DNA Methyltransferase Inhibitor 5-azacytidine Research Articles

Epigenetic modulation inhibits epithelial-mesenchymal transition-driven fibrogenesis and enhances characteristics of chemically-derived hepatic progenitors.

One of the novel cell sources of cell-based liver regenerative medicine is human chemically-derived hepatic progenitors (hCdHs). We previously established this cell by direct hepatocyte reprogramming with a combination of small molecules (hepatocyte growth factor, A83-01, CHIR99021). However, there have been several issues concerning the cell's stability and maintenance, namely the occurrences of epithelial-mesenchymal transition (EMT) that develop fibrotic phenotypes, resulting in the loss of hepatic progenitor characteristics. These hepatic progenitor attributes are thought to be regulated by SOX9, a transcription factor essential for hepatic progenitor cells and cholangiocytes. To suppress the fibrotic phenotype and improve our long-term hCdHs culture technology, we utilized the epigenetic modulating drugs DNA methyltransferase inhibitor (5-azacytidine) and histone deacetylase inhibitor (sodium butyrate) that have been reported to suppress and revert hepatic fibrosis. To confirm the essential role of SOX9 to our cell, we used clustered regularly interspaced short palindromic repeats-interference (CRISPRi) to repress the SOX9 expression. The treatment of only 5-azacytidine significantly reduces the fibrosis/mesenchymal marker and EMT-related transcription factor expression level in the early passages. Interestingly, this treatment also increased the hepatic progenitor markers expression, even during the reprogramming phase. Then, we confirmed the essential role of SOX9 by repressing the SOX9 expression with CRISPRi which resulted in the downregulation of several essential hepatic progenitor cell markers. These results highlight the capacity of 5-azacytidine to inhibit EMT-driven hepatic fibrosis and the significance of SOX9 on hepatic progenitor cell stemness properties.

Abstract 033: Innate Lymphoid Cells Mediate Hypertension In T Cell Deficient Hosts

T cells have been shown to contribute to hypertension, however, hypertension occurs in settings of T cell deficiency, such as human immunodeficiency virus infection and following chemotherapy. We studied two colonies of RAG-1 -/- mice with disparate responses to angiotensin II (Ang II) infusion. Ang II increased both systolic and diastolic pressure in RAG-1 -/- mice from Jackson labs (RAG-1 -/-US ) but had no effect in a colony of RAG-1 -/- mice housed at our Pozzilli facility (RAG-1 -/-POZ ) for the past 14 years (SBPs 169 ± 2 vs 114 ± 3 mmHg, p < 0.003). While both lacked T and B cells, the RAG-1 -/-US mice exhibited a striking expansion of natural killer and both type 1 and type 3 innate lymphoid cells, while the RAG-1 -/-POZ mice did not. In RAG-1 -/-US mice, NK cells and ILC1s produced IFNγ, while ILC3s produced IL-17A. Immunoclearing of IL-17A, but not IFNγ prevented the hypertension in RAG-1 -/-US mice. There were differences in global gene expression and DNA methylation status in NK1.1 + /NKp46 + cells between these two colonies and notably β2-AdR and IL-10 expression was significantly lower in RAG-1 -/-US mice. Knockdown of the β2-AdR using intra-splenic infusion of siRNA in RAG-1 -/-POZ mice led to a hypertensive response resembling that observed in wild type and RAG-1 -/-US mice. We also found striking differences in DNA methylation patterns in innate lymphoid cells of RAG-1 -/-US vs RAG-1 -/-POZ mice and treatment with the DNA methyltransferase inhibitor 5-azacytidine prevented hypertension in RAG-1 -/-US mice. Single cell sequencing showed that 5-azacytidine also reduced splenic populations of NK and ILC3 cells and reduced the presence of the transcription factor RORγt, which defines ILC3s. Ang II infusion caused only a transient elevation of blood pressure in NSG/MHCI/II -/- mice that lack T cells and innate lymphoid cells but led to sustained hypertension following adoptive transfer of either T cells or NK1.1 + /NKp46 + cells to these animals. We conclude that DNA methylation likely modulates expansion of NK and innate lymphoid cells. Either T cells or innate lymphoid cells can contribute to hypertension, and the latter can compensate by serving as a source of cytokines like IL-17A when T cells are deficient.

