Histone Methylation Levels Research Articles

Effect of histone demethylase KDM5B on long-term cognitive impairment in neonatal rats induced by sevoflurane

IntroductionWhether repeated inhalation of sevoflurane during the neonatal period causes long-term learning and memory impairments in humans is unclear. Some recent investigations have indicated that general anesthesia drugs affect histone methylation modification and may further affect learning and memory ability. This study aimed to explore the role and mechanism of histone methylation in long-term cognitive dysfunction caused by repeated inhalation of sevoflurane during the neonatal period.MethodsNeonatal SD rats were assigned into three groups. Sevoflurane group and sevoflurane +AS8351 group were exposed to 2% sevoflurane for 4 h on postnatal day 7 (P7), day 14 (P7) and day 21 (P21), and the control group was inhaled the air oxygen mixture at the same time. From postnatal day 22 to 36, rats in the +AS8351 group were treated with AS8351 while those in the Sevoflurane group and control group were treated with normal saline. Half of the rats were carried out Y-maze, Morris water maze (MWM), western blot and transmission electron microscope at P37, and the remaining rats were fed to P97 for the same experiment.ResultsNeonatal sevoflurane exposure affected histone demethylase expression in hippocampus, changed histone methylation levels, Down-regulated synapse-associated protein expression, impaired synaptic plasticity and long-term cognitive dysfunction and KDM5B inhibitors partially restored the negative reaction caused by sevoflurane exposure.DiscussionIn conclusion, KDM5B inhibitor can save the long-term learning and memory impairment caused by sevoflurane exposure in neonatal period by inhibiting KDM5B activity.

Ethionine-induced S-adenosylmethionine deficiency suppressed H3K27me3 and cell differentiation during neural tube development in mice.

S-adenosylmethionine (SAM) as a major methyl donor plays a key role in methylation modification in vivo, and its disorder was closely related to neural tube defects (NTDs). However, the exact mechanism between SAM deficiency and NTDs remained unclearly. Hence, we investigated the association between histone methylation modification and cell differentiation in NTDs mice induced by SAM deficiency. The levels of SAM and SAH (S-adenosylhomocysteine) were determined by enzyme linked immunosorbent assay (ELISA). The level of histone methylation, β-catenin were analyzed by Western blot, reversing transcription and quantitative PCR (RT-qPCR) and immunofluorescence. The results showed that the incidence rate of NTDs induced by ethionine were 46.2%. Post treatment of ethionine combined with SAM, the incidence rate of NTDs was reduced to 26.2%. The level of SAM was significantly decreased (p < 0.05) and a reduction in the SAM/SAH ratio was observed after entionine treatment. The SAM deficiency caused the reduction of H3K27me3 modifications and the elevated UTX activity (p < 0.05), and inhibited the expressions of β-catenin. The differentiations of NSCs into neurons and oligodendrocytes were inhibited under SAM deficiency (p < 0.05). These results indicated that the SAM deficiency led to reduce H3K27me3 modifications, prevented the β-catenin signaling pathway and NSCs differentiation, which provided an understanding of the novel function of epigenetic regulation in NTDs.

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.

MAT2A is essential for zygotic genome activation by maintaining of histone methylation in porcine embryos

Methionine adenosyltransferase 2A (MAT2A) is an essential enzyme in the methionine cycle that generates S-adenosylmethionine (SAM) by reacting with methionine and ATP. SAM acts as a methyl donors for histone and DNA methylation, which plays key roles in zygotic genome activation (ZGA). However, the effects of MAT2A on porcine ZGA remain unclear. To investigate the function of MAT2A and its underlying mechanism in porcine ZGA, MAT2A was knocked down by double-stranded RNA injection at the 1-cell stage. MAT2A is highly expressed at every stage of porcine embryo development. The percentages of four-cell-stage embryos and blastocysts were lower in the MAT2A-knockdown (KD) group than in the control group. Notably, depletion of MAT2A decreased the levels of H3K4me2, H3K9me2/3, and H3K27me3 at the four-cell stage, whereas MAT2A KD reduced the transcriptional activity of ZGA genes. MAT2A KD decreased embryonic ectoderm development (EED) and enhancer of zeste homolog 2 (EZH2) expression. Exogenous SAM supplementation rescued histone methylation levels and developmental arrest induced by MAT2A KD. Additionally, MAT2A KD significantly increased DNA damage and apoptosis. In conclusion, MAT2A is involved in regulating transcriptional activity and is essential for regulating histone methylation during porcine ZGA.

