knockout (KO) cell lines in HCT116 and HeLa S3 cells. In the current study, we used the HCT116 wildtype and KO pair for bulk RNA sequencing, differential gene expression, and gene set enrichment analyses (GSEA), which revealed an upregulation of gene markers and pathways potentially associated with metastasis and invasion in the HDAC10-KO cells, including S100A14, LAMA4, and TGF-beta. As the role of HDAC10 expression on cell migration and invasion has not been previously reported, we performed cell migration assays using our HDAC10 parental and knockout cell line pairs. In both HCT116 and HeLa systems, HDAC10 KO cells exhibited dramatically higher rates of cell migration in Boyden chamber and scratch assays. These same assays were performed in HDAC10-expressing cells in the presence of the HDAC10-specific inhibitor DKFZ-728, which similarly caused a dose-dependent increase in migration, strongly supporting a catalytic role for HDAC10 in repressing migration. In addition to these results, we present data investigating the role of HDAC10 in cell migration/invasion in representative prostate cancer cell lines (LNCAP and LAPC4), as the prostate tumor microenvironment is particularly rich in polyamines, including N 8-acetylated spermidine, the substrate of HDAC10. Finally, using our panel of HDAC10-KO cell lines and inhibitors, we analyze the contribution of commonly perturbed genes to the migratory phenotype with the goal of defining the precise mechanism of action. Overall, our studies provide functional evidence that loss of HDAC10 can increase cell migration, and thus, may lead to increased metastatic potential. These results emphasize the need for isoform-specific inhibitors and may have important clinical implications, as commonly utilized HDAC inhibitors, including vorinostat (SAHA), tubastatin A, and trichostatin A (TSA), also inhibit HDAC10. Our ongoing studies include incorporating HDAC10 proteolysis targeting chimeras (PROTACs) into our functional assays to examine differences between HDAC10 inhibition and PROTAC-mediated degradation, providing further rationale for modulating HDAC10 activity as an antineoplastic strategy. Citation Format: Ting-Ann Liu, Ashley Nwafor, Rajendra Kumar, Laura Sena, Raphael R. Steimbach, Aubry K. Miller, Robert A. Casero, Tracy Murray Stewart. Identification of histone deacetylase 10 as a repressor of cancer cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB210.

Molecular Mechanisms of Medicinal Plant Securinega suffruticosa-derived Compound Securinine against Spinal Muscular Atrophy based on Network Pharmacology and Experimental Verification.

Spinal Muscular Atrophy (SMA) is a severe motor neuronal disorder with high morbidity and mortality. Securinine has shown the potential to treat SMA; however, its anti-SMA role remains unclear. This study aims to reveal the anti-SMA mechanisms of securinine. Securinine-associated targets were acquired from Herbal Ingredients' Targets (HIT), Similarity Ensemble Approach (SEA), and SuperPred. SMA-associated targets were obtained from GeneCards and Dis- GeNET. Protein-protein Interaction (PPI) network was constructed using GeneMANIA, and hug targets were screened using cytoHubba. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using ClusterProfifiler. Molecular docking was conducted using Pymol and Auto- Dock. In vitro assays were used to verify the anti-SMA effects of securinine. Twenty-six intersection targets of securinine and SMA were obtained. HDAC1, HDAC2, TOP2A, PIK3R1, PRMT5, JAK2, HSP90AB1, TERT, PTGS2, and PAX8 were the core targets in PPI network. GO analysis demonstrated that the intersecting targets were implicated in the regulation of proteins, steroid hormones, histone deacetylases, and DNA transcription. KEGG analysis, pathway-pathway, and hub target-pathway networks revealed that securinine might treat SMA through TNF, JAK-STAT, Ras, and PI3K-Akt pathways. Securinine had a favorable binding affinity with HDAC1, HSP90AB, JAK2, PRMT5, PTGS2, and TERT. Securinine rescued viability suppression, mitochondria damage, and SMN loss in the SMA cell model. Furthermore, securinine increased HDAC1 and PRMT5 expression, decreased PTGS2 expression, suppressed the JAK2-STAT3 pathway, and promoted the PI3K-Akt pathway. Securinine might alleviate SMA by elevating HDAC1 and PRMT5 expression and reducing PTGS2 via JAK2-STAT3 suppression and PI3K-Akt activation.