5-Azacytidine promotes HCC cell metastasis by up-regulating RDH16 expression

The level of DNA methylation could affect the expression of tumor promoting and tumor suppressor genes. DNA methyltransferase inhibitors could reduce high methylation levels in cancer and inhibit the progression of a variety of cancers, including HCC. However, the pro-metastatic effect of DNA methyltransferase inhibitors in some cancers suggest the potential risk of their use. Whether DNA methyltransferase inhibitors also promote metastasis in HCC remains unclear. Our study will explore the effect of DNA methyltransferase inhibitor 5-Azacytidine on HCC metastasis. Our study found that 5-Azacytidine inhibited the proliferation of HCC cells while promoting in vitro and in vivo metastasis of HCC. Mechanistically, our study showed that 5-Azacytidine increased the expression of RDH16 by decreasing the methylation of RDH16 gene promoter. RDH16 is a highly methylated gene and its expression is very low in hepatocellular carcinoma. 5-Azacytidine promoted the migration of hepatocellular carcinoma cells by increasing the expression of RDH16. Our results suggest that 5-Azacytidine up-regulates the expression of RDH16 by decreasing the methylation level of RDH16, and then promoting HCC metastasis. These findings suggest that 5-Azacytidine and even other DNA methyltransferase inhibitors may have the risk of promoting metastasis in HCC treatment. RDH16 could be used as a pro-metastasis biomarker in the treatment of HCC with DNA methyltransferase inhibitors.

DNA methyltransferase inhibitor 5-azacytidine enhances neuroblastoma cell lysis by an oncolytic parainfluenza virus.

Studies with neuroblastoma have shown that the presence of aberrant DNA epigenetic modifications mediated by DNA methyltransferases correlates with poor prognosis, making these enzymes a target for therapeutics based on synthetic epigenetic modulators such as DNA methyltransferase inhibitors (DNMTi). Here, we have used a neuroblastoma cell line model to test the hypothesis that treatment with a DNMTi would enhance cell killing when used in combination with oncolytic Parainfluenza virus 5 (P/V virus), a cytoplasmic-replicating RNA virus. Pretreatment of SK-N-AS cells with the DNMTi 5-azacytidine substantially enhanced P/V virus-mediated cell death in a dose- and multiplicity of infection-dependent manner. Infection with the virus alone and the combination treatment with 5-azacytidine and P/V virus infection led to the activation of caspases-8, -9, and -3/7. Inhibition of caspases using a pan-caspase inhibitor minimally affected cell killing by P/V virus alone, but by contrast, largely reduced cell death mediated by 5-azacytidine treatment alone or in combination with P/V virus infection. 5-Azacytidine pretreatment dampened P/V virus gene expression and growth within the SK-N-AS cell population, which correlated with enhanced expression of important antiviral genes such as interferon-β and OAS2 . Taken together, our data support the role of combination treatment using 5-azacytidine and an oncolytic P/V virus for neuroblastoma therapy.

Epigenetic Reprogramming via Synergistic Hypomethylation and Hypoxia Enhances the Therapeutic Efficacy of Mesenchymal Stem Cell Extracellular Vesicles for Bone Repair.

Mesenchymal stem cells (MSCs) are a promising cell population for regenerative medicine applications, where paracrine signalling through the extracellular vesicles (EVs) regulates bone tissue homeostasis and development. MSCs are known to reside in low oxygen tension, which promotes osteogenic differentiation via hypoxia-inducible factor-1α activation. Epigenetic reprogramming has emerged as a promising bioengineering strategy to enhance MSC differentiation. Particularly, the process of hypomethylation may enhance osteogenesis through gene activation. Therefore, this study aimed to investigate the synergistic effects of inducing hypomethylation and hypoxia on improving the therapeutic efficacy of EVs derived from human bone marrow MSCs (hBMSCs). The effects of the hypoxia mimetic agent deferoxamine (DFO) and the DNA methyltransferase inhibitor 5-azacytidine (AZT) on hBMSC viability was assessed by quantifying the DNA content. The epigenetic functionality was evaluated by assessing histone acetylation and histone methylation. hBMSC mineralisation was determined by quantifying alkaline phosphate activity, collagen production and calcium deposition. EVs were procured from AZT, DFO or AZT/DFO-treated hBMSCs over a two-week period, with EV size and concentration defined using transmission electron microscopy, nanoflow cytometry and dynamic light scattering. The effects of AZT-EVs, DFO-EVs or AZT/DFO-EVs on the epigenetic functionality and mineralisation of hBMSCs were evaluated. Moreover, the effects of hBMSC-EVs on human umbilical cord vein endothelial cells (HUVECs) angiogenesis was assessed by quantifying pro-angiogenic cytokine release. DFO and AZT caused a time-dose dependent reduction in hBMSC viability. Pre-treatment with AZT, DFO or AZT/DFO augmented the epigenetic functionality of the MSCs through increases in histone acetylation and hypomethylation. AZT, DFO and AZT/DFO pre-treatment significantly enhanced extracellular matrix collagen production and mineralisation in hBMSCs. EVs derived from AZT/DFO-preconditioned hBMSCs (AZT/DFO-EVs) enhanced the hBMSC proliferation, histone acetylation and hypomethylation when compared to EVs derived from AZT-treated, DFO-treated and untreated hBMSCs. Importantly, AZT/DFO-EVs significantly increased osteogenic differentiation and mineralisation of a secondary hBMSC population. Furthermore, AZT/DFO-EVs enhanced the pro-angiogenic cytokine release of HUVECs. Taken together, our findings demonstrate the considerable utility of synergistically inducing hypomethylation and hypoxia to improve the therapeutic efficacy of the MSC-EVs as a cell-free approach for bone regeneration.