JMJD2A promotes the development of castration-resistant prostate cancer by activating androgen receptor enhancer and inhibiting the cGAS-STING pathway.

Exploring targets for inhibiting androgen receptor (AR) activity is an effective strategy for suppressing the development of castration-resistant prostate cancer (CRPC). Upregulation of histone demethylase JMJD2A activity is an important factor in increasing AR expression in CRPC. Based on our research, we found that the binding affinity between JMJD2A and AR increases in CRPC, while the level of AR histone methylation decreases and the H3K27ac level increases in the AR enhancer region. Further investigations revealed that overexpression of the histone demethylase JMJD2A increased the binding affinity between JMJD2A and AR, decreased AR histone methylation levels, upregulated H3K27ac in the AR enhancer region, and increased AR activity. Conversely, knocking down JMJD2A effectively reversed these effects. Additionally, in CRPC, JMJD2A expression was upregulated, the tumor-intrinsic immune cGAS-STING signaling pathway was suppressed, the tumor microenvironment was altered, and AR expression was upregulated. However, both knocking down JMJD2A and inhibiting the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS-STING) signaling pathway reversed these effects. In summary, our study indicates that in CRPC, JMJD2A can directly bind to AR and activate residual AR enhancers through its demethylation activity, thereby promoting AR expression. Furthermore, upregulation of JMJD2A expression inhibits the innate immune cGAS-STING signaling pathway of the tumor, leading to a decrease in antitumor immune function, and further promoting AR expression.

Lysine Methylation and Histone Modifications during Cold Stress of Insects: Freeze-Tolerant Eurosta solidaginis and Freeze-Avoiding Epiblema scudderiana.

Overwintering survival by insects, whether of the freeze-tolerant or freeze-avoiding types, is typically associated with a strong suppression of metabolic rate (e.g., entry into diapause) that involves the differential expression of many genes with regulation at the transcriptional, translational or post-translational levels. Epigenetic modifications have been suggested to play a vital role in regulating cold responses of insects. However, knowledge of the roles of epigenetic mechanisms in modulating gene expression for winter survival of the larvae of two goldenrod gall formers, the freeze-tolerant dipteran Eurosta solidaginis and the freeze-avoiding lepidopteran Epiblema scudderiana, remain unknown. The current study evaluates the role of cold-induced lysine methylation and histone modifications, with enzymes of lysine methylation (SETD8, SETD7, SUV39H1, SMYD2 and ASH2L), as well as relative levels of histone H3 acetylation (H3K9ac, H3K18ac, H3K27ac, H3K56ac) and methylation (H3K4me1, H3K9me3, H3K36me2) examined in two insects. Significant (p < 0.05) reductions were observed in most of the targets of histone methylation/acetylation for decreasing temperatures of Ep. scudderiana larvae, whereas selected histone methylation/acetylation targets were conversely elevated (p < 0.05) in E. solidaginis, particularly under conditions of 5 °C for 4 h. Histone H3 expression was found to be variable without statistical differences in larval goldenrod gall moths and gall flies. These results provide basic information on the patterns of epigenetic regulation involved in insect cold hardiness.

Revealing the Biophysics of Lamina-Associated Domain Formation by Integrating Theoretical Modeling and High-Resolution Imaging.