Class I HDAC inhibitors enhance antitumor efficacy and persistence of CAR-T cells by activation of the Wnt pathway

Epigenetic modification shapes differentiation trajectory and regulates the exhaustion state of chimeric antigen receptor T (CAR-T) cells. Limited efficacy induced by terminal exhaustion closely ties with intrinsic transcriptional regulation. However, the comprehensive regulatory mechanisms remain largely elusive. Here, we identify class I histone deacetylase inhibitors (HDACi) as boosters of CAR-T cell function by high-throughput screening of chromatin-modifying drugs, in which M344 and chidamide enhance memory maintenance and resistance to exhaustion of CAR-T cells that induce sustained antitumor efficacy both invitro and invivo. Mechanistically, HDACi decrease HDAC1 expression and enhance H3K27ac activity. Multi-omics analyses from RNA-seq, ATAC-seq, and H3K27ac CUT&Tag-seq show that HDACi upregulate expression of TCF4, LEF1, and CTNNB1, which subsequently activate the canonical Wnt/β-catenin pathway. Collectively, our findings elucidate the functional roles of class I HDACi in enhancing CAR-T cell function, which provides the basis and therapeutic targets for synergic combination of CAR-T cell therapy and HDACi treatment.

Clinicopathological Significance of Nucleosome Remodeling and Deacetylase Complex Expression in Endometrial Carcinoma.

Only a few studies have examined the expression of nucleosome remodeling and deacetylase complex in endometrial carcinoma (EC). The aim of this study was to analyze the expressions of histone deacetylase (HDAC1), HDAC2, and chromodomain helicase DNA-binding protein 4 (CHD4) in EC. Sixty cases of EC were categorized into two clusters based on the expression levels of the three proteins. Cluster 1 (C1) exhibited elevated expressions of HDAC2 and CHD4 compared with cluster 2 (C2). Notably, 75% of cases in C2 represented non-aggressive histological types, whereas 37.5% of cases in C1 manifested aggressive types. C2 exclusively comprised pathological tumor stage 1 (pT1) tumors, whereas C1 included pT2 and pT3 tumors. In C1, 25% of cases displayed aberrant p53 expression, contrasting with the absence of such expression in C2. Furthermore, only one patient in C2 experienced disease recurrence, whereas 20.8% of patients in C1 developed recurrent tumors. High HDAC2 and CHD4 expression may be associated with adverse clinicopathological characteristics in EC. Further studies are needed to validate these results.

Mitragynine (Kratom)-Withdrawal behaviour and cognitive impairments can be ameliorated by an epigenetic mechanism.

Kratom is a preparation from Mitragyna speciosa, which is used as a natural drug preparation for many purposes around the world. However, an overdose of Kratom may cause addiction-like problems including aversive withdrawal states resulting in cognitive impairments via unknown mechanisms. Its main psychoactive alkaloid is mitragynine, showing opioid-like properties. Here, we analysed the neuropharmacological effects of mitragynine compared with morphine withdrawal in rats and searched for a pharmacological treatment option that may reverse the occurring cognitive deficits that usually aggravate withdrawal. We found that withdrawal from 14-day mitragynine (1-10mg·kg-1·day-1) treatment caused dose-dependent behavioural withdrawal signs resembling those of morphine (5mg·kg-1·day-1) withdrawal. However, mitragynine (5 and 10mg·kg-1·day-1) withdrawal also induced impairments in a passive avoidance task. Mitragynine withdrawal not only reduced hippocampal field excitatory postsynaptic potential (fEPSP) amplitudes in basal synaptic transmission and long-term potentiation (LTP) but also reduced epigenetic markers, such as histone H3K9 and H4K12 expression. At the same time, it up-regulates HDAC2 expression. Targeting the epigenetic adaptations with the HDAC inhibitor, SAHA, reversed the effects of mitragynine withdrawal on epigenetic dysregulation, hippocampal input/output curves, paired-pulse facilitation, LTP and attenuated the cognitive deficit. However, SAHA amplified the effects of morphine withdrawal. The data from this work show that changes in histone expression and downstream hippocampal plasticity may explain mitragynine, but not morphine, withdrawal behaviours and cognitive impairments. Thus, it may provide a new treatment approach for aversive Kratom/mitragynine withdrawal and addiction.

