EZH1/2 dual inhibitors suppress HTLV-1-infected cell proliferation and hyperimmune response in HTLV-1-associated myelopathy.

Background: Human T-cell leukemia virus type 1 (HTLV-1) causes HTLV-1-associated myelopathy (HAM), adult T-cell leukemia/lymphoma (ATL), HTLV-1-associated uveitis, and pulmonary diseases. Although both HAM and ATL show proliferation of infected cells, their pathogeneses are quite different. In particular, the pathogenesis of HAM is characterized by hyperimmune responses to HTLV-1-infected cells. Recently, we demonstrated the overexpression of histone methyltransferase EZH2 in ATL cells and the cytotoxic effects of EZH2 inhibitors and EZH1/2 dual inhibitors on these cells. However, these phenomena have never been studied in HAM. Furthermore, what effect these agents have on the hyperimmune response seen in HAM is completely unknown. Methods: In this study, we investigated histone methyltransferase expression levels in infected cell populations (CD4+ and CD4+CCR4+ cells) from patients with HAM using microarray and RT-qPCR analyses. Next, using an assay system that utilizes the spontaneous proliferation characteristic of peripheral blood mononuclear cells derived from patients with HAM (HAM-PBMCs), we investigated the effects of EZH2 selective inhibitors (GSK126 and tazemetostat) and EZH1/2 dual inhibitors (OR-S1 and valemetostat, also known as DS-3201), particularly on cell proliferation rate, cytokine production, and HTLV-1 proviral load. We also examined the effect of EZH1/2 inhibitors on the proliferation of HTLV-1-infected cell lines (HCT-4 and HCT-5) derived from patients with HAM. Results: We found elevated expression of EZH2 in CD4+ and CD4+CCR4+ cells from patients with HAM. EZH2 selective inhibitors and EZH1/2 inhibitors significantly inhibited spontaneous proliferation of HAM-PBMC in a concentration-dependent manner. The effect was greater with EZH1/2 inhibitors. EZH1/2 inhibitors also reduced the frequencies of Ki67+ CD4+ T cells and Ki67+ CD8+ T cells. Furthermore, they reduced HTLV-1 proviral loads and increased IL-10 levels in culture supernatants but did not alter IFN-γ and TNF-α levels. These agents also caused a concentration-dependent inhibition of the proliferation of HTLV-1-infected cell lines derived from patients with HAM and increased annexin-V(+)7-aminoactinomycin D(-) early apoptotic cells. Conclusion: This study showed that EZH1/2 inhibitors suppress HTLV-1-infected cell proliferation through apoptosis and the hyperimmune response in HAM. This indicates that EZH1/2 inhibitors may be effective in treating HAM.

CDKN1C-mediated growth inhibition by an EZH1/2 dual inhibitor overcomes resistance of mantle cell lymphoma to ibrutinib.

Mantle cell lymphoma (MCL) is a rare subtype of non-Hodgkin's lymphoma, which is characterized by overexpression of cyclin D1. Although novel drugs, such as ibrutinib, show promising clinical outcomes, relapsed MCL often acquires drug resistance. Therefore, alternative approaches for refractory and relapsed MCL are needed. Here, we examined whether a novel inhibitor of enhancer of zeste homologs 1 and 2 (EZH1/2), OR-S1 (a close analog of the clinical-stage compound valemetostat), had an antitumor effect on MCL cells. In an ibrutinib-resistant MCL patient-derived xenograft (PDX) mouse model, OR-S1 treatment by oral administration significantly inhibited MCL tumor growth, whereas ibrutinib did not. In vitro growth assays showed that compared with an established EZH2-specific inhibitor GSK126, OR-S1 had a marked antitumor effect on MCL cell lines. Furthermore, comprehensive gene expression analysis was performed using OR-S1-sensitive or insensitive MCL cell lines and showed that OR-S1 treatment modulated B-cell activation, differentiation, and cell cycle. In addition, we identified Cyclin Dependent Kinase Inhibitor 1C (CDKN1C, also known as p57, KIP2), which contributes to cell cycle arrest, as a direct target of EZH1/2 and showed that its expression influenced MCL cell proliferation. These results suggest that EZH1/2 may be a potential novel target for the treatment of aggressive ibrutinib-resistant MCL via CDKN1C-mediated cell cycle arrest.

The Association between time spent in performing physical activity and physical function in outpatients with type 2 diabetes who may have diabetic neuropathy.