The interactions between chromatin and the nuclear lamina orchestrate cell type-specific gene activity by forming lamina-associated domains (LADs) which preserve cellular characteristics through gene repression. However, unlike the interactions between chromatin segments, the strength of chromatin-lamina interactions and their dependence on cellular environment are not well understood. Here, we develop a theory to predict the size and shape of peripheral heterochromatin domains by considering the energetics of chromatin-chromatin interactions, the affinity between chromatin and the nuclear lamina and the kinetics of methylation and acetylation9in human mesenchymal stem cells (hMSCs). Through the analysis of super-resolution images of peripheral heterochromatin domains using this theoretical framework, we determine the nuclear lamina-wide distribution of chromatin-lamina affinities. We find that the extracted affinity is highly spatially heterogeneous and shows a bimodal distribution, indicating regions along the lamina with strong chromatin binding and those exhibiting vanishing chromatin affinity interspersed with some regions exhibiting a relatively diminished chromatin interactions, in line with the presence of structures such as nuclear pores. Exploring the role of environmental cues on peripheral chromatin, we find that LAD thickness increases when hMSCs are cultured on a softer substrate, in correlation with contractility-dependent translocation of histone deacetylase 3 (HDAC3) from the cytosol to the nucleus. In soft microenvironments, chromatin becomes sequestered at the nuclear lamina, likely due to the interactions of HDAC3 with the chromatin anchoring protein LAP2 β ,increasing chromatin-lamina affinity, as well as elevated levels of the intranuclear histone methylation. Our findings are further corroborated by pharmacological interventions that inhibit contractility, as well as by manipulating methylation levels using epigenetic drugs. Notably, in the context of tendinosis, a chronic condition characterized by collagen degeneration, we observed a similar increase in the thickness of peripheral chromatin akin to that of cells cultured on soft substrates consistent with theoretical predictions. Our findings underscore the pivotal role of the microenvironment in shaping genome organization and highlight its relevance in pathological conditions.

Abstract 2865: Efficacy of MDM2 and epigenetic inhibitors treatments for neuroblastoma

Abstract Pediatric Neuroblastoma (NB) is one of the most prevalent solid tumors in children that arise from embryonic neural crest cells and poses a significant challenge in the field of oncology. There are about 700 to 800 new cases of neuroblastoma diagnosed each year in the United States, and accounts for approximately 15% of all childhood cancer mortalities. Since this malignancy predominantly affects infants and young children, it is imperative to decipher the underlying molecular factors driving its pathogenesis. The novel molecular therapeutic approach includes the use of epigenetic modifiers as drugs. This approach aims to reverse significant epigenetic events underlying cancer pathogenesis, particularly abnormalities in histone modifications and DNA methylation. The reduction of both DNA and histone methylation levels leads to the reactivation of tumor suppressor genes and inhibits cancer cell proliferation and cell growth. In this study, we used selective and reversible small molecule agents such as MDM2 inhibitor (RG7388) and epigenetic modifiers (CM272 and SAHA) to block cell proliferation by inducing cell cycle arrest via enforcing Epigenetic alterations. Our initial experiments were designed to understand the effects of RG7388, CM272, and SAHA treatments on NB and their impact on cell cycle progression. It was hypothesized that the decrease in DNA methylation and increase in histone modification- augment p21 expression via the use of MDM2 inhibitors and epigenetic modifiers. To test this hypothesis, the NB cells (SK-N-SK and IMR-32) were treated with RG7388, CM272, and SAHA for 24 hours. The extent of Cell death was determined for NB cells by using the Trypan Blue Dye Exclusion (TBDE) method, and CellTiter-Glo® Luminescent Cell Viability Assay. Induction of apoptosis was determined by using the Immunofluorescence staining method with DEVD-amc substrate. The results of our study suggested that RG7388, CM272, and SAHA were able to induce cell death in the SK-N-SH cells following 24-hour treatment. Furthermore, we analyzed the levels of Tumor suppressor (p53), cell cycle arrest (p21, p27, AurkB, CDC25A, CyclinD) cell death (Bax, Bcl2, LC3, Beclin, PARP, Cleaved PARP, RIP1, RIP3, MLKL, p-AKT), and Epigenetic biomarker (DNMT1, DNMT3A, DNMT3B, acetyl histoneH3, H4, and H2b) protein expression levels using western blot analysis. The results showed significant elevation of p53, p21, p27, and acetylation of histone family biomarkers after treatment when compared with the control group. In summary, our results indicate that RG7388, CM272, and SAHA treatments can effectively induce cellular responses and prevent cell growth and progression by enforcing epigenetic changes in the NB cells. (This research was supported by the National Pediatric Cancer Foundation, Tampa, Florida, and the Royal Dames of Cancer Research Inc. of Ft. Lauderdale, Florida) Citation Format: Tahani Momenah, Umamaheswari Natarajan, Shyam Jaganathan, Appu Rathinavelu. Efficacy of MDM2 and epigenetic inhibitors treatments for neuroblastoma [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 2865.