Abstract 6020: The role of TP53 mutation status in the response to DNA methyltransferase inhibitor treatment

Abstract The purpose of this study was to understand the effect of p53 mutation status in ovarian cancer cells on the expression of repetitive elements (REs) following epigenetic treatment. High-grade serous ovarian carcinoma is the most aggressive subtype of ovarian cancer and the 5 year survival rate has remained unchanged for over two decades. Over 90% of high-grade serous ovarian carcinoma (OC) patients have a mutation in the TP53 gene, which encodes the p53 protein. P53 is a transcription factor primarily involved in maintaining genome integrity, but it can also regulate the expression of REs in the genome. REs account for more than half of the genome and these repetitive genomic elements are epigenetically silenced in healthy cells to prevent genome instability. The expression of some REs, such as long interspersed nuclear elements and endogenous retroviruses, are upregulated in human OC patients and cell lines. The mechanism by which p53 regulates expression of REs is not well-understood; the literature documents some instances of p53 activating RE transcription and others of p53 repressing RE transcription, depending on the target elements. We have previously shown that RE expression can be induced by treating OC cells with low doses of DNA methyltransferase inhibitors (DNMTis). Recently, we showed that OC cell lines with mutant p53 have higher baseline expression of REs compared to wild type (WT) cell lines. Treatment of TP53 mutant OC cell lines with DNMTis had a muted effect on RE expression compared to treatment of WT OC cell lines. To explore the relationship between TP53 mutation status and RE expression after DNMTi treatment, OC cell lines with different p53 mutation statuses were treated with 500 nM of the DNMTi 5-azacytidine. We then analyzed the expression of several REs via RT-qPCR and western blot. We hypothesized that TP53 mutant cell lines would have lower DNMTi upregulation of REs than TP53 WT cell lines. While at the RNA level RE expression was variable, at the protein level, we found that DNMTi treatment increased protein levels of several REs in TP53 WT and mutant, but not null, OC cell lines. Future work will examine the feasibility of utilizing these RE proteins as immunotherapy targets in OC. Citation Format: Khadra K. Omar, Erin E. Grundy, Olivia Cox, Melissa Hadley, Katherine B. Chiappinelli. The role of TP53 mutation status in the response to DNA methyltransferase inhibitor treatment [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 6020.

Epigenetic Manipulation Induced Production of Immunosuppressive Chromones and Cytochalasins from the Mangrove Endophytic Fungus Phomopsis asparagi DHS-48.

A mangrove endophytic fungus Phomopsis asparagi DHS-48 was found to be particularly productive with regard to the accumulation of substantial new compounds in our previous study. In order to explore its potential to produce more unobserved secondary metabolites, epigenetic manipulation was used on this fungus to activate cryptic or silent genes by using the histone deacetylase (HDAC) inhibitor sodium butyrate and the DNA methyltransferase (DNMT) inhibitor 5-azacytidine (5-Aza). Based on colony growth, dry biomass, HPLC, and 1H NMR analyses, the fungal chemical diversity profile was significantly changed compared with the control. Two new compounds, named phaseolorin J (1) and phomoparagin D (5), along with three known chromones (2–4) and six known cytochalasins (6–11), were isolated from the culture treated with sodium butyrate. Their structures, including their absolute configurations, were elucidated using a combination of detailed HRESIMS, NMR, and ECD and 13C NMR calculations. The immunosuppressive and cytotoxic activities of all isolated compounds were evaluated. Compounds 1 and 8 moderately inhibited the proliferation of ConA (concanavalin A)-induced T and LPS (lipopolysaccharide)-induced B murine spleen lymphocytes. Compound 5 exhibited significant in vitro cytotoxicity against the tested human cancer cell lines Hela and HepG2, which was comparative to the positive control adriamycin and fluorouracil. Our finding demonstrated that epigenetic manipulation should be an efficient strategy for the induction of new metabolites from mangrove endophytic fungi.

