Inhibitor GSK-J4 Research Articles

Histone H3K27 Demethylase Negatively Controls the Memory Formation of Antigen-Stimulated CD8+ T Cells.

Although the methylation status of histone H3K27 plays a critical role in CD4+ T cell differentiation and its function, the role of Utx histone H3K27 demethylase in the CD8+ T cell-dependent immune response remains unclear. We therefore generated T cell-specific Utx flox/flox Cd4-Cre Tg (Utx KO) mice to determine the role of Utx in CD8+ T cells. Wild-type (WT) and Utx KO mice were infected with Listeria monocytogenes expressing OVA to analyze the immune response of Ag-specific CD8+ T cells. There was no significant difference in the number of Ag-specific CD8+ T cells upon primary infection between WT and Utx KO mice. However, Utx deficiency resulted in more Ag-specific CD8+ T cells upon secondary infection. Adoptive transfer of Utx KO CD8+ T cells resulted in a larger number of memory cells in the primary response than in WT. We observed a decreased gene expression of effector-associated transcription factors, including Prdm1 encoding Blimp1, in Utx KO CD8+ T cells. We confirmed that the trimethylation level of histone H3K27 in the Prdm1 gene loci in the Utx KO cells was higher than in the WT cells. The treatment of CD8+ T cells with Utx-cofactor α-ketoglutarate hampered the memory formation, whereas Utx inhibitor GSK-J4 enhanced the memory formation in WT CD8+ T cells. These data suggest that Utx negatively controls the memory formation of Ag-stimulated CD8+ T cells by epigenetically regulating the gene expression. Based on these findings, we identified a critical link between Utx and the differentiation of Ag-stimulated CD8+ T cells.

Comprehensive profiling of JMJD3 in gastric cancer and its influence on patient survival

Histone methylation is thought to control the regulation of genetic program and the dysregulation of it has been found to be closely associated with cancer. JMJD3 has been identified as an H3K27 demethylase and its role in cancer development is context specific. The role of JMJD3 in gastric cancer (GC) has not been examined. In this study, JMJD3 expression was determined. The prognostic significance of JMJD3 and its association with clinical parameters were evaluated. JMJD3 dysregulation mechanism and targets were analyzed. The effect of JMJD3 mutation was determined by functional study. Results showed that JMJD3 was overexpressed in different patient cohorts and also by bioinformatics analysis. High JMJD3 expression was correlated with shortened overall survival in patients with GC and was an independent prognosis predictor. Genetic aberration and DNA methylation might be involved in the deregulation of JMJD3 in GC. Downstream network of JMJD3 was analyzed and several novel potential targets were identified. Furthermore, functional study discovered that both demethylase-dependent and demethylase-independent mechanisms were involved in the oncogenic role of JMJD3 in GC. Importantly, histone demethylase inhibitor GSK-J4 could reverse the oncogenic effect of JMJD3 overexpression. In conclusion, our study report the oncogenic role of JMJD3 in GC for the first time. JMJD3 might serve as an important epigenetic therapeutic target and/or prognostic predictor in GC.

KDM6B overexpression activates innate immune signaling and impairs hematopoiesis in mice

AbstractKDM6B is an epigenetic regulator that mediates transcriptional activation during differentiation, including in bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs). Overexpression of KDM6B has been reported in BM HSPCs of patients with myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). Whether the overexpression of KDM6B contributes to the pathogenesis of these diseases remains to be elucidated. To study this, we generated a Vav-KDM6B mouse model, which overexpresses KDM6B in the hematopoietic compartment. KDM6B overexpression alone led to mild hematopoietic phenotype, and chronic innate immune stimulation of Vav-KDM6B mice with the Toll-like receptor (TLR) ligand lipopolysaccharide (LPS) resulted in significant hematopoietic defects. These defects recapitulated features of MDS and CMML, including leukopenia, dysplasia, and compromised repopulating function of BM HSPCs. Transcriptome studies indicated that KDM6B overexpression alone could lead to activation of disease-relevant genes such as S100a9 in BM HSPCs, and when combined with innate immune stimulation, KDM6B overexpression resulted in more profound overexpression of innate immune and disease-relevant genes, indicating that KDM6B was involved in the activation of innate immune signaling in BM HSPCs. Finally, pharmacologic inhibition of KDM6B with the small molecule inhibitor GSK-J4 ameliorated the ineffective hematopoiesis observed in Vav-KDM6B mice. This effect was also observed when GSK-J4 was applied to the primary BM HSPCs of patients with MDS by improving their repopulating function. These results indicate that overexpression of KDM6B mediates activation of innate immune signals and has a role in MDS and CMML pathogenesis, and that KDM6B targeting has therapeutic potential in these myeloid disorders.

