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Currently, two DNA methyltransferase inhibitors, azacitadine and decitabine, are the epigenetic agents used for AML treatment. As a result of DNA hipomethylation, these DNA methyltransferase inhibitors contribute to the reactivation of methylation silence tumor suppressor genes. Azacitadine and decitabine can increase life expectancy of older patients precluded from intensive chemotherapy. New studies are being conducted in order to determine the use of azacitadine and decitabine in prevention and treatment of AML relapse after allogeneic hematopoietic stem cell transplantation. The present paper manifests the mechanism of action of hypomethylating drugs, as well as provides a brief overview of some clinical trials concerning the use of azacitadine and decitabine in AML. The article also discusses some potential epigenetic drugs that are undergoing clinical trials.

Background Early-life stress increases the risk for posttraumatic stress disorder. However, the epigenetic mechanism of early-life stress-induced susceptibility to posttraumatic stress disorder in adulthood remains unclear. Methods Rat pups were exposed to maternal deprivation during postnatal days 1 to 14 for 3 hours daily and treated with the DNA methyltransferase inhibitor zebularine, L-methionine, or vehicle 7 days before contextual fear conditioning, which was used as a second stress and to mimic the reexperiencing symptom of posttraumatic stress disorder in adulthood. Long-term potentiation, dendritic spine density, DNA methyltransferase mRNA, Reelin gene methylation, and Reelin protein expression in the hippocampal CA1 were measured. Results Maternal deprivation enhanced contextual fear memory in adulthood. Meanwhile, maternal deprivation decreased DNA methyltransferase mRNA and Reelin gene methylation in the hippocampal CA1 on postnatal days 22 and 90. Reelin protein expression was increased in the hippocampal CA1 following contextual fear conditioning in adulthood. Furthermore, compared with rats that experienced maternal deprivation alone, rats also exposed to contextual fear conditioning showed an enhanced induction of hippocampal long-term potentiation and increased dendritic spine density in the hippocampal CA1 following contextual fear conditioning in adulthood. Zebularine pretreatment led to an enhancement of contextual fear memory, hypomethylation of the Reelin gene, and increased Reelin protein expression in adult rats, while L-methionine had the opposite effects. Conclusions Maternal deprivation can epigenetically program second-hit stress-induced Reelin expression and enhance the susceptibility to contextual fear memory in adulthood. These findings provide a new framework for understanding the cumulative stress hypothesis.

DNA methylation plays a pivotal role in the etiology of cancer by mediating epigenetic silencing of cancer-related genes. Since the relationship between aberrant DNA methylation and cancer has been understood, there has been an explosion of research at developing anti-cancer therapies that work by inhibiting DNA methylation. From the discovery of first DNA hypomethylating drugs in the 1980s to recently discovered second generation pro-drugs, exceedingly large number of studies have been published that describe the DNA hypomethylation-based anti-neoplastic action of these drugs in various stages of the pre-clinical investigation and advanced stages of clinical development. This review is a comprehensive report of the literature published in past 40 years, on so far discovered nucleosidic DNA methylation inhibitors in chronological order. The review will provide a complete insight to the readers about the mechanisms of action, efficacy to demethylate and re-express various cancer-related genes, anti-tumor activity, cytotoxicity profile, stability, and bioavailability of these drugs. The review further presents the far known mechanisms of primary and secondary resistance to azanucleoside drugs. Finally, the review highlights the ubiquitous role of DNA hypomethylating epi-drugs as chemosensitizers and/or priming agents, and recapitulate the combinatorial cancer preventive effects of these drugs with other epigenetic agents, conventional chemo-drugs, or immunotherapies. This comprehensive review analyzes the beneficial characteristics and drawbacks of nucleosidic DNA methylation inhibitors, which will assist the pre-clinical and clinical researchers in the design of future experiments to improve the therapeutic efficacy of these drugs and circumvent the challenges in the path of successful epigenetic therapy.

The DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT), can confer resistance to guanine O6-alkylating agents. Therefore, inhibition of resistant MGMT protein is a practical approach to increase the anticancer effects of such alkylating agents. Numerous small molecule inhibitors were synthesized and exhibited potential MGMT inhibitory activities. Although they were nontoxic alone, they also inhibited MGMT in normal tissues, thereby enhancing the side effects of chemotherapy. Therefore, strategies for tumor-specific MGMT inhibition have been proposed, including local drug delivery and tumor-activated prodrugs. Over-expression of MGMT in hematopoietic stem cells to protect bone marrow from the toxic effects of chemotherapy is also a feasible selection. The future prospects and challenges of MGMT inhibitors in cancer chemotherapy were also discussed.