Hippocampal HDAC6 promotes POCD by regulating NLRP3-induced microglia pyroptosis via HSP90/HSP70 in aged mice

BackgroundPostoperative Cognitive Dysfunction (POCD) has attracted increased attention, but its precise mechanism remains to be explored. This study aimed to figure out whether HDAC6 could regulate NLRP3-induced pyroptosis by modulating the functions of HSP70 and HSP90 in microglia to participate in postoperative cognitive dysfunction in aged mice. MethodsAnimal models of postoperative cognitive dysfunction in aged mice were established by splenectomy under sevoflurane anesthesia. Morris water maze was used to examine the cognitive function and motor ability. Sixteen-months-old C57BL/6 male mice were randomly divided into six groups: control group (C group), sham surgery group (SA group), splenectomy group (S group), splenectomy + HDAC6 inhibitor ACY-1215 group (ACY group), splenectomy + HDAC6 inhibitor ACY-1215 + HSP70 inhibitor Apoptozole group (AP group), splenectomy + solvent control group (SC group). The serum and hippocampus of mice were taken after mice were executed. The protein levels of HDAC6, HSP90, HSP70, NLRP3, GSDMD-N, cleaved-Caspase-1 (P20), IL-1β were detected by western blotting. Serum IL-1β, IL-6 and S100β were measured using ELISA assay, and cell localization of HDAC6 was detected by immunofluorescence. In vitro experiments, BV2 cells were used to validate whether this mechanism worked in microglia. The protein levels of HDAC6, HSP90, HSP70, NLRP3, GSDMD-N, P20, IL-1β were detected by western blotting and the content of IL-1β in the supernatant was measured using ELISA assay. The degree of acetylation of HSP90, the interaction of HSP70, HSP90 and NLRP3 were analyzed by coimmunoprecipitation assay. ResultsSplenectomy under sevoflurane anesthesia in aged mice could prolong the escape latency, reduce the number of crossing platforms, increase the expression of HDAC6 and activate the NLRP3 inflammasome to induce pyroptosis in hippocampus microglia. Using ACY-1215 could reduce the activation of NLRP3 inflammasome, the pyroptosis of microglia and the degree of spatial memory impairment. Apoptozole could inhibit the binding of HSP70 to NLRP3, reduce the degradation of NLRP3 and reverse the protective effect of HDAC6 inhibitors. The results acquired in vitro experiments closely resembled those in vivo, LPS stimulation led to the pyroptosis of BV2 microglia cells and the release of IL-1β due to the activation of the NLRP3 inflammasome, ACY-1215 showed the anti-inflammatory effect and Apoptozole exerted the opposite effect. ConclusionsOur findings suggest that hippocampal HDAC6 promotes POCD by regulating NLRP3-induced microglia pyroptosis via HSP90/HSP70 in aged mice.

Abstract 711: HDAC3 sustains resistance to hypofractionated radiotherapy in fusion positve rhabdomyosarcoma cells

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. The fusion-positive (FP)-RMS variant expressing chimeric oncoproteins such as PAX3-FOXO1 and PAX7-FOXO1 shows a dismal prognosis with 5-year survival of less than 30% compared to non-metastatic fusion-negative (FN)-RMS variant. In the last years, a lot of interest has focused on new targets identification to improve the radiotherapy (RT) efficacy. HDAC inhibitors (HDACi) radio-sensitize different cancer cell types including RMS. Recently, we reported that MS-275, a Class I and IV HDACi, in combination with RT affected cell survival, reduced colony formation ability, increased DNA damage repair inhibition and reactive oxygen species formation in FP-RMS cells. However, despite promising preclinical studies, HDAC inhibitors (HDACi) achieved only modest success in human clinical trials for solid tumors, frequently showing several toxicities, probably because of the limited specificity of many HDACi. Thus, a major effort is being directed toward identification of HDACi which are selective for HDAC isoforms often uniquely implicated in the radioresistance of specific cancers. In order to identify the class I HDAC responsible of radioresistance in RMS we knocked-down HDAC1, HDAC2, HDAC3, and HDAC8 expression in combination with RT. Interestingly, we observed an increased radiosensitivity only in HDAC3-depleted FP-RMS cells. Thus, we focused on HDAC3 to understand the mechanisms by which it promotes radioresistance in RMS. We observed that HDAC3 is overexpressed in RMS patients and cell lines compared to the normal counterpart. Furthermore, RMS cells are strongly dependent by HDAC3 expression while slight dependency has been observed with other class I HDACs in DepMap portal. We demonstrated that HDAC3 depletion by CRISPR in combination with RT in FP-RMS cells increases apoptosis, reduces colony formation ability, cancer stem cells population and anchorage independent growth and reduces cell growth in vivo and in vitro. Accordingly, HDAC3 depleted cells in combination with RT reduces levels and activation of key player of FP-RMS biology such as MYCN, ERK and AKT. Moreover, HDAC3 KD increases radiotherapy-induced DNA double strand break and impairs DNA repair mechanisms reducing levels and activation of both homologous recombination (HR) and non-homologous end joining (NHEJ) factors such as ATM, RAD51 and DNA-PKcs. Thus, we developed a new potent and highly specific HDAC3 inhibitor (HDAC3i). The new HDAC3i is highly specific in targeting FP-RMS cell growth in vitro while no effects have been observed in normal cells such as myoblasts and lung fibroblast. Moreover, the drug treatment in combination with radiotherapy phenotypically and molecularly recapitulated what observed in HDAC3 depleted cells.The study has been founded by Italian Association for Cancer Research (AIRC) to FM. Citation Format: Matteo Cassandri, Antonella Porrazzo, Silvia Pomella, Simona Camero, Francesca A. Aiello, Lucrezia D'Archivio, Clemens Zwergel, Beatrice Noce, Miriam Tomaciello, Francesca Vulcano, Luisa Milazzo, Francesca Pedini, Michele Signore, Alessandro Fanzani, Cinzia Marchese, Giuseppe Minniti, Sergio Valente, Antonello Mai, Francesca Megiorni, Rossella Rota, Francesco Marampon. HDAC3 sustains resistance to hypofractionated radiotherapy in fusion positve rhabdomyosarcoma cells [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 711.