Transgenerational toxicity induced by maternal AFB1 exposure in Caenorhabditis elegans associated with underlying epigenetic regulations

Aflatoxin B1 (AFB1), usually seriously contaminates in grain and oil foods or feed, displayed significant acute and chronic toxic effects in human and animal populations. However, little is known about the transgenerational toxic effects induced by a maternal AFB1 intake at a lower dose on offspring. In our study, only parental wild-type Caenorhabditis elegans was exposed to AFB1 (0–8 μg/ml) and the following three filial generations were grown on AFB1-free NGM. Results showed that the toxic effects of AFB1 on the growth (body length) and reproduction (brood size, generation time and morphology of gonad arm) can be transmitted through generations. Moreover, the levels of MMP and ATP were irreversibly inhibited in the filial generations. By using RNomics and molecular biology techniques, we found that steroid biosynthesis, phagosome, valine/leucine/isoleucine biosynthesis and oxidative phosphorylation (p < 0.05) were the core signaling pathways to exert the transgenerational toxic effects on nematodes. Also, notably increased histone methylation level at H3K36me3 was observed in the first generation. Taken together, our study demonstrated that AFB1 has notable transgenerational toxic effects, which were resulted from the complex regulatory network of various miRNAs, mRNAs and epigenetic modification in C. elegans.

Epigenetic modifications of 45S rDNA associates with the disruption of nucleolar organisation during Cd stress response in Pakchoi

The role of the nucleolus in Pakchoi response to Cd stress remains largely unknown. In this work, we focus on exploring the underling mechanism between nucleolus disruption and epigenetic modification in Cd stressed-Pakchoi. Our results indicated that the proportion of nucleolus disruption, decondensation of 45 S rDNA chromatin, and a simultaneous increase in 5' external transcribed spacer region (ETS) transcription were observed with increasing Cd concentration, accompanied by genome-wide alterations in the levels of histone acetylation and methylation. Further results showed that Cd treatment exhibited a significant increase in H3K9ac, H4K5ac, and H3K9me2 levels occurred in promoter regions of the 45 S rDNA. Additionally, DNA methylation assays in the 45 S rDNA promoter region revealed that individual site-specific hypomethylation may be engaged in the activation of 45 S rDNA transcription. Our study provides some molecular mechanisms for the linkage between Cd stress, rDNA epigenetic modifications, and nucleolus disintegration in plants.

Structural optimization of a lysine demethylase 5 inhibitor for improvement of its cellular activity

Lysine demethylase 5 (KDM5) subfamily proteins are important in epigenetic gene regulation. They are involved in the growth and drug resistance of cancer cells. Therefore, KDM5s are potential cancer therapeutic targets, and their inhibitors hold promise as anti-cancer drugs. Several KDM5 inhibitors, including KDM5-C49 (2a), have exhibited potent KDM5-inhibitory activities in in vitro enzyme assays. However, they do not show enough cellular activity despite being converted to their prodrugs. We hypothesized that their poor lipophilicity should prevent them from sufficiently penetrating the cell membrane, and introducing more lipophilic groups should improve cellular activities. In this study, we investigated 2a and KDM5-C70 (3a), a prodrug of 2a, and attempted to improve its cellular activity by replacing the N,N-dimethyl amino group of 3a with more lipophilic groups. N-Butyl, N-methyl amino compound 2e exhibited potent and selective KDM5-inhibitory activity equal to that of 2a. Furthermore, the cell membrane permeability of 3e, an ethyl ester prodrug of 2e, was six times higher than that of 3a in a parallel artificial membrane permeation assay. In addition, western blot analysis indicated that treating human lung cancer A549 cells with 3e increased histone methylation levels more strongly than that with 3a. Thus, we identified compound 3e as a more cell-active KDM5 inhibitor that has sufficient cell membrane permeability.