Somatic cell nuclear transfer (SCNT) derived embryos suffer from abnormal epigenetic reprogramming, which handicaps pre- and postimplantation development. It was hypothesized that epigenetic modifiers, including zebularine (DNA methyltransferase inhibitors) and BIX-01294 (histone methyltransferase inhibitors), could decrease the respective levels of 5-methylcytosine and H3K9me2 in reconstructed oocytes (RO). Accordingly, we investigated whether treating RO with zebularine and BIX-01294 for 16 hours after activation could improve developmental competence and quality of buffalo-bovine interspecies SCNT (iSCNT) embryos. Treatment of RO with zebularine but not BIX-01294 significantly increased two-cell formation at 16 hours postactivation. Conversely, early cleaved embryos had significantly lower rate of blastocyst formation in zebularine treated RO compared to their counterparts in control and BIX-01294 groups. Treatment of RO with zebularine and BIX-01294 did not improve blastocyst rate of buffalo-bovine iSCNT embryos compared to their control counterparts. However, these two epigenetic drugs might have some beneficial effects on buffalo-bovine iSCNT compared to bovine SCNT embryos. The quality of iSCNT blastocysts was improved due to significant expression of OCT4 and CDX2 in BIX-01294 and CDX2 in zebularine treated RO. Furthermore, treatment of RO with zebularine and BIX-01294 did not affect DNA fragmentation in derived blastocysts against control group. In conclusion, treatment with zebularine and BIX-01294 did not enhance developmental competence of iSCNT embryos, but may have some beneficial effects on epigenetic makeup and quality of derived blastocysts.

The paternally-imprinted genes insulin-like growth factor 2 (IGF2), H19, delta-like homologue 1 (DLK1), and maternally-expressed gene 3 (MEG3) are expressed from the tandem gene loci IGF2-H19 and DLK1-MEG3, which play crucial roles in initiating embryogenesis and development. The erasure of imprinting (EOI) at differentially methylated regions (DMRs) which regulate the expression of these genes maintains the developmental quiescence of primordial germ cells (PGCs) migrating through the embryo proper during embryogenesis and prevents them from forming teratomas. To address the potential involvement of the IGF2-H19 and DLK1-MEG3 loci in the pathogenesis of embryonal carcinoma (EC), we investigated their genomic imprinting at DMRs in the human PGC-derived EC cell line NTera-2 (NT2). We observed EOI at the IGF2-H19 locus and, somewhat to our surprise, a loss of imprinting (LOI) at the DLK1-MEG3 locus. As a result, NT2 cells express imprinted gene ratios from these loci such that there are i) low levels of the proliferation-promoting IGF2 relative to ii) high levels of the proliferation-inhibiting long noncoding RNA (lncRNA) H19 and iii) high levels of proliferation-promoting DLK1 relative to iv) low levels of the proliferation-inhibiting lncRNA MEG3. Consistent with this pattern of expression, the knockdown of DLK1 mRNA by shRNA resulted in decreased in vitro cell proliferation and in vivo tumor growth as well as decreased in vivo organ seeding by NT2 cells. Furthermore, treatment of NT2 cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-azaD) inhibited their proliferation. This inhibition was accompanied by changes in expression of both tandem gene sets: a decrease in the expression of DLK1 and upregulation of the proliferation-inhibiting lncRNA MEG3, and at the same time upregulation of IGF2 and downregulation of the lncRNA H19. These results suggest that the DLK1-MEG3 locus, and not the IGF2-H19 locus, drives the tumorigenicity of NT2 cells. Based on these results, we identified DLK1 as a novel treatment target for EC that could be downregulated by 5-azaD.

Radiotherapy is an important modality for treatment of carcinomas; however, radio‐resistance is still a difficult problem. Aberrant epigenetic alterations play an important role in cancer development. Among epigenetic parameters, DNA methylation has arguably attracted the most attention in the radio‐resistance process. To determine the role of DNA methylation in radiation resistance, several studies were conducted. We summarized previous studies on the role of DNA methylation in radiotherapy. We observed this significant role of DNA methylation in genes related to DNA repair, cell proliferation, cell cycle process, and re‐oxygenation. Furtherly, we also conclude the predictive effect of DNA methylation on tumor radio‐sensitivity and the using of DNA methyltransferase inhibitors in clinical practice. DNA methylation plays a pivotal role in the radio‐sensitivity of tumor radio‐therapy. While hyper‐methylation or hypo‐methylation of genes is related to gene functions.