Abstract 4667: HDAC9 as a potential regulator of lenvatinib resistance in hepatocellular carcinoma

Abstract Lenvatinib is used as the first-line therapy for advanced hepatocellular carcinoma (HCC) patients. However, poor response and compromised therapeutic effect with prolonged treatment are frequently observed in HCC patients. To elucidate the underlying mechanisms, lenvatinib responsive and non-responsive stages were modeled in liver tumor-bearing immunocompetent mice which were divided into lenvatinib and vehicle control treatment groups. Tumor tissues after short and prolonged lenvatinib treatment were harvested for single-cell RNA sequencing. Results from single-cell RNA sequencing revealed heterogenous tumor cell sub-populations associated with tumor differentiation and identified an upregulated expression of class IIa histone acetylase HDAC9 in lenvatinib-treated tumor at the non-responsive stage. Upregulation of HDAC9 was similarly observed in lenvatinib-resistant human patient-derived xenograft (PDX). Functional studies suggested that suppressing HDAC9 could re-sensitize lenvatinib response in both in vitro and in vivo models. In sum, we identified a potential regulator of lenvatinib treatment response in HCC. Citation Format: Yunong Xie, Minghe Zhang, Yan Liu, Linglin Liu, Alfred Sze-Lok Cheng, Stephanie Ma, Man Tong. HDAC9 as a potential regulator of lenvatinib resistance in hepatocellular carcinoma [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 4667.

Abstract 1101: A patient-derived xenograft in vitro platform identifies panobinostat as the most efficient drug to treat high-risk hepatoblastoma patients

Abstract Hepatoblastoma is the most common pediatric liver tumor, and the treatment of patients with preoperative chemotherapy and surgical resection of the tumor has led to survival rates of up to 83%. However, resistance of the primary tumor towards chemotherapy and the persistence of metastases still represents major challenges to achieve complete remission in high-risk patients, which is associated with poor prognosis. In order to identify new treatment options, we have used a newly developed in vitro drug-testing platform comprised of conventional cell lines and cultures from dissociated patient-derived xenografts of pediatric liver cancers to test standard-of-care medications used in the current PHITT trial as well as compounds targeting a wide range of different cellular pathways. We identified histone deacetylase (HDAC) inhibitors as a promising drug family to reduce tumor cell growth in a dose-dependent manner, with panobinostat being the most effective drug tested. Subsequent in vitro assays showed that panobinostat reduced short- and long-term proliferation, retarded three-dimensional spheroid growth and ultimately induced apoptosis of hepatoblastoma cells. RNA sequencing indicated that panobinostat results in downregulation of MYC target genes and upregulation of the dual specificity phosphatase 1. Of clinical relevance, the combination of panobinostat with doxorubicin revealed a strong synergistic effect, even stronger than the one of cisplatin and doxorubicin, which is currently given to high-risk hepatoblastoma patients. Moreover, increased expression of HDAC4 and HDAC11 in primary tumor tissues was significantly associated with metastatic disease. Our results strongly suggest panobinostat to be used in future therapeutic trials for high-risk hepatoblastoma patients, especially when combined with doxorubicin. Citation Format: Salih Demir, Emilie Indersie, Sophie Branchereau, Eiso Hiyama, Stefano Cairo, Roland Kappler. A patient-derived xenograft in vitro platform identifies panobinostat as the most efficient drug to treat high-risk hepatoblastoma patients [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 1101.