An epigenetic modifier enhances the generation of anti-phytopathogenic compounds from the endophytic fungus Chaetomium globosporum of Euphorbia humifusa

Endophytic fungi are striking resources rich in bioactive structures with agrochemical significance. In order to maximize the opportunity of search for bioactive compounds, chemical epigenetic manipulation was introduced to enhance the structural diversity of the fungal products, and an UPLC-ESIMS and bioassay-guided separation was used to detect novel bioactive metabolites. Consequently, four previously undescribed compounds including two cyclopentenones (globosporins A and B) and two monoterpenoid indole alkaloids (globosporines C and D), as well as three known compounds, were isolated from the endophytic fungus Chaetomium globosporum of Euphorbia humifusa by exposure to a DNA methyltransferase inhibitor 5-azacytidine. Their structures including the absolute configurations were elucidated by the analysis of NMR spectroscopic data, HRESIMS, and TD-DFT-ECD calculations. The indole alkaloids (globosporines C and D) showed antimicrobial activities against three phytopathogenic microbes (Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, and Pseudomonas syringae pv. lachrymans) with MICs in the range of 14–72 μg/mL. Mostly, globosporine D was proved to be potently anti-phytopathogenic against X. oryzae pv. oryzae in vitro and in vivo, which suggested that it has the potential to be developed as a candidate for the prevention of rice bacterial leaf blight. This work provides an efficient and environmentally friendly approach for expanding fungal products with agricultural importance.

DDEL-04. Pharmacokinetic/Pharmacodynamic analysis of the DNA methyltransferase inhibitor 5-azacytidine shows adequate brain tissue penetration with intravenous administration in a DIPG mouse patient-derived xenograft model.

One of the biggest obstacles in developing effective therapies for CNS tumors is drug delivery due to the enigmatic challenge of penetrating the blood-brain barrier. 5-azacytidine is a DNA methyltransferase inhibitor which was the first epigenetic targeting chemotherapeutic approved by the FDA. Altered DNA methylation is a hallmark of many cancers, including diffuse intrinsic pontine glioma (DIPG). 5-azacytidine has been shown to be active in DIPG cell lines, with only modest in-vivo activity. We have previously shown in a non-human primate model that intravenous (IV) administration of 5-azacitidine does not result in measurable CSF penetration, while intrathecal (IT) administration does and is well tolerated. To follow up these studies in a tumor-bearing mouse model (HSJD DIPG007), we performed pharmacokinetic (PK) and pharmacodynamic (PD) analysis following IV vs. IT administration. Forty mice were randomized to receive four weekly doses of IV 5-azacytidine (25 mg/kg), IT 5-azacytidine (40 μg), or corresponding vehicle controls. Four mice from each arm were sacrificed 30 minutes after the last dose and brain tissue was collected for PK/PD analysis. Drug concentration was quantified using ultra-high-performance liquid chromatography with tandem mass spectrometric detection, while pharmacodynamic methylation profiling was performed using the Infinium MethylationEPIC BeadChip (850K). Brain tissue concentrations were comparable between IV (7.6-58.0 pg/mg) and IT (6.9-63.9 pg/mg) dosing. Methylation profiling unexpectedly showed a significantly more pronounced pharmacodynamic effect with IV dosing vs. IT, with a mean decrease of 13.6% vs. 2.6% in global DNA methylation score (GDMS = percentage of highly methylated (beta ≥ 0.7) genomic loci) compared to vehicle controls. For the remaining mice, there was no significant difference in survival. Our results are encouraging that phenotypically relevant demethylating effects can be achieved in the CNS with IV 5-azacytidine administration; however, further research is needed to develop promising combination strategies in DIPG.

Bypassing reproductive barriers in hybrid seeds using chemically induced epimutagenesis.