Abstract A023: Nicotinamide Phosphoribosyltransferase (NAMPT) is required for trained macrophage-mediated antitumor immunity

Abstract Trained immunity or innate immune memory plays an active role in suppressing tumour growth and metastasis, however the specific metabolic mechanisms underlying such memory response-mediated antitumor activity have not been fully elucidated. Here, we show that the Nicotinamide Phosphoribosyltransferase/Nicotinamide Adenine Dinucleotide (NAMPT/NAD+) axis is required for the establishment of immune memory in macrophages against tumour. Chemical inhibition or genetic knockout of NAMPT in macrophages blocked trained immunity in vitro and in vivo, which could be rescued by NMN, an NAD+ precursor and a direct metabolite of NAMPT. Further studies revealed that myeloid-specific knockout of Nampt in mice significantly impaired the antitumor effect induced by in vivo β-glucan training, accompanied with decreased infiltration of intratumoral M1 macrophages. Interestingly, NAMPT deficiency did not impair β-glucan-induced interferon signalling or Akt/mTOR/HIF-1α signalling pathway, but markedly reduced histone methylation and acetylation levels, suggesting that NAMPT/NAD+ axis regulates trained immunity in macrophages via potential epigenetic mechanisms. Collectively, our study identifies that NAMPT/NAD+ metabolic axis-regulated trained immunity is a critical mechanism in early immune surveillance that suppresses tumour development via epigenetic regulation. Citation Format: Huan Jin, Yongxiang Liu, Xiaojun Xia. Nicotinamide Phosphoribosyltransferase (NAMPT) is required for trained macrophage-mediated antitumor immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A023.

Photoaged Polystyrene Nanoplastics Result in Transgenerational Reproductive Toxicity Associated with the Methylation of Histone H3K4 and H3K9 in Caenorhabditis elegans.

Polystyrene nanoplastics (PS-NPs) are emerging environmental contaminants that are ubiquitously detected in various environments and have toxic effects on various organisms. Nevertheless, the transgenerational reproductive toxicity and underlying mechanisms of PS-NPs remain largely unknown, especially for photoaged PS-NPs under ultraviolet irradiation. In this study, only the parental generation (P0) was exposed to virgin and aged PS-NPs at environmentally relevant concentrations (0.1-100 μg/L), and subsequent generations (F1-F4) were cultured under normal conditions. Ultraviolet irradiation induced the generation of environmentally persistent free radicals and reactive oxygen species, which altered the physical and chemical characteristics of PS-NPs. The results of toxicity testing suggested that exposure to aged PS-NPs caused a more severe decrease in brood size, egg ejection rate, number of fertilized eggs, and hatchability than did the virgin PS-NPs in the P0, F1, and F2 generations. Additionally, a single maternal exposure to aged PS-NPs resulted in transgenerational effects on fertility in the F1 and F2 generations. Increased levels of H3K4 and H3K9 methylation were observed in the F1 and F2 generations, which were concomitant with the transgenerational downregulation of the expression of associated genes, such as spr-5, set-17, and met-2. On the basis of correlation analyses, the levels of histone methylation and the expression of these genes were significantly correlated to transgenerational reproductive effects. Further research showed that transgenerational effects on fertility were not observed in spr-5(by134), met-2(n4256), and set-17(n5017) mutants. Overall, maternal exposure to aged PS-NPs induced transgenerational reproductive effects via H3K4 and H3K9 methylation, and the spr-5, met-2, and set-17 genes were involved in the regulation of transgenerational toxicity. This study provides new insights into the potential risks of photoaging PS-NPs in the environment.

Epigenetic control of plant senescence and cell death and its application in crop improvement.