TSPYL2 Regulates the Expression of EZH2 Target Genes in Neurons

Testis-specific protein, Y-encoded-like 2 (TSPYL2) is an X-linked gene in the locus for several neurodevelopmental disorders. We have previously shown that Tspyl2 knockout mice had impaired learning and sensorimotor gating, and TSPYL2 facilitates the expression of Grin2a and Grin2b through interaction with CREB-binding protein. To identify other genes regulated by TSPYL2, here, we showed that Tspyl2 knockout mice had an increased level of H3K27 trimethylation (H3K27me3) in the hippocampus, and TSPYL2 interacted with the H3K27 methyltransferase enhancer of zeste 2 (EZH2). We performed chromatin immunoprecipitation (ChIP)-sequencing in primary hippocampal neurons and divided all Refseq genes by k-mean clustering into four clusters from highest level of H3K27me3 to unmarked. We confirmed that mutant neurons had an increased level of H3K27me3 in cluster 1 genes, which consist of known EZH2 target genes important in development. We detected significantly reduced expression of genes including Gbx2 and Prss16 from cluster 1 and Acvrl1, Bdnf, Egr3, Grin2c, and Igf1 from cluster 2 in the mutant. In support of a dynamic role of EZH2 in repressing marked synaptic genes, the specific EZH2 inhibitor GSK126 significantly upregulated, while the demethylase inhibitor GSKJ4 downregulated the expression of Egr3 and Grin2c. GSK126 also upregulated the expression of Bdnf in mutant primary neurons. Finally, ChIP showed that hemagglutinin-tagged TSPYL2 co-existed with EZH2 in target promoters in neuroblastoma cells. Taken together, our data suggest that TSPYL2 is recruited to promoters of specific EZH2 target genes in neurons, and enhances their expression for proper neuronal maturation and function.

Critical role of histone demethylase Jumonji domain-containing protein 3 in the regulation of neointima formation following vascular injury.

Jumonji domain-containing protein 3 (JMJD3), also called lysine specific demethylase 6B (KDM6b), is an inducible histone demethylase which plays an important role in many biological processes, however, its function in vascular remodelling remains unknown. We aim to demonstrate that JMJD3 mediates vascular neointimal hyperplasia following carotid injury, and proliferation and migration in platelet-derived growth factor BB (PDGF-BB)-induced vascular smooth muscle cells (VSMCs). By using both genetic and pharmacological approaches, our study provides the first evidence that JMJD3 controls PDGF-BB-induced VSMCs proliferation and migration. Furthermore, our in vivo mouse and rat intimal thickening models demonstrate that JMJD3 is a novel mediator of neointima formation based on its mediatory effects on VSMCs proliferation, migration, and phenotypic switching. We further show that JMJD3 ablation by small interfering RNA or inhibitor GSK J4 can suppress the expression of NADPH oxidase 4 (Nox4), which is correlated with H3K27me3 enrichment around the gene promoters. Besides, deficiency of JMJD3 and Nox4 prohibits autophagic activation, and subsequently attenuates neointima and vascular remodelling following carotid injury. Above all, the increased expression of JMJD3 and Nox4 is further confirmed in human atherosclerotic arteries plaque specimens. JMJD3 is a novel factor involved in vascular remodelling. Deficiency of JMJD3 reduces neointima formation after vascular injury by a mechanism that inhibits Nox4-autophagy signalling activation, and suggesting JMJD3 may serve as a perspective target for the prevention and treatment of vascular diseases.