The triploid block, which prevents interploidy hybridizations in flowering plants, is characterized by a failure in endosperm development, arrest in embryogenesis, and seed collapse. Many genetic components of triploid seed lethality have been successfully identified in the model plant Arabidopsis thaliana, most notably the paternally expressed genes (PEGs), which are upregulated in tetraploid endosperm with paternal excess. Previous studies have shown that the paternal epigenome is a key determinant of the triploid block response, as the loss of DNA methylation in diploid pollen suppresses the triploid block almost completely. Here, we demonstrate that triploid seed collapse is bypassed in Arabidopsis plants treated with the DNA methyltransferase inhibitor 5-Azacytidine during seed germination and early growth. We identified strong suppressor lines showing stable transgenerational inheritance of hypomethylation in the CG context, as well as normalized expression of PEGs in triploid seeds. Importantly, differentially methylated loci segregate in the progeny of “epimutagenized” plants, which may allow epialleles involved in the triploid block response to be identified in future studies. Finally, we demonstrate that chemically induced epimutagenesis facilitates hybridization between different Capsella species, thus potentially emerging as a strategy for producing triploids and interspecific hybrids with high agronomic interest.

Chemical epigenetic manipulation triggers the production of sesquiterpenes from the deep-sea derived Eutypella fungus

Chemical epigenetic manipulation of a deep-sea-derived Eutypella sp. fungus by the co-treatment with a histonedeacetylase inhibitor (suberohydroxamic acid, SBHA) and a DNA methyltransferase inhibitor (5-azacytidine, 5-Aza), resulted in the activation of a sesquiterpene-related biosynthetic gene cluster. Chromatographic separation of the elicitor-treated cultures led the isolation of 21 sesquiterpenes, including 17 undescribed compounds, eutypeterpenes A-Q. Their structures were identified by the extensive analysis of the spectroscopic data, including the single-crystal X-ray diffraction, chemical conversion, and the calculated NMR and ECD data for configurational assignments. Eutypeterpene A is a first bergamotene-type sesquiterpene incorporated with a dioxolanone unit, and eutypeterpenes O-Q with a cyclopentane ring represent an undescribed subtype of sesquiterpenes. The bioassay results showed that most compounds exert inhibitory effects against the lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophages, and eutypeterpene N is the most active. This study demonstrates that the epigenetic manipulation is an effective approach to trigger the production of cryptic metabolites from deep-sea derived fungus. The significant inhibition against LPS-induced NO production in vitro suggests eutypeterpenes to be potential for the development as anti-inflammatory agents.

DNA methylation is involved in acclimation to iron‐deficiency in rice (Oryza sativa)

Iron (Fe) is an essential micronutrient in plants, and Fe limitation significantly affects plant growth, yield and food quality. While many studies have reported the transcriptomic profile and pursue molecular mechanism in response to Fe limitation, little is known if epigenetic factors play a role in response to Fe-deficiency. In this study, whole-genome bisulfite sequencing analysis, high-throughput RNA-Seq of mRNA, small RNA and transposable element (TE) expression with root and shoot organs of rice seedlings under Fe-sufficient and Fe-deficient conditions were performed. The results showed that widespread hypermethylation, especially for the CHH context, occurred after Fe-deficiency. Integrative analysis of methylation and transcriptome revealed that the transcript abundance of Fe-deficiency-induced genes was negatively correlated with nearby TEs and positively with the 24-nucleotide siRNAs. The ability of methylation to affect the physiology and molecular response to Fe-deficiency was tested using an exogenous DNA methyltransferase inhibitor (5-azacytidine), and genetically using a mutant for domains rearranged methyltransferase 2 (DRM2), that lacks CHH methylation. Both approaches resulted in decreased growth and Fe content in rice plants. Thus, alterations in specific methylation patterns, directed by siRNAs, play an important role in acclimation of rice to Fe-deficient conditions. Furthermore, comparison with other reports suggests this may be a universal mechanism to acclimate to limited nutrient availability.

The DNA cytosine-5-methyltransferase 3 (DNMT3) involved in regulation of CgIL-17 expression in the immune response of oyster Crassostrea gigas