AIMS: To determine the relationship between physical activity time and physical function according to the Japanese Clinical Practice Guideline for Diabetes, which recommended 150 min of activity for outpatients with type 2 diabetes who may have diabetic neuropathy. METHODS: We examined a cross-sectional study with 79 outpatients with Type 2 diabetes participated. A short version, Japanese language edition of the International Physical Activity Questionnaire (IPAQ) was used to evaluate physical activity. Isometric knee extensor strength, grip strength, maximum 10-m walking speed, the Michigan Neuropathy Screening Instrument score, and the Short Physical Performance Battery (SPPB). Each evaluation item was compared between time spent performing physical activity ≥150 min group and <150 min group, and multiple regression analysis was performed to determine the factors associated with time spent performing physical activity. Further, the correlation between time spent performing physical activity and isometric knee extensor strength was examined. RESULTS: The ≥150 min group had significantly higher isometric knee extensor strength than the <150 min group. In addition, the ≥150 min group had significantly faster maximum 10-m walking speed and sit-to-stand time than the <150 min group. Isometric knee extensor strength was determined to be an independent factor associated with the IPAQ score. A positive correlation was found between the IPAQ score and isometric knee extensor strength. CONCLUSIONS: Among the patients with type 2 diabetes who may have diabetic neuropathy, those who performed physical activity for ≥150 min per week were suggested to have higher physical function than those with <150 min of activity.

Targeting Excessive EZH1 and EZH2 Activities for Abnormal Histone Methylation and Transcription Network in Malignant Lymphomas.

Although global H3K27me3 reprogramming is a hallmark of cancer, no effective therapeutic strategy for H3K27me3-high malignancies harboring EZH2WT/WT has yet been established. We explore epigenome and transcriptome in EZH2WT/WT and EZH2WT/Mu aggressive lymphomas and show that mutual interference and compensatory function of co-expressed EZH1 and EZH2 rearrange their own genome-wide distribution, thereby establishing restricted chromatin and gene expression signatures. Direct comparison of leading compounds introduces potency and a mechanism of action of the EZH1/2 dual inhibitor (valemetostat). The synthetic lethality is observed in all lymphoma models and primary adult T cell leukemia-lymphoma (ATL) cells. Opposing actions of EZH1/2-polycomb and SWI/SNF complexes are required for facultative heterochromatin formation. Inactivation of chromatin-associated genes (ARID1A, SMARCA4/BRG1, SMARCB1/SNF5, KDM6A/UTX, BAP1, KMT2D/MLL2) and oncovirus infection (HTLV-1, EBV) trigger EZH1/2 perturbation and H3K27me3 deposition. Our study provides the mechanism-based rationale for chemical dual targeting of EZH1/2 in cancer epigenome.

Dual inhibition of enhancer of zeste homolog 1/2 overactivates WNT signaling to deplete cancer stem cells in multiple myeloma.

Multiple myeloma (MM) is an incurable hematological malignancy caused by accumulation of abnormal clonal plasma cells. Despite the recent development of novel therapies, relapse of MM eventually occurs as a result of a remaining population of drug-resistant myeloma stem cells. Side population (SP) cells show cancer stem cell-like characteristics in MM; thus, targeting these cells is a promising strategy to completely cure this malignancy. Herein, we showed that SP cells expressed higher levels of enhancer of zeste homolog (EZH) 1 and EZH2, which encode the catalytic subunits of Polycomb repressive complex 2 (PRC2), than non-SP cells, suggesting that EZH1 as well as EZH2 contributes to the stemness maintenance of the MM cells and that targeting both EZH1/2 is potentially a significant therapeutic approach for eradicating myeloma stem cells. A novel orally bioavailable EZH1/2 dual inhibitor, OR-S1, effectively eradicated SP cells and had a greater antitumor effect than a selective EZH2 inhibitor in vitro and in vivo, including a unique patient-derived xenograft model. Moreover, long-term continuous dosing of OR-S1 completely cured mice bearing orthotopic xenografts. Additionally, PRC2 directly regulated WNT signaling in MM, and overactivation of this signaling induced by dual inhibition of EZH1/2 eradicated myeloma stem cells and negatively affected tumorigenesis, suggesting that repression of WNT signaling by PRC2 plays an important role in stemness maintenance of MM cells. Our results show the role of EZH1/2 in the maintenance of myeloma stem cells and provide a preclinical rationale for therapeutic application of OR-S1, leading to significant advances in the treatment of MM.