Abstract 954: Predicting synergistic drug combinations and resistance mechanisms from genomic features and single-agent response profiles

Abstract Drug combinations promise to improve clinical responses and/or forestall drug resistance. To capitalize on this promise, we need to know which drugs to combine, and which patients to give them to based on the genetic or pathological features of their disease. However, progress towards this goal has been hindered by the infeasibility of performing comprehensive drug-combination studies across thousands of cellular contexts. We hypothesized that the basal gene-transcription state of cancer cell lines, in concert with the cell-viability profiles of single-agent small molecules, might be leveraged to nominate specific synergistic drug combinations and identify mechanisms of drug resistance, eliminating the need to test all possible drug/drug combinations across cellular models. Specifically, we predicted that inhibiting the protein product of transcripts associated with drug resistance to a given small molecule might induce drug synergy. To test this notion, we analyzed nearly 400,000 drug-sensitivity profiles in >800 cancer cell lines to identify candidate compound-gene pairs. We identified over 100 examples where outlier expression of a single transcript was correlated with resistance to a small molecule. Of these gene/drug pairs, 9 genes represented imminently druggable targets, including established clinically-relevant relationships between the alkylating agent temozolomide and MGMT expression, and between a subset of chemotherapeutics including paclitaxel and the efflux pump ABCB1. Inhibition of candidate “co-targets”, which included 3 previously characterized relationships and 6 novel relationships, resulted in cell-line-specific synergistic cell killing across multiple cell-line models. For validated compound-gene pairs, exogenous expression of the “co-target” was sufficient to confer resistance. For example, we found that high expression of MGLL, encoding monoglyceride lipase, was uniquely associated with lack of response to the histone lysine demethylase inhibitor GSK-J4. Endogenous or exogenous MGLL expression conferred resistance to GSK-J4, while MGLL-proficient cell lines could be sensitized to GSK-J4 up to 50-fold by co-treatment with an irreversible MGLL inhibitor. These initial studies highlight the potential of integrating basal gene expression features with small-molecule response to nominate rational candidates for drug combinations. As public repositories of single agent response data from diverse cellular contexts continue to expand, so too will our repertoire of therapeutic combinations. Moreover, this approach permits the parallel identification of genomic features that indicate which patient populations are most likely to benefit from such combinations. Citation Format: Matthew G. Rees, Lisa Brenan, Patrick Duggan, Cory M. Johannessen. Predicting synergistic drug combinations and resistance mechanisms from genomic features and single-agent response profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 954.

Cystathionine-γ-lyase ameliorates the histone demethylase JMJD3-mediated autoimmune response in rheumatoid arthritis.

Cystathionine-γ-lyase (CSE), an enzyme associated with hydrogen sulfide (H2S) production, is an important endogenous regulator of inflammation. Jumonji domain-containing protein 3 (JMJD3) is implicated in the immune response and inflammation. Here, we investigated the potential contribution of JMJD3 to endogenous CSE-mediated inflammation in rheumatoid arthritis (RA). Upregulated CSE and JMJD3 were identified in synovial fibroblasts (SFs) from RA patients as well as in the joints of arthritic mice. Knocking down CSE augmented inflammation in IL-1β-induced SFs by increasing JMJD3 expression. In addition, CSE-/- mice with collagen-induced arthritis (CIA) developed severe joint inflammation and bone erosion. Conversely, overexpressing CSE inhibited JMJD3 expression by the transcription factor Sp-1 and was accompanied by reduced inflammation in IL-1β-treated SFs. Furthermore, JMJD3 silencing or the administration of the JMJD3 inhibitor GSK-J4 significantly decreased the inflammatory response in IL-1β-treated SFs, mainly by controlling the methylation status of H3K27me3 at the promoter of its target genes. GSK-J4 markedly attenuated the severity of arthritis in CIA mice. In conclusion, suppressing JMJD3 expression by the transcription factor Sp-1 is likely responsible for the ability of CSE to negatively modulate the inflammatory response and reduce the progression of RA.

JMJD3 inhibition protects against isoproterenol-induced cardiac hypertrophy by suppressing β-MHC expression.

Jumonji domain-containing protein D3 (JMJD3), a histone 3 lysine 27 (H3K27) demethylase, has been extensively studied for their participation in development, cellular physiology and a variety of diseases. However, its potential roles in cardiovascular system remain unknown. In this study, we found that JMJD3 played a pivotal role in the process of cardiac hypertrophy. JMJD3 expression was elevated by isoproterenol (ISO) stimuli both in vitro and in vivo. Overexpression of wild-type JMJD3, but not the demethylase-defective mutant, promoted cardiomyocyte hypertrophy, as implied by increased cardiomyocyte surface area and the expression of hypertrophy marker genes. In contrary, JMJD3 silencing or its inhibitor GSK-J4 suppressed ISO-induced cardiac hypertrophy. Mechanistically, JMJD3 was recruited to demethylate H3K27me3 at the promoter of β-MHC to promote its expression and cardiac hypertrophy. Thus, our results reveal that JMJD3 may be a key epigenetic regulator of β-MHC expression in cardiomyocytes and a potential therapeutic target for cardiac hypertrophy.