Abstract 1730: Unraveling the oncogenic potential of KDM2A with gastric organoids

Abstract Background: Gastric adenocarcinoma (GAC), prevalent and often lethal, imposes a serious global health challenge. Therefore, it is critical to identify novel oncogenes (drivers) that can be therapeutically targeted. Of epigenetic modifications, histone lysine methylation is a hallmark of epigenetic and transcriptional regulation of gene expression. Therefore, investigating the involvement of histone methylation modifiers in GAC is paramount to gaining a comprehensive understanding of the disease but also to discover novel targets. Our analysis of the GAC genome indicates that these epigenetic modifiers are amplified in GAC. Objective and Hypothesis: The overall objective is to define the oncogenic role of KDM2A in GAC pathogenesis and progression. The central hypothesis is that KDM2A promotes GAC by upregulating the expression of oncogenic proteins via the activation of epigenomic signatures/pathways.KDM2A (FBXL11) is a histone lysine demethylase that demethylates H3K36me2 and represses target genes involved in various biological processes, including cell proliferation, DNA damage repair, and stem cell maintenance. Previously, we reported that KDM2A was necessary for the tumorigenic and metastatic capabilities of non-small lung cancer cells through transcriptionally repressing the expression of DUSP3 and HDAC3, which led to activation of ERK signaling and upregulation of cell cycle-associated genes. Results: Our analysis of the GAC genome indicates that KDM2A is one of the top demethylases frequently amplified and overexpressed (14%). In addition, KDM2A expression was significantly higher in GAC samples than in normal tissues, and high expression of KDM2A was associated with poor survival in GAC patients. To examine the effect of KDM2A knockdown on characteristics of GAC, we used gastric organoids generated from KDM2Aflox/flox mouse stomach. We expressed wild-type (Flag-KDM2A) and catalytic mutant (Flag-KDM2A-mut) in these organoids. Ectopic expression of KDM2A wild-type, but not mutant, increased the proliferation of organoids. We showed that KDM2A knockdown decreased the proliferation and invasion of two GAC cells, GA0518 and AGS. In addition, we determine the effect of overexpression of KDM2A on cell proliferation by comparing the control organoids to KDM2A overexpressed using IHC staining of Ki-67 (a proliferation marker) and other activated oncogenic signaling PI3KCA/mTOR/AKT, EGFR. We showed that GA5018 cells were very sensitive to the KDM2A inhibitor (daminozide) alone and in combination with erlotinib (EGFRi inhibitor) and RAD001 (RAD1 mTOR inhibitor). Significance and Innovation: In contrast to advances in cellular signaling pathways in GAC pathogenesis, how GAC development is epigenetically regulated remains largely unknown. Our results from using organoids indicate that KDM2A is an oncoprotein. Citation Format: Shilpa S. Dhar, Calena Brown, Constantin Zod, Jaffer A. Ajani, Min Gyu Lee. Unraveling the oncogenic potential of KDM2A with gastric organoids [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 1730.

Novel mechanistic study of HDAC6 regulation of rheumatoid arthritis via CMA: exploring potential therapeutic targets.

Rheumatoid arthritis (RA) is a systemic autoimmune disease. Its pathogenesis has not yet been clarified, so it is urgent to explore therapeutic targets. Here, we clarified the role of HDAC6 in the mechanism of action of RA through mediating chaperone-mediated autophagy (CMA) to provide a clinical treatment of RA. We used rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and collagen-induced arthritis mice (CIA mice) as models of RA and pharmacological inhibitors as well as genetic interference with adeno-associated viruses to reduce the expression of HDAC6. We explored the influence of CAY10603 on RA-FLS proliferation and inflammation, as well as the expression of proteins related to the CMA signaling pathway. CIA model was constructed using DBA/1J mice. Arthritis symptoms in CIA mice were evaluated, and the expression and localization of CMA-related proteins in mouse ankle joints were examined. CAY10603 inhibited proliferation as well as the level of the molecular chaperone autophagy in RA-FLS. HDAC6 shRNA significantly reduced the clinical signs of arthritis in CIA mice, as did the expression of HDAC6 in the serum and ankle synovial tissues of CIA mice. Finally, it significantly inhibited the level of Hsc70 and LAMP-2A, which are involved in the CMA signaling pathway, in ankle joint tissues. Downregulation of HDAC6 may inhibit CMA and thereby ameliorate RA.