Nerve injury-induced hyperactivity of primary sensory neurons in the dorsal root ganglion (DRG) contributes to chronic pain development, but the underlying epigenetic mechanisms remain poorly understood. Here we determined genome-wide changes in DNA methylation in the nervous system in neuropathic pain. Spinal nerve ligation (SNL), but not paclitaxel treatment, in male Sprague Dawley rats induced a consistent low-level hypomethylation in the CpG sites in the DRG during the acute and chronic phases of neuropathic pain. DNA methylation remodeling in the DRG occurred early after SNL and persisted for at least 3 weeks. SNL caused DNA methylation changes at 8% of CpG sites with prevailing hypomethylation outside of CpG islands, in introns, intergenic regions, and repetitive sequences. In contrast, SNL caused more gains of methylation in the spinal cord and prefrontal cortex. The DNA methylation changes in the injured DRGs recapitulated developmental reprogramming at the neonatal stage. Methylation reprogramming was correlated with increased gene expression variability. A diet deficient in methyl donors induced hypomethylation and pain hypersensitivity. Intrathecal administration of the DNA methyltransferase inhibitor RG108 caused long-lasting pain hypersensitivity. DNA methylation reprogramming in the DRG thus contributes to nerve injury-induced chronic pain. Restoring DNA methylation may represent a new therapeutic approach to treat neuropathic pain.SIGNIFICANCE STATEMENT Epigenetic mechanisms are critically involved in the transition from acute to chronic pain after nerve injury. However, genome-wide changes in DNA methylation in the nervous system and their roles in neuropathic pain development remain unclear. Here we used digital restriction enzyme analysis of methylation to quantitatively determine genome-wide DNA methylation changes caused by nerve injury. We showed that nerve injury caused DNA methylation changes at 8% of CpG sites with prevailing hypomethylation outside of CpG islands in the dorsal root ganglion. Reducing DNA methylation induced pain hypersensitivity, whereas increasing DNA methylation attenuated neuropathic pain. These findings extend our understanding of the epigenetic mechanism of chronic neuropathic pain and suggest new strategies to treat nerve injury-induced chronic pain.

Bone-Marrow Stromal Cells (BMSCs)-derived vascular endothelial cells (VECs) is regarded as an important therapeutic strategy for spinal cord injury, disc degeneration, cerebral ischemic disease and diabetes. The change in DNA methylation level is essential for stem cell differentiation. However, the DNA methylation related mechanisms underlying the endothelial differentiation of BMSCs are not well understood. In this study, DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC) significantly elevated the endothelial markers expression (CD31/PECAM1, CD105/ENG, eNOS and VE-cadherin), as well as promoted the capacity of angiogenesis on Matrigel. The result of Alexa 488-Ac-LDL uptake assay indicated that the differentiation ratio of BMSCs into VECs was 68.7% in 5-azaz-dC induced differentiation. And then we screened differentiation inducers with altered expression patterns and DNA methylation levels in four important families (VEGF, ANG, FGF and ETS). By integrating these data, five endothelial differentiation inducers (VEGFA, ANGPT2, FGF2, FGF9 and ETS1) which were directly upregulated by 5-aza-dC and five indirect factors (FGF1, FGF3, ETS2, ETV1 and ETV4) were identified. These data suggested that 5-aza-dC is an excellent chemical molecule for BMSCs differentiation into functional VECs and also provided essential clues for DNA methylation related signaling during 5-aza-dC induced endothelial differentiation of BMSCs.

Background Urothelial bladder cancer (UBC) accounts for 10,000 new diagnoses and 5000 deaths annually in the UK (Cancer Research UK, http://www.cancerresearchuk.org/health-professional/cancer-statistics/statistics-by-cancer-type/bladder-cancer, Cancer Research UK, Accessed 26 Mar 2018). Cisplatin-based chemotherapy is standard of care therapy for UBC for both palliative first-line treatment of advanced/metastatic disease and radical neoadjuvant treatment of localised muscle invasive bladder cancer. However, cisplatin resistance remains a critical cause of treatment failure and a barrier to therapeutic advance in UBC. Based on supportive pre-clinical data, we hypothesised that DNA methyltransferase inhibition would circumvent cisplatin resistance in UBC and potentially other cancers. Methods The addition of SGI-110 (guadecitabine, a DNA methyltransferase inhibitor) to conventional doublet therapy of gemcitabine and cisplatin (GC) is being tested within the phase Ib/IIa SPIRE clinical trial. SPIRE incorporates an initial, modified rolling six-dose escalation phase Ib design of up to 36 patients with advanced solid tumours followed by a 20-patient open-label randomised controlled dose expansion phase IIa component as neoadjuvant treatment for UBC. Patients are being recruited from UK secondary care sites. The dose escalation phase will determine a recommended phase II dose (RP2D, primary endpoint) of SGI-110, by subcutaneous injection, on days 1–5 for combination with GC at conventional doses (cisplatin 70 mg/m2, IV infusion, day 8; gemcitabine 1000 mg/m2, IV infusion, days 8 and 15) in every 21-day cycle. In the dose expansion phase, patients will be randomised 1:1 to GC with or without SGI-110 at the proposed RP2D. Secondary endpoints will include toxicity profiles, SGI-110 pharmacokinetics and pharmacodynamic biomarkers, and pathological complete response rates in the dose expansion phase. Analyses will not be powered for formal statistical comparisons and descriptive statistics will be used to describe rates of toxicity, efficacy and translational endpoints by treatment arm. Discussion SPIRE will provide evidence for whether SGI-110 in combination with GC chemotherapy is safe and biologically effective prior to future phase II/III trials as a neoadjuvant therapy for UBC and potentially in other cancers treated with GC. Trial Registration EudraCT Number: 2015–004062-29 (entered Dec 7, 2015) ISRCTN registry number: 16332228 (registered on Feb 3, 2016) Electronic supplementary material The online version of this article (10.1186/s13063-018-2586-7) contains supplementary material, which is available to authorized users.