DNA methyltransferase, a key enzyme mediating DNA methylation, is involved in numerous processes including genomic imprinting, X chromosome inactivation, transposable element suppression, and immune defense in vertebrates. In the present study, a DNA cytosine-5-methyltransferase 3 was identified from oyster Crassostrea gigas (designed as CgDNMT3). There were a PWWP domain, a PHD domain and a DNA-methylase domain in the deduced amino acid sequences of CgDNMT3, and the conserved motifs I, IV, VI, Ⅷ, IX and X were identified in its C-terminal catalytic DNA-methylase domain. The mRNA transcripts of CgDNMT3 were detected in haemocytes, mantle, gill, adductor muscle, digestive gland and labial palp, with higher expression level in haemocytes (6.54 folds of those in gill, p < 0.01). The expression level of CgDNMT3 mRNA in haemocytes increased significantly after LPS primed (2.87 folds of that in control group, p < 0.05) in vitro or Vibrio splendidus challenging (1.94 folds of that in control group, p < 0.05) in vivo. Immunocytochemical analysis revealed that CgDNMT3 protein was distributed mainly in cytoplasm and partial in nucleus of oyster haemocytes. After CgDNMT3 was transfected and expressed in HEK293T cells, the DNA 5-methylcytosine (5-mc) level in the transfected group was significantly increased, which was 1.22 folds (p < 0.05) of the pcDNA-3.1 group. The expressions of oyster CgIL17-1, CgIL17-2 and CgIL17-5 in haemocytes increased (13.05 folds, 4.78 folds and 9.41 folds of that in control group, respectively) at 12 h after V. splendidus challenging, but the increase were significantly inhibited when the oysters were pre-treated with DNA methyltransferase inhibitor 5-Azacytidine, which were 9 folds, 1.93 folds and 3.22 folds of that in control group, respectively. These results collectively suggested that CgDNMT3 was a conserved member of DNA methyltransferase 3 family in oyster, and participated in regulating the expression of cytokines during immune response.

Genome-Wide Circular RNA Expression Patterns Reflect Resistance to Immunomodulatory Drugs in Multiple Myeloma Cells.

Simple SummaryMultiple myeloma (MM) constitutes the second most common hematological malignancy and is caused by aberrant plasma cell proliferation in the bone marrow. While recent improvements in the treatment of MM has been observed using immunomodulatory drugs (IMiDs), patients often relapse due to acquired drug resistance and no cure for the disease is currently available. In this report, we profile circular RNA (circRNA) expression patterns in cultured MM cells being sensitive to IMiDs and their resistant counterparts. CircRNAs constitute a large class of non-coding RNA molecules with emerging roles in cancer development and progression, but have not previously been explored in this context. We found that global circRNA expression patterns reflect IMiD sensitivity, but the most downregulated circRNA in IMiD resistant MM cells did not seem to be a direct driver of IMiD resistance. Future studies should investigate other circRNA candidates identified here in the context of IMiD resistance.Immunomodulatory drugs (IMiDs), such as lenalidomide and pomalidomide, may induce significant remissions in multiple myeloma (MM) patients, but relapses are frequently observed and the underlying molecular mechanisms for this are not completely understood. Circular RNAs (circRNAs) constitute an emerging class of non-coding RNAs with important roles in cancer. Here, we profiled genome-wide expression patterns of circRNAs in IMiD-sensitive MM cells and their resistant counterparts as well as in IMiD-resistant cells treated with specific epigenetic drugs alone or in combination. We found that genome-wide circRNA expression patterns reflect IMiD sensitivity and ciRS-7 (also known as CDR1as) was the most downregulated circRNA upon acquired resistance. The depletion of ciRS-7 correlated with increased methylation levels of the promoter CpG island of its host gene, LINC00632. Expression of LINC00632 and ciRS-7 was partly restored by treatment with a combination of an EZH2 inhibitor (EPZ-6438) and a DNA methyl transferase inhibitor (5-azacytidine), which also restores the IMiD sensitivity of the cells. However, knockdown of ciRS-7 did not affect IMiD sensitivity and we found that ciRS-7 also becomes epigenetically silenced after prolonged cell culture without drug-exposure. In conclusion, we found that genome-wide circRNA expression patterns reflect IMiD sensitivity in an in vitro model of acquired resistance.

VHL gene methylation contributes to excessive erythrocytosis in chronic mountain sickness rat model by upregulating the HIF-2α/EPO pathway

AimsHypoxia-inducible factors (HIFs) play important roles in the pathogenesis of erythrocytosis in chronic mountain sickness (CMS). von Hippel-Lindau (VHL) is a key regulator of hypoxia that can direct the poly-ubiquitylation and degradation of HIFs. Epigenetic mechanisms are believed to contribute toward adaption to chronic hypoxia. Here, we investigated the contribution and mechanism of VHL methylation in rats with erythrocytosis in CMS. Main methodsThe methylation status of VHL was measured via bisulfite sequencing PCR, while VHL, DNMT1, DNMT3α, and DNMT3β expression were assessed using real-time reverse transcription PCR and western blotting. HIF-2α and EPO expression levels in bone marrow were determined via immunohistochemical staining, and erythroid hyperplasia in bone marrow sections were observed with hematoxylin and eosin staining. Key findingsWe found that chronic hypoxia triggered erythroid hyperplasia in the bone marrow and increased the quantity of peripheral red blood cells in CMS rats. Chronic hypoxia significantly induced methylation at the CpG site in the VHL promoter, decreased VHL expression, and increased HIF-2α and EPO expression. Chronic hypoxia increased DNMT3α and DNMT3β expression, consistent with the decrease in VHL expression. The DNA methyltransferase inhibitor 5-azacytidine reduced chronic hypoxia-induced erythroid proliferation in the bone marrow of rats with CMS by suppressing VHL methylation and DNMTs expression. SignificanceOur study suggests that VHL methylation contributes toward excessive erythrocytosis in CMS by upregulating the HIF-2α/EPO pathway in the bone marrow of rats. We demonstrated that the DNMT inhibitor 5-azacytidine can attenuate erythroid hyperplasia in the bone marrow by demethylating the VHL promoter.