Plant senescence is the last stage of plant development and a type of programmed cell death, occurring at a predictable time and cell. It involves the functional conversion from nutrient assimilation to nutrient remobilization, which substantially impacts plant architecture and plant biomass, crop quality, and horticultural ornamental traits. In past two decades, DNA damage was believed to be a main reason for cell senescence. Increasing evidence suggests that the alteration of epigenetic information is a contributing factor to cell senescence in organisms. In this review, we summarize the current research progresses of epigenetic and epitranscriptional mechanism involved in cell senescence of plant, at the regulatory level of DNA methylation, histone methylation and acetylation, chromatin remodeling, non-coding RNAs and RNA methylation. Furthermore, we discuss their molecular genetic manipulation and potential application in agriculture for crop improvement. Finally we point out the prospects of future research topics.

Natural products modulating epigenetic mechanisms by affecting histone methylation/demethylation: targeting cancer cells.

Many natural products can exert anticancer or chemopreventive activity by interfering with the cellular epigenetic machinery. While a plethora of studies indicate the relevance of affecting DNA methylation and histone acetylation, the influence on the mechanisms related to histone methylation is often overlooked. This may be associated with the lagging evidence that changes in the action of histone methylation writers and erasers and subsequent alterations in the profile of histone methylation are causally related with carcinogenesis. However, recent animal studies have shown that targeting histone methylation/demethylation affects the course of experimentally induced carcinogenesis. Existing data suggest that numerous natural compounds from different chemical groups, including green tea polyphenols and other flavonoids, curcuminoids, stilbene derivatives, phenolic acids, isothiocyanates, alkaloids and terpenes, can affect the expression and activity of crucial enzymes involved in the methylation and demethylation of histone lysine and arginine residues. These activities have been associated with the modulation of cancer-related gene expression and functional changes, including reduced cell proliferation and migration, and enhanced apoptosis in various cancer models. Most studies focused on the modulation of the expression and/or activity of two proteins - EZH2 (a H3K27 methyltransferase) and LSD1 (a H3K4/9 demethylase), or the effects on the global levels of histone methylation marks by the phytochemicals, but data regarding other histone methyltransferases or demethylases are scarce. While the field still remains relatively unexplored, this review aims to explore the impact of natural products on the enzymes related to histone methylation/demethylation, showing their relevance to carcinogenesis and cancer progression.

SAT178 EZH2 Upregulation And Lysine 27 Trimethylation at Histone 3 Is Associated With Severe Sporadic Primary Hyperparathyroidism

Abstract Disclosure: P. Kumari: None. A.K. Arya: None. D. Dahiya: None. N. Sachdeva: None. J. Kaur: None. U.N. Saikia: None. S. Bhadada: None. S.D. Rao: None. Introduction: The enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that regulates gene activity through histone modifications of lysine (K) at H3 histone and has a crucial role in tumor development. However, the level of methylation at H3 histone and its association with EZH2 expression in sporadic primary hyperparathyroidism (PHPT) are not known. This study was designed to investigate how global levels of histone methylation were associated with altered expression of EZH2 in promoting parathyroid tumorigenesis. Methodology: A total of 30 parathyroid tumors (15 non-severe, 15 severe, and 5 normal parathyroid tissues) were collected during 2020-2021. The global histone methylation of major methylation sites at lysine (K) K4, K9, K27, K36, and K79 was evaluated from blood and tissue samples using a calorimetric ELISA (H3 global modification kit, Epegentek, USA). Gene and protein expression of EZH2 were performed by quantitative real-time PCR and immunohistochemistry respectively. Histone methylation and expression data were correlated with indices of disease severity. Results: We found that the global levels of H3K27me1 and me3 were significantly upregulated, whereas, H3K4me3 and H3K36me2 were significantly downregulated in tumors from patients with severe PHPT compared to tumors from non-severe PHPT and controls. Moreover, H3K27me3 showed the highest upregulation (8-fold) in severe PHPT. Additionally, EZH2 gene expression was significantly upregulated in severe PHPT [Mean ± S.E; (9.1 ± 1.6 vs. 1.2 ± 0.3, p = 0.005), but not in non-severe (4.2 ± 2.7 vs. 1.2 ± 0.3, p = 0.4) PHPT, compared to normal parathyroid tissues. Furthermore, the protein expression was also consistent with the gene expression, with 86% (n= 13/15) nuclear positivity for EZH2 in tumors from severe PHPT and 40% (6 of 15) in tumors from non-severe PHPT compared to normal parathyroid tissue (negative staining). Correlation analysis revealed that EZH2 gene expression is positively related with S.Ca (r = 0.44, p = 0.003), PTH (r = 0.59, p = 0.000), tumor weight (r = 0.455, p = 0.002), EZH2 protein expression (r = 0.7, p = 0.000), and Ki67 proliferative marker (r = 0.66, p = 0.002). Conclusions: Increased global trimethylation of K27 at histone 3 (H3K27me3) was associated with increased EZH2 expression in sporadic PHPT and was predictive of a more severe clinical phenotype. Sources of research support: (ICMR), New Delhi Extramural Research Grant 2020. Presentation: Saturday, June 17, 2023