Novel orally bioavailable EZH1/2 dual inhibitors with greater antitumor efficacy than an EZH2 selective inhibitor.

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 and represses gene expression to regulate cell proliferation and differentiation. Enhancer of zeste homolog 2 (EZH2) or its close homolog EZH1 functions as a catalytic subunit of PRC2, so there are two PRC2 complexes containing either EZH2 or EZH1. Tumorigenic functions of EZH2 and its synthetic lethality with some subunits of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes have been observed. However, little is known about the function of EZH1 in tumorigenesis. Herein, we developed novel, orally bioavailable EZH1/2 dual inhibitors that strongly and selectively inhibited methyltransferase activity of both EZH2 and EZH1. EZH1/2 dual inhibitors suppressed trimethylation of histone H3 lysine 27 in cells more than EZH2 selective inhibitors. They also showed greater antitumor efficacy than EZH2 selective inhibitor in vitro and in vivo against diffuse large B-cell lymphoma cells harboring gain-of-function mutation in EZH2. A hematological cancer panel assay indicated that EZH1/2 dual inhibitor has efficacy against some lymphomas, multiple myeloma, and leukemia with fusion genes such as MLL-AF9, MLL-AF4, and AML1-ETO. A solid cancer panel assay demonstrated that some cancer cell lines are sensitive to EZH1/2 dual inhibitor in vitro and in vivo. No clear correlation was detected between sensitivity to EZH1/2 dual inhibitor and SWI/SNF mutations, with a few exceptions. Severe toxicity was not seen in rats treated with EZH1/2 dual inhibitor for 14 days at drug levels higher than those used in the antitumor study. Our results indicate the possibility of EZH1/2 dual inhibitors for clinical applications.

Synthesis and biological evaluation of pyrethroid insecticide-derivatives as a chemical inducer for Bdnf mRNA expression in neurons.

Brain-derived neurotrophic factor (BDNF) plays a fundamental role in neuronal synaptic plasticity. A decrease of plasticity in the brain may be related to the pathogenesis of neurodegenerative or psychiatric disorders. Pyrethroid insecticides, which affect sodium channels in neurons, are widely used to control insect pests in agriculture and in the home. We previously found that deltamethrin (DM), a type II pyrethroid, increased Bdnf mRNA expression in cultured rat cortical neurons. However, the cyano group at the α-position of type II pyrethroids is likely susceptible to hydrolytic degradation and, its degraded product, hydrogen cyanide, could generate a cellular toxicity in the human body. To determine if the cyano group is required for the Bdnf exon IV-IX (Bdnf eIV-IX) mRNA expression induced by type II pyrethroids, for this study we synthesized a series of derivatives, in which the cyano group at the α-position was replaced with an ethynyl group. Then we added various substituents at the terminal position of the ethynyl group, and biologically evaluated the effects of these derivatives on Bdnf eIV-IX mRNA expression. These ethynyl derivatives induced the Bdnf eIV-IX mRNA expression in a concentration-dependent manner, at varying levels but lower levels than that evoked by DM. The mechanisms for the Bdnf induction and the morphological changes of neurons were the same whether the cyano or ethynyl group was included in the compounds.

Deltamethrin, a type II pyrethroid insecticide, has neurotrophic effects on neurons with continuous activation of the Bdnf promoter.

Pyrethroids, widely used insecticides with low acute toxicity in mammals, affect sodium channels in neurons. In a primary culture of rat cortical neurons, deltamethrin (DM), a type II pyrethroid, markedly enhanced the expression of brain-derived neurotrophic factor (BDNF) exon IV-IX (Bdnf eIV-IX) mRNA. In this study, we found that DM has a neurotrophic effect on cultured neurons and investigated the mechanisms responsible for it. One µM DM increased cell survival, neurite complexity and length. Neurite complexity and length were reduced not only by a blockade of cellular excitation with GABA or Ca(2+) influx via L-type voltage-dependent calcium channels with nicardipine, but also by a blockade of TrkB, a specific receptor for BDNF, with TrkB/Fc. These data indicate DM has neurotrophic actions. DM-induced Bdnf eIV-IX mRNA expression through the calcineurin and ERK/MAPK pathways, the increase of which was reduced by GABA(A) receptor activation. Using a promoter assay, we found that Ca(2+)-responsive elements including a CRE are involved in the DM-induced activation of the Bdnf promoter IV (Bdnf-pIV). The intracellular concentration of Ca(2+) and activation of Bdnf-pIV remained elevated for, at least, 1 and 24 h, respectively. Moreover, GABA(A) receptor activation or a blockade of Ca(2+) influx even after starting the incubation with DM reduced the elevated activity of Bdnf-pIV. These data demonstrated that the prolonged activation of Bdnf-pIV occurred because of this continuous increase in the intracellular Ca(2+) concentration. Thus, DM has neurotrophic effects on neurons, likely due to prolonged activation of Bdnf promoter in neurons. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