DNA methyltransferase inhibitor 5-azacytidine in high dose promotes ultrastructural maturation of cardiomyocyte.

The adult human heart muscle cells, cardiomyocytes are not capable of regenerate after injury. Stem cells are a powerful means for future regenerative medicine because of their capacity for self-renewal and multipotency. Several studies have reported the cardiogenic potential in human adipose tissue-derived stem cells (ADSCs) differentiation, but there is still no efficient protocol for the induction of cardiac differentiation by 5-azacytidine (5-Aza). The present study involves characterization and mainly, the ultrastructure of ADSCs derived cardiomyocyte-like cells. The cultured ADSCs were treated with 50 µM 5-Aza for 24 hours, followed by a 10-week extension. At different time points, cardiomyocyte-like cells were assessed by qRT-PCR and were evaluated by transmission electron microscopy at 10th week. The expression of cardiac-specific markers entailing cardiac troponin I (cTnI), connexin 43, myosin light chain-2v (Mlc-2v), increased over 10 weeks and the highest expression was at 10th week. The expression of the β-myosin heavy chain (β-MHC) increased significantly over 5 weeks and then decreased. At the ultrastructural level myofibrils, transverse tubules (T-tubules), sarcoplasmic reticular membrane, and intercalated discs were present. These data suggest that treatment with 5-Aza in high dose could promote differentiation of ADSCs into cardiomyocyte-like cells. These differentiated cells could be used for regeneration of damaged cardiomyocytes with the 3D scaffold for delivery of the cells.

Generation of Cochlear Hair Cells from Sox2 Positive Supporting Cells via DNA Demethylation

Regeneration of auditory hair cells in adult mammals is challenging. It is also difficult to track the sources of regenerated hair cells, especially in vivo. Previous paper found newly generated hair cells in deafened mouse by injecting a DNA methyltransferase inhibitor 5-azacytidine into the inner ear. This paper aims to investigate the cell sources of new hair cells. Transgenic mice with enhanced green fluorescent protein (EGFP) expression controlled by the Sox2 gene were used in the study. A combination of kanamycin and furosemide was applied to deafen adult mice, which received 4 mM 5-azacytidine injection into the inner ear three days later. Mice were followed for 3, 5, 7 and 14 days after surgery to track hair cell regeneration. Immunostaining of Myosin VIIa and EGFP signals were used to track the fate of Sox2-expressing supporting cells. The results show that (i) expression of EGFP in the transgenic mice colocalized the supporting cells in the organ of Corti, and (ii) the cell source of regenerated hair cells following 5-azacytidine treatment may be supporting cells during 5–7 days post 5-azacytidine injection. In conclusion, 5-azacytidine may promote the conversion of supporting cells to hair cells in chemically deafened adult mice.

High-throughput analyses and Bayesian network modeling highlight novel epigenetic Adverse Outcome Pathway networks of DNA methyltransferase inhibitor mediated transgenerational effects

A number of epigenetic modulating chemicals are known to affect multiple generations of a population from a single ancestral exposure, thus posing transgenerational hazards. The present study aimed to establish a high-throughput (HT) analytical workflow for cost-efficient concentration-response analysis of epigenetic and phenotypic effects, and to support the development of novel Adverse Outcome Pathway (AOP) networks for DNA methyltransferase (DNMT) inhibitor-mediated transgenerational effects on aquatic organisms. The model DNMT inhibitor 5-azacytidine (5AC) and the model freshwater crustacean Daphnia magna were used to generate new experimental data and served as prototypes to construct AOPs for aquatic organisms. Targeted HT bioassays (DNMT ELISA, MS-HRM and qPCR) in combination with multigenerational ecotoxicity tests revealed concentration-dependent transgenerational (F0-F3) effects of 5AC on total DNMT activity, DNA promoter methylation, gene body methylation, gene transcription and reproduction. Top sensitive toxicity pathways related to 5AC exposure, such as apoptosis and DNA damage responses were identified in both F0 and F3 using Gaussian Bayesian network modeling. Two novel epigenetic AOP networks on DNMT inhibitor mediated one-generational and transgenerational effects were developed for aquatic organisms and assessed for the weight of evidence. The new HT analytical workflow and AOPs can facilitate future ecological hazard assessment of epigenetic modulating chemicals.