The diapausing mosquito Culex pipiens exhibits reduced levels of H3K27me2 in the fat body.

Culex pipiens, the northern house mosquito, is a major vector of West Nile virus. To survive the severe winter, adult mosquitoes enter a diapause programme. Extended lifespan and an increase in lipid storage are key indicators of diapause. Post-translational modifications to histone proteins impact the expression of genes and have been linked to the lifespan and energy utilisation of numerous insects. Here, we investigated the potential contribution of epigenetic alterations in initiating diapause in this mosquito species. Multiple sequence alignment of H3 sequences from other insect species demonstrates a high conservation of the H3 histone in Cx. pipiens throughout evolution. We then compared the levels of histone methylation in the ovaries and fat body tissues of diapausing and non-diapausing Cx. pipiens using western blots. Our data indicate that histone methylation levels in the ovaries of Cx. pipiens do not change during diapause. In contrast, H3K27me2 levels decrease more than twofold in the fat body of diapausing mosquitoes relative to non-diapausing counterparts. H3K27 methylation plays a crucial role in chromosome activation and inactivation during development in many insect species. This is predominantly governed by polycomb repressor complex 2. Intriguingly, a previous ChIP-seq study demonstrated that the transcription factor FOXO (Forkhead box O) targets the genes that comprise this complex. In addition, H3K27me2 exhibits dynamic abundance throughout the diapause programme in Cx. pipiens, suggesting its potential role in the initial activation of the diapause programme. This study expands our understanding of the relationship between alterations in epigenetic regulation and diapause.

Role of histone methylation in skin cancers: Histone methylation-modifying enzymes as a new class of targets for skin cancer treatment.

Histone methylation, one of the most prominent epigenetic modifications, plays a vital role in gene transcription, and aberrant histone methylation levels cause tumorigenesis. Histone methylation is a reversible enzyme-dependent reaction, and histone methyltransferases and demethylases are involved in this reaction. This review addresses the biological and clinical relevance of these histone methylation-modifying enzymes for skin cancer. In particular, the roles of histone lysine methyltransferases, histone arginine methyltransferase, lysine-specific demethylases, and JmjC demethylases in skin cancer are discussed in detail. In addition, we summarize the efficacy of several epigenetic inhibitors targeting histone methylation-modifying enzymes in cutaneous cancers, such as basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma. In conclusion, we propose histone methylation-modifying enzymes as novel targets for next-generation pharmaceuticals in the treatment of skin cancers and further provide a rationale for the development of epigenetic drugs (epidrugs) that target specific histone methylases/demethylases in cutaneous tumors.

High-dose synthetic phenolic antioxidant propyl gallate impairs mouse oocyte meiotic maturation through inducing mitochondrial dysfunction and DNA damage.

Propyl gallate (PG) is one of the most widely used antioxidants in food products, cosmetics and pharmaceutical industries. Increased research has suggested that exposure to PG influences reproductive health in humans and animals. However, until now, it has not yet been confirmed whether PG would impact oocyte quality. In this study, the hazardous effects of PG on oocyte meiotic maturation were investigated in mice. The findings showed that PG exposure compromises oocyte meiosis by inducing mitochondrial stress which activates apoptosis to trigger oocyte demise. Moreover, DNA damage was significantly induced in PG-treated oocytes, which might be another cause of oocyte developmental arrest and degeneration. Besides, the level of histone methylation (H3K27me2 and H3K27me3) in oocyte was also significantly increased by PG exposure. Furthermore, PG-induced oxidative stress was validated by the increased level of reactive oxygen species (ROS), which might be the underlying reason for these abnormities. In conclusion, the foregoing findings suggested that PG exposure impaired oocyte meiotic maturation by yielding mitochondrial stress to activate apoptosis, inducing DNA damage and oxidative stress, and altering histone methylation level.