Revealing Novel Genes Related to Parkinson's Disease Pathogenesis and Establishing an associated Model

Parkinson's disease (PD) represents a multifaceted neurological disorder whose genetic underpinnings warrant comprehensive investigation. This study focuses on identifying genes integral to PD pathogenesis and evaluating their diagnostic potential. Initially, we screened for differentially expressed genes (DEGs) between PD and control brain tissues within a dataset comprising larger number of specimens. Subsequently, these DEGs were subjected to weighted gene co-expression network analysis (WGCNA) to discern relevant gene modules. Notably, the yellow module exhibited a significant correlation with PD pathogenesis. Hence, we conducted a detailed examination of the yellow module genes using a cytoscope-based approach to construct a protein–protein interaction (PPI) network, which facilitated the identification of central hub genes implicated in PD pathogenesis. Employing two machine learning techniques, including XGBoost and LASSO algorithms, along with logistic regression analysis, we refined our search to three pertinent hub genes: FOXO3, HIST2H2BE, and HDAC1, all of which demonstrated a substantial association with PD pathogenesis. To corroborate our findings, we analyzed two PD blood datasets and clinical plasma samples, confirming the elevated expression levels of these genes in PD patients. The association of the genes with PD, as reflected by the area under the curve (AUC) values for FOXO3, HIST2H2BE, and HDAC1, were moderate for each gene. Collectively, this research substantiates the heightened expression of FOXO3, HIST2H2BE, and HDAC1 in both PD brain and blood samples, underscoring their pivotal contribution to the pathogenesis of PD.

Exosomal miR-223-3p from bone marrow mesenchymal stem cells targets HDAC2 to downregulate STAT3 phosphorylation to alleviate HBx-induced ferroptosis in podocytes.

Background: Hepatitis B virus associated-glomerulonephritis (HBV-GN) is one of the major secondary renal diseases in China, and microRNAs (miRNAs) in bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exo) can attenuate HBV-X protein (HBx)-induced ferroptosis in renal podocytes, but the exact mechanism remains unclear. This study aimed to investigate the protective mechanism of miR-223-3p in BMSC-Exo in HBx-induced ferroptosis in podocytes. Methods: The study employed human renal podocyte cells (HPCs), bone marrow-derived mesenchymal stem cells (BMSCs), as well as kidney tissue from C57BL/6 mice and HBx transgenic mice. Initially, the correlation between STAT3 phosphorylation and ferroptosis was authenticated through the administration of signal transducer and activator of transcription 3 (STAT3) phosphorylation inhibitors in both in vivo and in vitro settings. Furthermore, the effect of HDAC2 overexpression on STAT3 phosphorylation was examined. Subsequently, the association between BMSC-Exo carrying miR-223-3p, HDAC2, and the phosphorylation of STAT3 in HPCs ferroptosis and injury induced by HBx was assessed. The interaction between miR-223-3p and HDAC2 was confirmed via RNA immunoprecipitation assay. Various techniques such as cell counting kit-8 assay, western blot, RT-qPCR, immunofluorescence, flow cytometry, lipid peroxidation assay kit, iron assay kit, transmission electron microscopy, and hematoxylin-eosin staining were employed to visualize the extent of HBx-induced podocyte injury and ferroptosis in both in vivo and in vitro. Results: The attenuation of podocyte ferroptosis can be achieved by inhibiting the phosphorylation of STAT3 in podocytes induced by HBx. Conversely, the upregulation of HDAC2 can enhance STAT3 phosphorylation, thereby promoting podocyte ferroptosis. MiR-223-3p was capable of directly exerting negative regulation on HDAC2 expression. BMSC-Exo carrying miR-223-3p can effectively suppress the expression of HDAC2, ultimately leading to reduce HBx-induced ferroptosis in podocytes by targeting HDAC2 with miR-223-3p and downregulating STAT3 phosphorylation. Conclusion: This study evidences the potential of BMSC-Exo mediated delivery of miR-223-3p in mitigating HBx-induced ferroptosis in podocytes, thereby offering a novel therapeutic target and approach for treating HBV-GN and alleviating renal injury.

Searching for Essential Genes and Targeted Drugs Common to Breast Cancer and Osteoarthritis.