Inhibition of the Histone H3K27 Demethylase UTX Enhances Tumor Cell Radiosensitivity.

The processes mediating the repair of DNA double-strand breaks (DSB) are critical determinants of radiosensitivity and provide a source of potential targets for tumor radiosensitization. Among the events required for efficient DSB repair are a variety of post-translational histone modifications, including methylation. Because trimethylation of histone H3 on lysine 27 (H3K27me3) has been associated with chromatin condensation, which can influence DSB repair, we determined the effects of radiation on H3K27me3 levels in tumor and normal cell lines. Irradiation of tumor cells resulted in a rapid loss of H3K27me3, which was prevented by the siRNA-mediated knockdown of the H3K27 demethylase UTX. Knockdown of UTX also enhanced the radiosensitivity of each tumor cell line. Treatment of tumor cells with the H3K27 demethylase inhibitor GSKJ4 immediately before irradiation prevented the radiation-induced decrease in H3K27me3 and enhanced radiosensitivity. As determined by neutral comet analysis and γH2AX expression, this GSKJ4 treatment protocol inhibited the repair of radiation-induced DSBs. Consistent with in vitro results, treatment of mice bearing leg tumor xenografts with GSKJ4 significantly enhance radiation-induce tumor growth delay. In contrast with results generated from tumor cell lines, radiation had no effect on H3K27me3 levels in normal fibroblast cell lines and GSKJ4 did not enhance their radiosensitivity. These data suggest that H3K27me3 demethylation contributes to DSB repair in tumor cells and that UTX, the demethylase responsible, provides a target for selective tumor cell radiosensitization. Mol Cancer Ther; 17(5); 1070-8. ©2018 AACR.

AURKA Suppresses Leukemic THP-1 Cell Differentiation through Inhibition of the KDM6B Pathway.

Aberrations in histone modifications are being studied in mixed-lineage leukemia (MLL)-AF9-driven acute myeloid leukemia (AML). In this study, we focused on the regulation of the differentiation of the MLL-AF9 type AML cell line THP-1. We observed that, upon phorbol 12-myristate 13-acetate (PMA) treatment, THP-1 cells differentiated into monocytes by down-regulating Aurora kinase A (AURKA), resulting in a reduction in H3S10 phosphorylation. We revealed that the AURKA inhibitor alisertib accelerates the expression of the H3K27 demethylase KDM6B, thereby dissociating AURKA and YY1 from the KDM6B promoter region. Using Flow cytometry, we found that alisertib induces THP-1 differentiation into monocytes. Furthermore, we found that treatment with the KDM6B inhibitor GSK-J4 perturbed the PMA-mediated differentiation of THP-1 cells. Thus, we discovered the mechanism of AURKA–KDM6B signaling that controls the differentiation of THP-1 cells, which has implications for biotherapy for leukemia.

Inhibition of histone H3K27 demethylases selectively modulates inflammatory phenotypes of natural killer cells

Natural killer (NK) cells are innate lymphocytes, important in immune surveillance and elimination of stressed, transformed, or virus-infected cells. They critically shape the inflammatory cytokine environment to orchestrate interactions of cells of the innate and adaptive immune systems. Some studies have reported that NK cell activation and cytokine secretion are controlled epigenetically but have yielded only limited insight into the mechanisms. Using chemical screening with small-molecule inhibitors of chromatin methylation and acetylation, further validated by knockdown approaches, we here identified Jumonji-type histone H3K27 demethylases as key regulators of cytokine production in human NK cell subsets. The prototypic JMJD3/UTX (Jumonji domain–containing protein 3) H3K27 demethylase inhibitor GSK-J4 increased global levels of the repressive H3K27me3 mark around transcription start sites of effector cytokine genes. Moreover, GSK-J4 reduced IFN-γ, TNFα, granulocyte–macrophage colony-stimulating factor (GM-CSF), and interleukin-10 levels in cytokine-stimulated NK cells while sparing their cytotoxic killing activity against cancer cells. The anti-inflammatory effect of GSK-J4 in NK cell subsets, isolated from peripheral blood or tissue from individuals with rheumatoid arthritis (RA), coupled with an inhibitory effect on formation of bone-resorbing osteoclasts, suggested that histone demethylase inhibition has broad utility for modulating immune and inflammatory responses. Overall, our results indicate that H3K27me3 is a dynamic and important epigenetic modification during NK cell activation and that JMJD3/UTX-driven H3K27 demethylation is critical for NK cell function.