5‐Hydroxymethylcytosine (5hmC) is produced from 5‐methylcytosine (5mC) by Ten‐eleven translocation (TET) dioxygenases. The epigenetic modification 5hmC has crucial roles in both cellular development and differentiation. The 5hmC level is particularly high in the brain. While 5mC is generally associated with gene silencing/reduced expression, 5hmC is a more permissive epigenetic mark. To understand its physiological function, an easy and accurate quantification method is required. Here, we have developed a novel LC‐MS/MS‐based approach to quantify both genomic 5mC and 5hmC contents. The method is based on the liberation of nucleobases by formic acid. Applying this method, we characterized the levels of DNA methylation and hydroxymethylation in mouse brain and liver, primary hepatocytes, and various cell lines. Using this approach, we confirm that the treatment of different cell lines with the DNA methyltransferase inhibitor 5‐aza‐2′‐deoxycytidine leads to a decrease in 5mC content. This decrease was accompanied by an increase in 5hmC levels in cell lines of hematopoietic origin. Finally, we showed that ascorbate elevates the levels of 5hmC and augments the effect of 5‐aza‐2′‐deoxycytidine without significantly influencing 5mC levels.

Esophageal cancer (EC) is the eighth most common highly aggressive cancer worldwide. The purpose of this study was to investigate the effect of the DNA methyltransferase inhibitor RG108 on the radiosensitivity of EC cells. MTT and clonogenic assays were performed to assess the effect of RG108 on the proliferation and radiosensitivity of Eca‑109 and TE‑1 human EC cells. The cell cycle progression and alterations in apoptosis were analyzed by flow cytometry. For the in vivo analysis, the Eca‑109 cells were inoculated into nude mice to establish tumors. Tissues from xenografts were obtained to detect changes to microvessels and tumor growth by immunohistochemistry (IHC). RNA-seq was used to identify differentially expressed genes. We found that RG108 increased the radiosensitivity of EC cells. Apoptosis and G2/M-phase arrest were induced by X-ray irradiation and were significantly enhanced by RG108. In addition, growth of tumor xenografts from the Eca‑109 cells was significantly inhibited by irradiation in combination with RG108. The RNA-seq analysis revealed that, compared with radiation alone, X-ray irradiation in combination with RG108 altered the expression of 121 genes in multiple pathways, including the TGF-β signaling pathway and the Epstein-Barr virus infection pathway. In conclusion, RG108 induced radiosensitivity in EC cells both in vitro and in vivo.

Lung cancer is the most common cause of cancer death in both men and women worldwide. In North America alone, more than 257,000 new cases are expected in 2019 (1, 2). About 85% of these cases are nonsmall cell lung cancer (NSCLC), and despite recent treatment advances, long-term prognosis remains poor. For NSCLC patients with locally advanced disease involving mediastinal lymph nodes, concomitant radiotherapy and chemotherapy followed by immunotherapy represents a backbone of curative-intent treatment (3). Despite aggressive multimodality therapy, disease recurrence is common with ∼30% local and 45% distant failure rates reported at 2 y (4, 5). To date, efforts to improve these outcomes with additional combinations (e.g., cetuximab) or increased radiation dose have not been successful (4). Novel strategies that modulate DNA damage repair pathways in response to cytotoxic chemotherapy and radiation in lung cancer may provide a path forward. In PNAS, Abbotts et al. (6) report the use of an epigenetic modifier to enhance antitumor response to these DNA-damaging therapeutics. The investigators find using cell lines and mouse models that DNA methyltransferase inhibitors (DNMTi) sensitize NSCLC to poly(ADP ribose) polymerase (PARP) inhibition and radiotherapy by down-regulating critical DNA damage repair pathways (6). PARP inhibition first gained real traction in cancer therapy in the context of BRCA-deficient tumors (7⇓–9). PARP1 participates in a key pathway required for efficient repair of single-strand DNA breaks. PARP inhibition prevents resolution of single-strand breaks, leading to acquisition of double-strand breaks (DSBs) in replication that require homologous recombination (HR) for high-fidelity repair. BRCA1 and BRCA2 mutations disrupt the HR repair … [↵][1]1To whom correspondence may be addressed. Email: rudinc{at}mskcc.org. [1]: #xref-corresp-1-1