Preparation of enzymatically active human Myc-tagged-NCre recombinase exhibiting immunoreactivity with anti-Myc antibody.

The Cre-loxP system has been recognized as a tool for conditional gene targeting in mice. However, most anti-Cre antibodies fail to react with Cre expressed in vivo. In an attempt to directly detect Cre by antibodies in vivo, we constructed the tagged-NCre (NCreMH) gene by connecting the human Myc and His tag sequences to the 3' end of the NCre gene carrying a nuclear localizing signal (NLS) sequence. The production of NCre protein and the recombinase activity were detected after co-transfection with pCMV-NCreMH and pCETZ-17 carrying the loxP-flanked lacZ gene into NIH3T3 cells. This activity was also confirmed in vivo after gene transfer of pCMV-NCreMH and pCRTEIL-6 carrying loxP-flanked HcRed1 and EGFP cDNAs, into oviductal epithelium by electroporation. Immunohistochemical staining using anti-Myc antibody demonstrated that the area positive for enhanced green fluorescent protein (EGFP) fluorescence was immunostained with the antibody. These findings indicate that NCreMH is useful as an alternative to NCre for gene targeting.

Interaction of a small heat shock protein with light-harvesting cyanobacterial phycocyanins under stress conditions.

Phycobiliproteins such as phycocyanins are the most abundant proteins found in cyanobacteria which are assembled to form the phycobilisome. Here, we showed that a small heat shock protein, HspA, interacts directly with phycocyanins from the cyanobacterium Synechococcus sp. strain PCC 7942 in vitro and suppresses inactivation of their light-harvesting functions due to heat denaturation in the presence of hydrogen peroxide. Under the denaturing conditions, phycobilisomes were de-assembled to lighter complexes and then aggregated. HspA associated with phycocyanins in the dissociated complexes, and suppressed the aggregation. The specific interaction between a small heat shock protein and phycocyanins was further supported by the fact that HspA and alpha-crystallin protected isolated phycocyanins from denaturation, while HtpG and lysozyme did not. The maximum protection was observed at a molar ratio of four HspA monomer per one phycocyanin (alpha beta) monomer.

Light-driven hydrogen production by a hybrid complex of a [NiFe]-hydrogenase and the cyanobacterial photosystem I.

In order to generate renewable and clean fuels, increasing efforts are focused on the exploitation of photosynthetic microorganisms for the production of molecular hydrogen from water and light. In this study we engineered a 'hard-wired' protein complex consisting of a hydrogenase and photosystem I (hydrogenase-PSI complex) as a direct light-to-hydrogen conversion system. The key component was an artificial fusion protein composed of the membrane-bound [NiFe] hydrogenase from the beta-proteobacterium Ralstonia eutropha H16 and the peripheral PSI subunit PsaE of the cyanobacterium Thermosynechococcus elongatus. The resulting hydrogenase-PsaE fusion protein associated with PsaE-free PSI spontaneously, thereby forming a hydrogenase-PSI complex as confirmed by sucrose-gradient ultracentrifuge and immunoblot analysis. The hydrogenase-PSI complex displayed light-driven hydrogen production at a rate of 0.58 mumol H(2).mg chlorophyll(-1).h(-1). The complex maintained its accessibility to the native electron acceptor ferredoxin. This study provides the first example of a light-driven enzymatic reaction by an artificial complex between a redox enzyme and photosystem I and represents an important step on the way to design a photosynthetic organism that efficiently converts solar energy and water into hydrogen.

Ultrastructural stability under high temperature or intensive light stress conferred by a small heat shock protein in cyanobacteria.