Low HOX gene expression in PML-RARα-positive leukemia results from suppressed histone demethylation

ABSTRACTHomeobox (HOX) genes are frequently dysregulated in leukemia. Previous studies have shown that aberrant HOX gene expression accompanies leukemogenesis and affects disease progression and leukemia patient survival. Patients with acute myeloid leukemia (AML) bearing PML-RARα fusion gene have distinct HOX gene signature in comparison to other subtypes of AML patients, although the mechanism of transcription regulation is not completely understood. We previously found an association between the mRNA levels of HOX genes and those of the histone demethylases JMJD3 and UTX in PML-RARα- positive leukemia patients. Here, we demonstrate that the release of the PML-RARα-mediated block in PML-RARα-positive myeloid leukemia cells increased both JMJD3 and HOX gene expression, while inhibition of JMJD3 using the specific inhibitor GSK-J4 reversed the effect. This effect was driven specifically through PML-RARα fusion protein since expression changes did not occur in cells with mutated RARα and was independent of differentiation. We confirmed that gene expression levels were inversely correlated with alterations in H3K27me3 histone marks localized at HOX gene promoters. Furthermore, data from chromatin immunoprecipitation followed by sequencing broaden a list of clustered HOX genes regulated by JMJD3 in PML-RARα-positive leukemic cells. Interestingly, the combination of GSK-J4 and all-trans retinoic acid (ATRA) significantly increased PML-RARα-positive cell apoptosis compared with ATRA treatment alone. This effect was also observed in ATRA-resistant NB4 clones, which may provide a new therapeutic opportunity for patients with acute promyelocytic leukemia (APL) resistant to current treatment. The results of our study reveal the mechanism of HOX gene expression regulation and contribute to our understanding of APL pathogenesis.

Activated Braf induces esophageal dilation and gastric epithelial hyperplasia in mice.

Germline mutations in BRAF are a major cause of cardio-facio-cutaneous (CFC) syndrome, which is characterized by heart defects, characteristic craniofacial dysmorphology and dermatologic abnormalities. Patients with CFC syndrome also commonly show gastrointestinal dysfunction, including feeding and swallowing difficulties and gastroesophageal reflux. We have previously found that knock-in mice expressing a Braf Q241R mutation exhibit CFC syndrome-related phenotypes, such as growth retardation, craniofacial dysmorphisms, congenital heart defects and learning deficits. However, it remains unclear whether BrafQ241R/+ mice exhibit gastrointestinal dysfunction. Here, we report that BrafQ241R/+ mice have neonatal feeding difficulties and esophageal dilation. The esophagus tissues from BrafQ241R/+ mice displayed incomplete replacement of smooth muscle with skeletal muscle and decreased contraction. Furthermore, the BrafQ241R/+ mice showed hyperkeratosis and a thickened muscle layer in the forestomach. Treatment with MEK inhibitors ameliorated the growth retardation, esophageal dilation, hyperkeratosis and thickened muscle layer in the forestomach in BrafQ241R/+ mice. The esophageal dilation with aberrant skeletal-smooth muscle boundary in BrafQ241R/+ mice were recovered after treatment with the histone H3K27 demethylase inhibitor GSK-J4. Our results provide clues to elucidate the pathogenesis and possible treatment of gastrointestinal dysfunction and failure to thrive in patients with CFC syndrome.

The pharmacological role of histone demethylase JMJD3 inhibitor GSK-J4 on glioma cells.

Glioma is regarded as the most prevalent malignant carcinoma of the central nervous system, and lack of effective treatment. Thus, the development of new therapeutic strategies targeting glioma is of significant clinical importance. In the present study, histone H3K27 demethylase jumonji domain-containing protein 3 (JMJD3) was investigated as target for glioma treatment. The mRNA of JMJD3 was overexpressed in glioblastoma tissues compared to normal brain tissues (P<0.05). The content of JMJD3 was also higher in glioma cells than in human brain microvascular endothelial cell (hCMEC), and the corresponding level of H3K27me3 was decreased (P<0.05). The treatment with JMJD3 specific inhibitor GSK-J4 can increase the content of H3K27me3 in glioma cells, which means the activity of JMJD3 was inhibited. GSK-J4 can inhibit glioma cell proliferation in a concentration dependent and time-dependent manner (P<0.05). GSK-J4 also induced glioma cell apoptosis and inhibited cell migration (P<0.05). But there was no obvious effect of GSK-J4 on hCMEC cells. All together, these data suggest that GSK-J4 has important potential in the gliomas treatment.