Inhibition of NF-κB is required for oleanolic acid to downregulate PD-L1 by promoting DNA demethylation in gastric cancer cells.

Gastric cancer is one of the most common causes of cancer-related death worldwide. Immunotherapy via programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) blockade has shown benefits for gastric cancer. Epigenetic DNA methylation critically regulates cancer immune checkpoints. We investigated how the natural compound oleanolic acid (OA) affected PD-L1 expression in gastric cancer cells. Interleukin-1β (IL-1β) at 20 ng/mL was used to stimulate human gastric cancer MKN-45 cells. IL-1β significantly increased PD-L1 expression, which was abolished by OA. Next, OA-treated MKN-45 cells were co-cultured with activated and PD-1-overexpressing Jurkat T cells. OA restored IL-2 levels in the co-culture system and increased T cell killing toward MKN-45 cells. Overexpression of PD-L1 eliminated OA-enhanced T cell killing capacity; however, PD-1 blocking antibody abrogated the cytotoxicity of T cells. Moreover, OA abolished IL-1β-increased DNA demethylase activity in MKN-45 cells. DNA methyltransferase inhibitor 5-azacytidine rescued OA-reduced PD-L1 expression; whereas DNA demethylation inhibitor gemcitabine inhibited PD-L1 expression, and, in combination with OA, provided more potent inhibitory effects. Furthermore, OA selectively reduced the expression of DNA demethylase TET3 in IL-1β-treated MKN-45 cells, and overexpression of TET3 restored OA-reduced PD-L1 expression. Finally, OA disrupted nuclear factor κB (NF-κB) signaling IL-1β-treated MKN-45 cells, and overexpression of NF-κB restored OA downregulation of TET3 and PD-L1. The cytotoxicity of T cells toward MKN-45 cells was also weakened by NF-κB overexpression. Altogether, OA blocked the IL-1β/NF-κB/TET3 axis in gastric cancer cells, leading to DNA hypomethylation and downregulation of PD-L1. Our discoveries suggested OA as an epigenetic modulator for immunotherapy or an adjuvant therapy against gastric cancer.

Oxymatrine reduces expression of programmed death-ligand 1 by promoting DNA demethylation in colorectal cancer cells.

Colorectal cancer (CRC) represents an important neoplasm with high mortality. Although PD-L1/PD-1 system-based immunotherapy has benefits for a certain type of CRC, many efforts should be made to enhance the responses to anti-PD-1/PD-L1 drugs. DNA methylation has been critically implicated in the regulation of tumor immunity. Here, we examined the effects of the natural alkaloid oxymatrine on PD-L1 expression in CRC cells and to elucidate the underlying mechanism. Human CRC SW620 and HCT116 cells were treated with interferon γ (IFNγ) and/or oxymatrine. Cell viability was determined using MTT assays. PD-L1 expression was detected by real-time PCR and Western blot analyses. DNA demethylase activity was measured using kits. Oxymatrine did not apparently affect the viability of normal human intestinal epithelial cells. IFNγ at 20ng/ml increased the viability of CRC cells, but oxymatrine concentration-dependently reduced the viability in the absence or presence of IFNγ. IFNγ increased the mRNA and protein expression of PD-L1 in the two cell lines, but oxymatrine significantly abolished IFNγ-elevated PD-L1 levels at both mRNA and protein levels. Furthermore, DNA demethylase activity was remarkably increased in IFNγ-treated CRC cells, which was abolished by oxymatrine concentration-dependently. In addition, DNA methyltransferase inhibitor 5-azacytidine considerably abrogated oxymatrine-induced downregulation of PD-L1 mRNA and protein levels in IFNγ-stimulated CRC cells. Oxymatrine suppressed viability and reduced PD-L1 expression in IFNγ-stimulated CRC cells, which was attributed to enhanced DNA demethylation. Our current discoveries suggested oxymatrine as an epigenetic modulatory agent for immunotherapy against CRC via PD-1/PD-L1 blockade.