INTRODUCTION: Transforming growth factor-β (TGF-β), a ubiquitous molecule in pancreatic ductal adenocarcinoma (PDA) tumors, acts as a potent inducer of tumor invasion by regulating proteins involved in metastasis and driving epithelial-mesenchymal transition (EMT). We hypothesized that TGF-β signaling-induced EMT is regulated by DNA methylation. To evaluate this, we investigated the association between EMT gene promoter methylation status and gene expression in the TCGA PDA dataset. We also investigated vimentin gene (a mesenchymal marker)-specific changes in DNA methylation due to TGF-β treatment in PDA cells. METHODS: The methylation status of EMT-related genes (ZEB2, CDH2, Vimentin) was interrogated in the TCGA PDA data set. β-values (proportion of methylated gene probes in a location) were compared to gene expression levels. Next, a PDA cell line (Panc-1) was treated with TGF-β (10ng/ml), vehicle control, DNA methyltransferase inhibitor (5-azacytidine, AZA, 10mM), or TGF-β plus AZA to evaluate the effects on the DNA methylation status of the vimentin gene with methylation-specific PCR (MSP) against the promoter region of vimentin. Unmethylation and methylation levels at the vimentin promoter region were then compared. RESULTS: We found that β values of promotor regions of ZEB2, CDH2, and Vimentin were moderately negatively correlated with gene expression (Pearson r varies from 0.45 to 0.63) in the PDA TCGA data set. In Panc-1 cells, vimentin gene expression was increased following treatment with AZA compared to controls suggesting that DNA demethylation increases vimentin gene expression. MSP demonstrated that TGF-β treatment increased unmethylated vimentin gene levels compared to vehicle control treatment. TGF-β treatment increased unmethylated vimentin gene levels more so than methylated gene levels. CONCLUSIONS: These results demonstrate that gene expression of EMT-related genes may be at least partially regulated by DNA methylation. We demonstrate that TGF-β treatment leads to increased Vimentin promotor demethylation (lower β value) and increased gene expression in Panc-1 cells. This observation is consistent with our findings in the TCGA data set that lower β-values are associated with increased expression of mesenchymal genes, indicative of EMT. Further studies are underway to investigate the relationship between DNA methylation and TGF-β-induced EMT in general. Citation Format: Manjul Rana, Abul Elahi, Abidemi O. Ajidahun, Rita G. Kansal, Anders E. Berglund, David Shibata, Evan S. Glazer. TGF-β induced EMT gene expression is associated with promoter demethylation in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-090.

Multiple Myeloma (MM) is a malignancy of the antibody-producing plasma cells. Despite improvements to treatment throughout the years, it remains an incurable and fatal disease [1]. Therefore, novel innovative therapies are needed for relapsed/refractory MM. The anti-apoptotic BCL-2 family of proteins (BCL-2, BCL-XL and MCL-1) are critical regulators of the intrinsic apoptotic pathway and determine the survival of human MM cells [2,3]. The anti-apoptotic BCL-2 proteins represent attractive therapeutic targets in MM. Recently, ABT-199, a selective BCL-2 inhibitor, was FDA approved for the treatment of CLL and AML. The aim of the study is to develop a biomarker to identify MM patients that are reliant on BCL-2 and could be treated with ABT-199. To assess anti-apoptotic dependence, we used BH3 profiling, a functional assay that interrogates BCL-2 protein interactions using synthetic BH3 peptides to measure the loss of mitochondrial membrane potential. We screened a panel of BH3 mimetics in MM cells and patient bone marrow samples; ABT-199 (selective BCL-2 inhibitor), ABT-263 (BCL-2, BCL-xL and BCL-W inhibitor), WEHI-539 (BCL-XL inhibitor) and AMG-176 (selective MCL-1 inhibitor). The BH3 profile data and BH3 mimetics sensitivity data revealed that there is a diverse anti-apoptotic dependence in MM cell lines and primary patient samples. In addition, we aim to identify novel combination treatments that will induce BCL-2 dependence in MM cells, enhancing sensitivity to ABT-199 treatment. We performed a small molecule screen to identify epigenetic modifiers that could induce BCL-2 dependence in two MM cell lines. The screen included the following classes of epigenetic drugs: histone deacetylase inhibitors, histone methyltransferase inhibitors, DNA methyltransferase inhibitors and BET inhibitors. Interestingly, two classes of the epigenetic drugs, were synergistic with ABT-199 in three MM cell lines, (CI

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 (p < 0.05). 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 oysters, and participated in regulating the expression of cytokines during immune response.