RNA sequencing reveals resistance of TLR4 ligand-activated microglial cells to inflammation mediated by the selective jumonji H3K27 demethylase inhibitor

Persistent microglial activation is associated with the production and secretion of various pro-inflammatory genes, cytokines and chemokines, which may initiate or amplify neurodegenerative diseases. A novel synthetic histone 3 lysine 27 (H3K27) demethylase JMJD3 inhibitor, GSK-J4, was proven to exert immunosuppressive activities in macrophages. However, a genome-wide search for GSK-J4 molecular targets has not been undertaken in microglia. To study the immuno-modulatory effects of GSK-J4 at the transcriptomic level, triplicate RNA sequencing and quantitative real-time PCR analyses were performed with resting, GSK-J4-, LPS- and LPS + GSK-J4-challenged primary microglial (PM) and BV-2 microglial cells. Among the annotated genes, the transcriptional sequencing of microglia that were treated with GSK-J4 revealed a selective effect on LPS-induced gene expression, in which the induction of cytokines/chemokines, interferon-stimulated genes, and prominent transcription factors TFs, as well as previously unidentified genes that are important in inflammation was suppressed. Furthermore, we showed that GSK-J4 controls are important inflammatory gene targets by modulating STAT1, IRF7, and H3K27me3 levels at their promoter sites. These unprecedented results demonstrate that the histone demethylase inhibitor GSK-J4 could have therapeutic applications for neuroinflammatory diseases.

The H3K27me3-demethylase KDM6A is suppressed in breast cancer stem-like cells, and enables the resolution of bivalency during the mesenchymal-epithelial transition.

The deposition of the activating H3K4me3 and repressive H3K27me3 histone modifications within the same promoter, forming a so-called bivalent domain, maintains gene expression in a repressed but transcription-ready state. We recently reported a significantly increased incidence of bivalency following an epithelial-mesenchymal transition (EMT), a process associated with the initiation of the metastatic cascade. The reverse process, known as the mesenchymal-epithelial transition (MET), is necessary for efficient colonization. Here, we identify numerous genes associated with differentiation, proliferation and intercellular adhesion that are repressed through the acquisition of bivalency during EMT, and re-expressed following MET. The majority of EMT-associated bivalent domains arise through H3K27me3 deposition at H3K4me3-marked promoters. Accordingly, we show that the expression of the H3K27me3-demethylase KDM6A is reduced in cells that have undergone EMT, stem-like subpopulations of mammary cell lines and stem cell-enriched triple-negative breast cancers. Importantly, KDM6A levels are restored following MET, concomitant with CDH1/E-cadherin reactivation through H3K27me3 removal. Moreover, inhibition of KDM6A, using the H3K27me3-demethylase inhibitor GSK-J4, prevents the re-expression of bivalent genes during MET. Our findings implicate KDM6A in the resolution of bivalency accompanying MET, and suggest KDM6A inhibition as a viable strategy to suppress metastasis formation in breast cancer.

Inhibitor of H3K27 demethylase JMJD3/UTX GSK-J4 is a potential therapeutic option for castration resistant prostate cancer.

Androgen receptor (AR) mediates initiation and progression of prostate cancer (PCa); AR-driven transcription is activated by binding of androgens to the ligand-binding domain (LBD) of AR. Androgen ablation therapy offers only a temporary relief of locally advanced and metastatic PCa, and the disease eventually recurs as a lethal castration-resistant PCa (CRPC) as there is no effective treatment for CRPC patients. Thus, it is critical to identify novel targeted and combinatorial regimens for clinical management of CRPC.Reduction of the repressive epigenetic modification H3K27me2/3 correlates with PCa aggressiveness, while corresponding demethylases JMJD3/UTX are overexpressed in PCa. We found that JMJD3/UTX inhibitor GSK-J4 reduced more efficiently proliferation of AR-ΔLBD cells (CRPC model) compared with isogenic AR-WT cells. Inhibition of JMJD3/UTX protects demethylation of H3K27Me2/3, thus reducing levels of H3k27Me1. We observed that the reduction dynamics of H3K27Me1 was faster and achieved at lower inhibitor concentrations in AR-ΔLBD cells, suggesting that inhibition of JMJD3/UTX diminished proliferation of these cells by hindering AR-driven transcription. In addition, we observed synergy between GSK-J4 and Cabazitaxel, a taxane derivative that is approved for CRPC treatment. Collectively, our results point at the H3K27 demethylation pathway as a new potential therapeutic target in CRPC patients.