Abstract 543: Preclinical characteristic of HMPL-306, a CNS-penetrable dual inhibitor of mutant IDH1 and IDH2

Abstract Background: Mutations in isocitrate dehydrogenase (IDH) 1/2 are frequently identified in various cancers, such as AML, cholangiocarcinoma, chondrosarcoma and glioma. Mutant IDHs (mIDHs) cause accumulated 2-HG, leading to blockage of cell differentiation, thereby inducing malignant transformation. Rare cases were identified carrying co-existing mutations in IDH1 and IDH2. mIDH isoform switching, from mutant IDH1 to mutant IDH2 and vice versa, have been reported as a mechanism of acquired resistance to IDH inhibition in AML and cholangiocarcinoma. Thus, simultaneous inhibition on both mIDH1 and mIDH2 may be a promising strategy to overcome resistance and improve clinical efficacy. HMPL-306, a dual inhibitor of mIDH1/mIDH2, developed by HUTCHMED, is being evaluated in clinical trials. Methods: The inhibition of HMPL-306 on IDH enzymes, including mutant and wild type, was determined by fluorescence-based assay. The selectivity of HMPL-306 was evaluated in 322 kinases (SelectScreenTM) and 88 proteins (Cerep). For cellular activities of HMPL-306, 2-HG production and differentiation were detected in cells harboring mIDH. Human tumor xenograft models carrying IDH1 or 2 mutations were established for evaluating mIDH inhibition by detecting 2-HG in plasma and tumor, and anti-tumor efficacies. Results: HMPL-306 inhibited mutant IDH enzyme activities including IDH1R132H, IDH2R140Q and IDH2R172K, while showed weaker inhibition on IDH1/2 wild type enzymes. HMPL-306 had a superior selectivity profile in a kinase panel and a safety panel, while enasidenib, an approved mIDH2 inhibitor, inhibited Adenosine-A3 with IC50 of 12 nM. In cellular assays, HMPL-306 displayed comparable activities to enasidenib and ivosidenib (approved mIDH1 inhibitor) and suppressed 2-HG through inhibition of mIDH1 or mIDH2 at similar level, indicating an equal potency against mIDH1 and 2. Moreover, in both mIDH1/2 cells, HMPL-306 reduced the levels of histone methylation, and promoted hemoglobin γ and Kruppel1 gene expression, which led to differentiation from immature malignant cells to mature normal cells. Oral administration of HMPL-306 remarkably decreased 2-HG level in plasma and tumor tissues in xenograft models carrying mIDH1 or mIDH2 and the inhibition is more potent and durable than either ivosidenib or enasidenib at the same dose. Pharmacokinetics (PK) study in rodents showed high exposures of HMPL-306 in brain and cerebrospinal fluid, a desirable feature for glioma therapy. Combination treatment of HMPL-306 and azacitidine synergized in releasing the differentiation block in mIDH AML cells. HMPL-306 also significantly improved in vivo anti-tumor efficacy of chemotherapy drugs in solid tumor models with mIDH1/2. Conclusion: HMPL-306 is a potent, dual inhibitor of IDH1/2 mutation. The strong activity and favorable PK profiles support further clinical evaluation. Citation Format: Na Yang, Jia Hu, Tingwen Li, Juntao Yu, Dongxia Shi, Min Cheng, Zeyu Zhong, Jian Wang, Yang Sai, Weiguo Qing, Guangxiu Dai, Yongxin Ren, Michael Shi, Weiguo Su. Preclinical characteristic of HMPL-306, a CNS-penetrable dual inhibitor of mutant IDH1 and IDH2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 543.