Procaine is a specific inhibitor of DNA methyltransferase (DNMT). In the present work, the Schiff base ligands were synthesized from procaine with the addition of salicylaldehyde and naphthaldehyde as metal precursor for procaine-based transition metal complexes (L1 and L2) with palladium (Pd). The compounds were checked and characterized for identity and purity using elemental analysis, SEM, melting points, FT-IR, and NMR spectral data. Among the compounds, L1-Pd and L2-Pd were found to display significant cytotoxicity with IC50 values of 10.21 µM and 10.79 µM against human gastric cancer cell line MKN-45. Novel synthesized procaine derivatives target molecules and their palladium complexes had an inhibitory effect on colony formation and wound healing on MKN-45 cells. Furthermore, these compounds were evaluated for their apoptotic activity and DNMT activity in MKN-45 cells. Western blotting and qPCR studies demonstrated that compound L1-Pd and L2-Pd induced apoptosis and a slight decrease in anti-apoptotic Bcl-2 protein/gene expression and highly increased pro-apoptotic Bax protein/gene expression in MKN-45 cells treated. Also, total DNMT enzyme activity and protein expression (DNMT1, DNMT3a, and DNMT3b) levels were decreased in L1-Pd and L2-Pd treated cells. The L1-Pd and L2-Pd complexes showed that they are potential new DNMT inhibitors to prevent cancer-induced DNA hypermethylation and can be used in the treatment of gastric cancer.

Testicular germ cell tumors (TGCTs) are the most frequent solid cancers of males between the ages of 16 to 39 and have the highest mortality in this age group. Testicular cancer is highly curable with platinum-based chemotherapeutics. However, 15-20% of patients are resistant and succumb to their disease. The mechanisms for cisplatin sensitivity/resistance in testicular cancer is unclear. Previously we showed that cisplatin refractory testicular cancer is highly sensitive to the DNA methyltransferase inhibitors (DNMTi), 5-Aza-2'-deoxycytidine (5-AZA-DC) and guadecitabine in vivo and in vitro. High expression of DNMT3B correlates with sensitivity to DNMTi in TGCT cells. Based on these preclinical findings we recently completed a phase 1 clinical trial which demonstrated the combination of guadecitabine and cisplatin was tolerable and effective in treating patients with platinum refractory germ cell cancer. To better understand the mechanism of chemotherapy sensitivity/resistance in testicular cancer we generated a series of cisplatin resistant cell models. Genome wide methylome array and de novo transcriptional profiling of cisplatin resistant TGCT cells revealed decreased global H3K27 methylation due to suppression of the polycomb repressive complex 2 (PRC2 ). To better understand the relationship between cisplatin and 5-AZA-DC sensitivity, 5-AZA-DC resistant testicular cancer cells were also generated. Remarkably, there was a reciprocal relationship between cisplatin and 5-AZA-DC resistance, with cisplatin resistance associated with increased sensitivity to 5-AZA-DC and 5-AZA-DC resistance associated with increased sensitivity to cisplatin. Transcriptomics profiling and other studies in 5-AZA-DC resistant cells revealed increased H3K27me3, repression of polycomb target genes and a dramatic decreased in DNMT3B expression, the exact opposite of the situation in cisplatin resistant cells. Further knockdown of DNMT3B resulted in induction of H3K27me3, decreased sensitivity to 5-AZA-DC and increased sensitivity to cisplatin. These data suggest that DNA methylation and polycomb signaling are interconnected drivers of chemotherapy and epigenetic therapy responses in TGCTs. Ongoing H3K27 me3 ChIP-Seq, global methylation analysis, and validation studies will be described to further elucidate these mechanisms with the goal of devising novel epigenetic-based therapies for testicular and potentially other cancers. Citation Format: Ratnakar Singh, Emmanuel Bikorimana, Zeeshan Fazal, Cliff Yerby, Hannah Baldwin, Aleyah Destiny Hattab, Megan Tomlin, Andrea K. Corbet, Raya Iman Boyd, Khadeeja Shahid, Doha Naguib Ahmed Mohamed Shokry, Sarah Joy Spinella, Michael J. Spinella. Polycomb signaling reciprocally regulates sensitivity of testicular germ cell tumors cells to cisplatin and DNA hypomethylating agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2127.