The role and sub-cellular localization of the small heat shock protein HspA under stress conditions was investigated comparing the cyanobacterium Synechococcus strain ECT16-1, which constitutively expresses HspA, with the reference strain ECT. The ultrastructure of ECT cells under elevated temperature or intensive light stress exhibited severe damage including aggregation of cytosol and disordered thylakoid membranes, but in ECT16-1 cells these ultrastructural changes were much less conspicuous. Immunocytochemical studies showed that the main localization of HspA in the ECT16-1 cells shifted from the thylakoid area to the cytoplasm, then back to thylakoid area during the heat stress. Expression of HspA stabilized the morphology of nucleoids. The results are discussed, in particular with respect to the unique property of HspA to associate with thylakoid membranes.

A novel method for the production of transgenic cloned pigs: electroporation-mediated gene transfer to non-cultured cells and subsequent selection with puromycin.

Puromycin N-acetyl transferase gene (pac), of which the gene product catalyzes antibiotic puromycin (an effective inhibitor of protein synthesis), has been widely used as a dominant selection marker in embryonic stem (ES) cell-mediated transgenesis. The present study is the first to report on the usefulness of puromycin for production of enhanced green fluorescent protein (EGFP) transgenic piglets after somatic cell cloning and embryo transfer. Somatic cells isolated from porcine fetuses at 73 days of gestation were immediately electroporated with a transgene (pCAG-EGFPac) carrying both EGFP cDNA and pac. This procedure aims to avoid aging effects thought to be generated during cell culture. The recombinant cells were selected with puromycin at a low concentration (2 microg/ml), cultured for 7 days, and then screened for EGFP expression before somatic cell cloning. The manipulated embryos were transplanted into the oviducts of 14 foster mother sows. Four of the foster sows became pregnant and nine piglets were delivered. Of the nine piglets, eight died shortly after birth and one grew healthy after weaning. Results indicate that puromycin can be used for the selection of recombinant cells from noncultured cells, and moreover, may confer the production of genetically engineered newborns via nuclear transfer techniques in pigs.

Analysis of a full-length cDNA library constructed from swine olfactory bulb for elucidation of expressed genes and their transcription initiation sites.

The olfactory system is indispensable to the survival of animals in finding foods and for the reproductive process. Odorant signals are conveyed through olfactory sensory neurons to the olfactory bulb, which modifies the signals and relays them to the neocortex. In the present study, a "full-length" cDNA library was constructed from the main and accessory olfactory bulbs of 5-week-old male pigs, in order to elucidate the expressed genes. The average insert size of the library was estimated to be 1.7 kb based on 54 randomly-selected clones. One thousand randomly selected clones were subjected to sequencing, and the resulting 883 sequences were then clustered into 753 sequences based on similarity. Since 723 of the 753 sequences had sufficient sequence information for homology analysis, the 723 sequences were subjected to BLAST analysis against GenBank/EMBL/DDBJ; 655 out of the 723 sequences showed similarities with known genes, and the remaining 68 were indicated to be novel sequences. The full-length rate of the library was estimated to be ca. 80%, using 70 sequences corresponding to human full-length cDNAs. The full-length cDNA sequences of a single gene appearing more than 6 times in the analysis were aligned to determine major transcription initiation sites for SLC25A, CKB, TUBB4, TUBB, YWHAH, TUBB2, and CNP genes.

Cloning and characterization of porcine common gamma chain gene.

The common gamma chain, which was originally identified as a component of interleukin-2 receptors (IL-2R), plays a key role in differentiation of T lymphocytes and natural killer (NK) cells. In the present study, cDNA of the porcine common gamma chain gene and its genomic DNA were molecularly cloned and characterized. The porcine common gamma chain gene was found to consist of 8 exons, spanning approximately 3.7 kb, and to encode a 368-amino acid polypeptide. The amino acid sequence showed 82.4%, 71.1%, 86.1%, and 84.8% similarities with that of human, murine, bovine, and canine chains, respectively. The common gamma chain gene was assigned to swine chromosome Xq13 by FISH analysis and was consistent with the result of radiation hybrid (RH) mapping. When various porcine tissues were examined for the expression of this gene, the expression was observed in lymphocytes and lymphocyte-related tissues. Since GATA, T cell factor-1 (TCF-1), Ets-1, activated protein2 (AP-2), and Ikaros2 binding motifs were demonstrated in the 5' upstream region of this gene, promoter activity was investigated using luciferase gene as a reporter. The results indicate that the Ets-1 binding motif in the segment from -95 to -59 (major transcription initiation site: +1) was an essential cis-acting regulatory element for the common gamma chain gene in lymphoid cells.