It is documented that osteoarthritis can promote the progression of breast cancer (BC). This study aims to search for the essential genes associated with breast cancer (BC) and osteoarthritis (OA), explore the relationship between epithelial-mesenchymal transition (EMT)- related genes and the two diseases, and identify the candidate drugs. The genes related to both BC and OA were determined by text mining. Protein-protein Interaction (PPI) analysis was carried out, and as a result, the exported genes were found to be related to EMT. PPI and the correlation of mRNA of these genes were also analyzed. Different kinds of enrichment analyses were performed on these genes. A prognostic analysis was performed on these genes for examining their expression levels at different pathological stages, in different tissues, and in different immune cells. Drug-gene interaction database was employed for potential drug discovery. A total number of 1422 genes were identified as common to BC and OA and 58 genes were found to be related to EMT. We found that HDAC2 and TGFBR1 were significantly poor in overall survival. High expression of HDAC2 plays a vital role in the increase of pathological stages. Four immune cells might play a role in this process. Fifty-seven drugs were identified that could potentially have therapeutic effects. EMT may be one of the mechanisms by which OA affects BC. Using the drugs can have potential therapeutic effects, which may benefit patients with both diseases and broaden the indications for drug use.

Histone deacetylase-mediated silencing of PSTPIP2 expression contributes to aristolochic acid nephropathy-induced PANoptosis.

Aristolochic acid nephropathy (AAN) is a progressive kidney disease caused by using herbal medicines. Currently, no therapies are available to treat or prevent aristolochic acid nephropathy. Histone deacetylase (HDAC) plays a crucial role in the development and progression of renal disease. We tested whether HDAC inhibitors could prevent aristolochic acid nephropathy and determined the underlying mechanism. HDACs expression in the aristolochic acid nephropathy model was examined. The activation of PANoptosis of mouse kidney and renal tubular epithelial cell were assessed after exposure to HDAC1 and HDAC2 blockade. Kidney-specific knock-in of proline-serine-threonine-phosphatase-interacting protein 2 (PSTPIP2) mice were used to investigate whether PSTPIP2 affected the production of PANoptosome. Aristolochic acid upregulated the expression of HDAC1 and HDAC2 in the kidneys. Notably, the HDAC1 and HDAC2 specific inhibitor, romidepsin (FK228, depsipeptide), suppressed aristolochic acid-induced kidney injury, epithelial cell pyroptosis, apoptosis and necroptosis (PANoptosis). Moreover, romidepsin upregulated PSTPIP2 in renal tubular epithelial cells, which was enhanced by aristolochic acid treatment. Conditional knock-in of PSTPIP2 in the kidney protected against aristolochic acid nephropathy. In contrast, the knockdown of PSTPIP2 expression in PSTPIP2-knock-in mice restored kidney damage and PANoptosis. PSTPIP2 function was determined in vitro using PSTPIP2 knockdown or overexpression in mouse renal tubular epithelial cells (mTECs). Additionally, PSTPIP2 was found to regulate caspase 8 in aristolochic acid nephropathy. HDAC-mediated silencing of PSTPIP2 may contribute to aristolochic acid nephropathy. Hence, HDAC1 and HDAC2 specific inhibitors or PSTPIP2 could be valuable therapeutic agents for preventing aristolochic acid nephropathy.

DOP61 Up-regulated Gremlin 1 in fibroblasts from Crohn’s Disease fibrotic strictures: A potential therapeutic target

Abstract Background Fibrosis is a common complication in Crohn’s disease (CD), often leading to intestinal strictures that are refractory to available biologic drugs and require surgery. Addressing this complication remains a significant unmet need. This study aims to explore the transcriptomic signature of fibrostenotic ileal CD to identify novel therapeutic targets. Methods 9 CD patients undergoing surgery for fibrotic strictures were prospectively recruited. All patients underwent magnetic resonance enterography, ileoscopy, and assessment of C-reactive protein and faecal calprotectin. A multidisciplinary team adjudicated the presence of fibrotic strictures, warranting surgical resection. Resected specimens were harvested directly from the operating theatre and delivered fresh to pathologists, who dissected samples from the strictured area, and the proximal and distal non-strictured regions. RNA was extracted from all samples for bulk-transcriptomic and single-cell RNA sequencing (scRNAseq) analysis, using the 10x Chromium Controller® platform. Differentially expressed genes (adj. p-value &amp;lt;0.05) were identified. Machine learning analyses, including PCA and PLS-DA, were employed to compare gene expression patterns among different tissue segments. Pathway enrichment analysis pinpointed relevant pathways. Further, the random forest model was constructed to evaluate the predictive significance of genes in relation to strictures, and the resulting ranked list of genes was subsequently validated through qPCR, confirming the significance of selected genes. Results Bulk-transcriptomic analysis revealed 64 significantly up-regulated genes in strictures, associated with inflammation, matrix remodeling, adipogenesis and cellular stress. Furthermore, 17 down-regulated genes were linked to epithelial barrier integrity. Normalised gene expression of GREM1, SERPINE1, FGF2, HDAC1 AND LY96 showed high predictive value for strictures compared to distal and proximal segments. Notably, qPCR confirmed the significant up-regulation of GREM1 expression exclusively in the ileal stricture samples (non-stricture vs. stricture: AUC 0.88, 95% CI 0.77-0.95). scRNAseq linked up-regulated GREM1 exclusively to the fibroblast cell population (THY1+, COL1A1+,ACTA2+). Conclusion Bulk and single-cell transcriptomics, along with qPCR validation of resected CD ileal strictures, showed the up-regulation of GREM1 in fibroblasts within stricture compared to adjacent proximal and distal intestinal samples. These findings confirmed in ileal CD previous reports from animal models, highlighting the role of GREM1 in promoting intestinal fibrosis. Therefore, GREM1 emerges an attractive target for the treatment of fibrotic strictures in CD.