BackgroundTriple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and lacks definite treatment targets. Tumor immune microenvironment (TIME) heterogeneity has a profound impact on the immunotherapy response. Tumors with non-inflamed TIME derive limited benefit from immunotherapy. However, what drives the formation of the non-inflamed TIME in TNBC remains unclear.MethodsUsing our multiomics database of TNBC, we conducted an analysis to explore the key genomic events driving the formation of the non-inflamed TIME in TNBC. In vitro and in vivo studies further revealed potential mechanisms and the efficacy of combination treatment with immunotherapy.ResultsWith transcriptomic and genomic data, we systematically analyzed the TIME of TNBC and revealed that the classical basal-like subtype of TNBC consisted of two distinct microenvironment phenotypes, defined as the ‘inflamed’ and ‘non-inflamed’ subtypes. We performed further screening and demonstrated that MYC amplification and overexpression led to low immune infiltration and cytolytic activity in TIME. Mechanistically, MYC bound to DNMT1 promoter and activated DNMT1 transcription in TNBC cells, thus suppressing the Cyclic GMP-AMP synthase (cGAS)-STING pathway via an epigenetic regulatory way. In MYC-overexpressing TNBC, decitabine, an Food and Drug Administration (FDA)-approved DNA methyltransferase inhibitor, converted tumors from non-inflamed to inflamed tumors by enhancing T cell infiltration. Furthermore, the combination of decitabine with programmed cell death protein 1 (PD-1) inhibitor reversed T cell exhaustion and improved T cell function in mouse models, which elicited potent antitumor activity in MYC-overexpressing TNBC.ConclusionsOur work elucidates that the classic oncogene MYC induces immune evasion by repressing innate immunity. Furthermore, we provide a rationale for combining DNA methyltransferase inhibition with immunotherapy for the treatment of MYC-overexpressing TNBC.

// Hui Li 1 , Guoqing Liu 2 , Ke Pan 2 , Xiongying Miao 2 and Yong Xie 2 1 Department of Anesthesia, Second Xiangya Hospital, Central South University, Changsha, Hunan, China 2 Department of General Surgery, Second Xiangya Hospital, Central South University, Changsha, Hunan, China Correspondence to: Yong Xie, email: xiangyaxieyong@163.com Keywords: gastric cancer; microRNA; methylation; LIM and SH3 protein 1 Received: May 12, 2017 Accepted: August 21, 2017 Published: September 30, 2017 ABSTRACT MicroRNAs (miRs) have been demonstrated to play promoting or tumor suppressive roles in various human cancers, but the regulatory mechanism of miR-29b underlying gastric cancer development and progression still remains largely unclear. In the present study, we found that miR-29b was significantly downregulated in gastric cancer tissues and cell lines. Low expression of miR-29b was significantly associated with DNA methylation, and treatment with DNA methyltransferase inhibitor 5-Aza-20-deoxycytidine upregulated miR-29b in gastric cancer cells. In addition, both reduced miR-29b expression and miR-29b methylation were associated with disease progression and poor prognosis in gastric cancer. Restoration of miR-29b caused a reduction in gastric cancer cell proliferation, migration, and invasion, and inhibited tumor growth in vivo . LASP1 was then identified as a target gene of miR-29b in gastric cancer cells. Moreover, upregulation of LASP1 was significantly associated with gastric cancer progression and poor prognosis. Knockdown of LASP1 also suppressed the proliferation, migration, and invasion of gastric cancer cells. Moreover, overexpression of LASP1 impaired the suppressive effects of miR-29b on the malignant phenotypes of gastric cancer cells, suggesting that miR-29b may inhibit gastric cancer growth and metastasis via targeting LASP1. According to these data, miR-29b may be used as a potential therapeutic candidate for gastric cancer.

Targeting DNA hypermethylation, using nucleoside analogs, is an efficient approach to reprogram cancer cell epigenome leading to reduced proliferation, increased differentiation, recognition by the immune system, and ultimately cancer cell death. DNA methyltransferase inhibitors have been approved for the treatment of myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myelogenous leukemia. To improve clinical efficacy and overcome mechanisms of drug resistance, a second generation of DNA methyltransferase inhibitors has been designed and is currently in clinical trials. Although efficient in monotherapy against hematologic malignancies, the potential of DNA methyltransferase inhibitors to synergize with small molecules targeting chromatin or immunotherapy will provide additional opportunities for their future clinical application against leukemia and solid tumors.

DNA methyltransferase inhibitors, so-called hypomethylating agents (HMAs), are the only drugs approved for the treatment of higher-risk myelodysplastic syndromes and are widely used in this context. However, it is still unclear why some patients respond to HMAs, whereas others do not. Recent sequencing efforts have identified molecular disease entities that may be specifically sensitive to these drugs, and many attempts are being made to clarify how HMAs affect the malignant clone during treatment. Here, we review the most recent data on the clinical effects of HMAs in myeloid malignancies.