Lysine demethylase inhibition protects pancreatic β cells from apoptosis and improves β-cell function

Transcriptional changes control β-cell survival in response to inflammatory stress. Posttranslational modifications of histone and non-histone transcriptional regulators activate or repress gene transcription, but the link to cell-fate signaling is unclear. Inhibition of lysine deacetylases (KDACs) protects β cells from cytokine-induced apoptosis and reduces type 1 diabetes incidence in animals. We hypothesized that also lysine demethylases (KDMs) regulate β-cell fate in response to inflammatory stress.Expression of the demethylase Kdm6B was upregulated by proinflammatory cytokines suggesting a possible role in inflammation-induced β-cell destruction. Inhibition of KDM6 demethylases using the selective inhibitor GSK-J4 protected insulin-producing cells and human and mouse islets from cytokine-induced apoptosis by blunting nuclear factor (NF)-κB signaling and endoplasmic reticulum (ER) stress response gene expression. GSK-J4 furthermore increased expression of insulin gene and glucose-stimulated insulin secretion. Expression of genes regulating purinergic and cytokine ligand-receptor interactions was downregulated following GSK-J4 exposure, while expression of genes involved in cell maintenance and survival was upregulated. These data suggest that KDMs are important regulators of inflammation-induced β-cell dysfunction and death.

HDAC8 Prevents Anthrax Lethal Toxin-induced Cell Cycle Arrest through Silencing PTEN in Human Monocytic THP-1 Cells.

Anthrax lethal toxin (LeTx) is a cytotoxic virulence factor that causes cell cycle arrest and cell death in various cell types. However, susceptibility to the cytotoxic effects varies depending on cell types. In proliferating monocytes, LeTx has only transient cytotoxic effects due to activation of the phosphoinositide 3-kinase (PI3K)-AKT-mediated adaptive responses. To date, the mechanism of LeTx in activating PI3K-AKT signaling axis is unknown. This study shows that the histone deacetylase 8 (HDAC8) is involved in activating PI3K-AKT signaling axis through down-regulating the phosphatase and tensin homolog 1 (PTEN) in human monocytic THP-1 cells. The HDAC8-specific activator TM-2-51 and inhibitor PCI-34051 enhanced and prevented, respectively, AKT activation and cell cycle progression in LeTx-treated cells. Furthermore, HDAC8 induced tri-methylation of histone H3 lysine 27 (H3K27me3), which is known to suppress PTEN expression, through at least in part down-regulating the H3K27me3 eraser Jumonji Domain Containing (JMJD) 3. Importantly, the JMJD3-specific inhibitor GSK-J4 induced AKT activation and protected cell cycle arrest in LeTx-treated cells, regardless the presence of HDAC8 activity. Collectively, this study for the first time demonstrated that HDAC8 activity determines susceptibility to cell cycle arrest induced by LeTx, through regulating the PI3K-PTEN-AKT signaling axis.

Inhibition of H3K27me3 Histone Demethylase Activity Prevents the Proliferative Regeneration of Zebrafish Lateral Line Neuromasts.

The H3K27 demethylases are involved in a variety of biological processes, including cell differentiation, proliferation, and cell death by regulating transcriptional activity. However, the function of H3K27 demethylation in the field of hearing research is poorly understood. Here, we investigated the role of H3K27me3 histone demethylase activity in hair cell regeneration using an in vivo animal model. Our data showed that pharmacologic inhibition of H3K27 demethylase activity with the specific small-molecule inhibitor GSK-J4 decreased the number of regenerated hair cells in response to neomycin damage. Furthermore, inhibition of H3K27me3 histone demethylase activity dramatically suppressed cell proliferation and activated caspase-3 levels in the regenerating neuromasts of the zebrafish lateral line. GSK-J4 administration also increased the expression of p21 and p27 in neuromast cells and inhibited the ERK signaling pathway. Collectively, our findings indicate that H3K27me3 demethylation is a key epigenetic regulator in the process of hair cell regeneration in zebrafish and suggest that H3K27me3 histone demethylase activity might be a novel therapeutic target for the treatment of hearing loss.