Trifluoromethyl quinoline derivative targets inhibiting HDAC1 for promoting the acetylation of histone in cervical cancer cells

Cervical cancer is the leading cause of death among gynecological malignant tumors, especially due to the poor prognosis of patients with advanced tumors due to recurrence, metastasis, and chemotherapy resistance. Therefore, exploring new antineoplastic drugs with high efficacy and low toxicity may bring new expectations in patients with cervical cancer. Natural products and their derivatives exert an antitumor activity. Therefore, in this work, combined with network pharmacology analysis and experimental validation, we investigated the anti-cervical cancer activity and molecular mechanism of a new trifluoromethyl quinoline (FKL) derivative in vivo and in vitro. FKL117 inhibited the proliferation of cervical cancer cells in a dose and time-dependent manner, induced apoptosis in HeLa cells, arrested the cell cycle in the G2/M phase, and regulated the expression of the apoptotic and cell cycle-related proteins Bcl-2, Bax, cyclin B1, and CDC2. We used online databases to obtain HDAC1 as one of the possible targets of FKL117 and the target binding and binding affinity were modeled by molecular docking. The results showed that FKL117 formed a hydrogen bond with HDAC1 and had good binding ability. We found that FKL117 targeted to inhibit the expression and function of HDAC1 and increased the acetylation of histone H3 and H4, which was also confirmed in vivo. The migration of HMGB1 from the nucleus to the cytoplasm further verified the above results. In conclusion, our study suggested that FKL117 might be used as a novel candidate for targeting the inhibition of HDAC1 against cervical cancer.

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.

Altered histone acetylation patterns in pancreatic cancer cell lines induce subtype‑specific transcriptomic and phenotypical changes.

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at advanced tumor stages with chemotherapy as the only treatment option. Transcriptomic analysis has defined a classical and basal‑like PDAC subtype, which are regulated by epigenetic modification. The present study aimed to determine if drug‑induced epigenetic reprogramming of pancreatic cancer cells affects PDAC subtype identity and chemosensitivity. Classical and basal‑like PDAC cell lines PaTu‑S, Capan‑1, Capan‑2, Colo357, PaTu‑T, PANC‑1 and MIAPaCa‑2, were treated for a short (up to 96h) and long (up to 30weeks) period with histone acetyltransferase (HAT) and histone deacetylase (HDAC) inhibitors. The cells were analyzed using gene expression approaches, immunoblot analysis, and various cell assays to assess cell characteristics, such as proliferation, colony formation, cell migration and sensitivity to chemotherapeutic drugs. Classical and basal‑like PDAC cell lines showed pronounced epigenetic regulation of subtype‑specific genes through acetylation of lysine 27 on Histone H3 (H3K27ac). Moreover, classical cell lines revealed a significantly decreased expression of HDAC2 and increased total levels of H3K27ac in comparison with the basal‑like cell lines. Following HAT inhibitor treatment, classical cell lines exhibited a loss of epithelial marker gene expression, decreased chemotherapy response gene score and increased cell migration invitro, indicating a tumor‑promoting phenotype. HDAC inhibitor treatment, however, exerted minimal reprogramming effects in both subtypes. Epigenetic reprogramming of classical and basal‑like tumor cells did not have a major impact on gemcitabine response, although the gemcitabine transporter gene SLC29A1 (solute carrier family 29 member 1) was epigenetically regulated.