In breast cancer (BC), silencing of miRNA genes due to miRNA gene promoter methylation are the important mechanisms directly contributing to tumorigenesis and tumor progression. miRNA-495 (miR-495) has been reported to be a tumor suppressor gene in various cancers, but its role and regulation in BC remains unclear. In the present study, the level of miR-495 was inversely correlated with the expression of STAT-3 in BC tissues and cell lines. miR-495 can directly target 3'-UTR of STAT-3 mRNA and thereby decrease the expression of STAT-3 in MCF-7 and HCC1973 cells by Targetscan and Dual-luciferase assay. We further analyzed miR-495 promoter methylation by sodium bisulfite sequencing method (BSP), and found DNA methyltransferase inhibitor, 5-AzaC concomitantly upregulated expression of miR-495 and downregulated its target gene STAT-3 and its downstream target VEGF. Furthermore, we further observed that 5-AzaC treatment, miR-495 mimics and STAT-3 knockdown significantly inhibited cell function in breast cancer by Transwell assay, EdU flow cytometry, Annexin V-FITC/PI combined with flow cytometry and Hoechst staining. Taken together, our data are first to demonstrate that the miR-495 is silenced due to promoter methylation in breast cancer. DNA methyltransferase inhibitor 5-AzaC could reverse miR‑495 (suppressor gene) and STAT-3 (oncogene). The anticancer properties of 5-AzaC were preliminarily confirmed in breast cancer.

DNA methylation and demethylation is part of the essential biological processes regulating gene expression in normal cell development. Abnormal methylation status of specific genes and their irregularly translated proteins are normally associated with certain kinds of diseases or cancer. The rapid development of innovative DNA methylation mapping techniques provides a better understanding of DNA methylation pattern and its mechanisms in the human genome and its correlation with numerous diseases. These new techniques can lead us to develop new epigenetic medications, such as DNA methyltransferase inhibitors. As part of the approaches to probe DNA methylation and evaluate the effects of epigenetic therapy, mass spectrometry has been taking an important role in the identification, validation, and quantification of DNA methylation and demethylation. In this review, we will briefly summarize the current breadth of knowledge on the topic of DNA methylation and its occurrence in diseases, DNA methylation drugs, and mass spectrometry based approaches used to study DNA methylation.

It has been clearly demonstrated that DNA methyltransferase inhibition can induce embryonic stem cells to differentiate into cells of specific tissues by altering the genes expression. Chicken embryonic fibroblasts (CEFs) are mesenchymal stem cell (MSC)-like multipotent progenitor cells and can both self-renew and undergo differentiation. In this study, we hypothesize that DNA demethylation may lead to cell differentiation and the re-expression of development-related genes in chicken embryo fibroblasts. Thus, we investigated which development-related genes are affected when CEFs are treated with DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine (5-Aza-dC) and how those genes are affected. In this study, we analyzed the expression profiles of genes by RNA sequencing of CEFs treated with 5-Aza-dC. A total of 40 differentially expressed genes (DEGs) (>2-fold change) related to chicken development were identified. Co-expression cluster analysis revealed that these genes are mainly related to development of bone, feather follicle, epidermis, and blood vessels. Moreover, the molecular interaction network analysis showed that canonical Wnt signaling tended to play a central role in development associated with these genes. These findings will help us to understand the molecular regulatory mechanisms of development.

Diacylglycerol kinases (DGKs) are important regulators of cell signaling and have been implicated in human malignancies. Whether epigenetic alterations are involved in the dysregulation of DGKs in cancer is unknown, however. We therefore analyzed methylation of the promoter CpG islands of DGK genes in colorectal cancer (CRC) cell lines. We found that DGKG, which encodes DGKγ, was hypermethylated in all CRC cell lines tested (n = 9), but was not methylated in normal colonic tissue. Correspondingly, DGKG expression was suppressed in CRC cell lines but not in normal colonic tissue, and was restored in CRC cells by treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). DGKG methylation was frequently observed in primary CRCs (73/141, 51.8%) and was positively associated with KRAS and BRAF mutations and with the CpG island methylator phenotype (CIMP). DGKG methylation was also frequently detected in colorectal adenomas (89 of 177, 50.3%), which suggests it is an early event during colorectal tumorigenesis. Ectopic expression of wild-type DGKγ did not suppress CRC cell proliferation, but did suppress cell migration and invasion. Notably, both constitutively active and kinase-dead DGKγ mutants exerted inhibitory effects on CRC cell proliferation, migration and invasion, and the wild-type and mutant forms of DGKγ all suppressed Rac1 activity in CRC cells. These data suggest DGKG may play a tumor suppressor role in CRC.