Effect Of DNA Methyltransferase Inhibitor Research Articles

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.

Effect of zebularine on apoptotic pathways in hepatocellular carcinoma cell lines

The alteration of DNA cytosine methylation is one of the most common epigenetic changes that can play a significant role in human cancers. The enzymes involved in DNA methylation of promoter regions of the genes are DNA methyltransferases (DNMTs). The therapeutic activities and apoptotic effects of DNA methyltransferase inhibitors (DNMTIs) have been reported in various cancers. This study was assigned to assess the effect of zebularine on intrinsic and extrinsic pathways, DNAT 1, 3a, and 3b, p21, and p53, viability, and apoptosis in hepatocellular carcinoma (HCC) cell lines.Hepatocellular carcinoma cell lines (HCCLM3, MHCC97H, and MHCC97L) were purchased from the National Cell Bank of Iran, Pasteur Institute, treated with zebularine, and the MTT assay was performed. Then, flow cytometry assay and real-time RT-PCR analysis were performed with zebularine. Statistical comparisons between groups were made using GraphPad Prism software version 8.0. A significant difference was considered as P < 0.05.Zebularine up-regulated DR4, DR5, FAS, FAS-L, TRAIL, Bax, Bak, Bim, p21WAF/CIP1 (p21), and p53 and down-regulated DNMTs (DNAT 1, 3a, and 3b), Bcl-2, Bcl-xL, and Mcl-1, significantly resulting in apoptosis induction in HCC cell lines. Maximal and minimal apoptosis was seen in HCCLM3 and MHCC97L cell lines, respectively.Our findings indicated that DNMTI zebularine can induce apoptosis and inhibit cell growth through both pathways (extrinsic and intrinsic) in HCC cell lines HCCLM3, MHCC97H, and MHCC97L.

Epigenetic reprogramming in cloned mouse embryos following treatment with DNA methyltransferase and histone deacetylase inhibitors

We examined the effects of DNA methyltransferase inhibitor – RG108, and histone deacetylase inhibitor – SAHA, on the reprogramming parameters of cloned mouse embryos produced by somatic cell nuclear transfer into oocytes. The programming parameters studied included dynamics of histone reacetylation, developmental rate, DNA methylation, and transcript levels of genes, all of which are pivotal to lineage specification and blastocyst formation. At the pronuclear stage, somatic nucleus-transplanted oocytes treated with 5 µM SAHA presented higher histone acetylation at H3K9, H3K14, H4K16 and H4K12, compared to untreated clones (p < 0.05). At the morula stage, cloned embryos treated with 5 μM RG108 or 5 μM SAHA presented lower DNA methylation intensity compared to untreated clones (p < 0.05), resembling the intensity levels of fertilized embryos. However, these effects were not observed when RG108 and SAHA were used in combination. The rate of morula formation was significantly higher in cloned embryos treated with 5 µM SAHA than in untreated clones, whereas treatment with RG108 resulted in no obvious effects on morula formation rates. On the other hand, the combined treatment with RG108 and SAHA resulted in inferior rates of cloned morula formation, compared to untreated clones. At the blastocyst stage, the aberrant expression levels of key developmental genes Oct4 and Cdx2, but not Nanog, were corrected in cloned embryos by the treatment with RG108. This is similar to the intensity levels seen in fertilized embryos. The expression of Rpl7l1 gene was significantly higher in embryos treated with both RG108 and SAHA than in untreated and in control groups. In summary, the present study showed that SAHA and RG108, when applied separately, improve the rate and quality of cloned mouse embryos.

5-Aza-2'-deoxycytidine induces a greater inflammatory change, at the molecular levels, in normoxic than hypoxic tumor microenvironment.

Hypoxia is associated with tumor aggressiveness and poor prognosis, including breast cancer. Low oxygen levels induces global genomic hypomethylation and hypermethylation of specific loci in tumor cells. DNA methylation is a reversible epigenetic modification, usually associated with gene silencing, contributing to carcinogenesis and tumor progression. Since the effects of DNA methyltransferase inhibitor are context-dependent and as there is little data comparing their molecular effects in normoxic and hypoxic microenvironments in breast cancer, this study aimed to understand the gene expression profiles and molecular effects in response to treatment with DNA methyltransferase inhibitor in normoxia and hypoxia, using the breast cancer model. For this, a cDNA microarray was used to analyze the changes in the transcriptome upon treatment with DNA methyltransferase inhibitor (5-Aza-2'-deoxycytidine: 5-Aza-2'-dC), in normoxia and hypoxia. Furthermore, immunocytochemistry was performed to investigate the effect of 5-Aza-2'-dC on NF-κB/p65 inflammation regulator subcellular localization and expression, in normoxia and hypoxia conditions. We observed that proinflammatory pathways were upregulated by treatment with 5-Aza-2'-dC, in both conditions. However, treatment with 5-Aza-2'-dC in normoxia showed a greater amount of overexpressed proinflammatory pathways than 5-Aza-2'-dC in hypoxia. In this sense, we observed that the NF-κB expression increased only upon 5-Aza-2'-dC in normoxia. Moreover, nuclear staining for NF-κB and NF-κB target genes upregulation, IL1A and IL1B, were also observed after 5-Aza-2'-dC in normoxia. Our results suggest that 5-Aza-2'-dC induces a greater inflammatory change, at the molecular levels, in normoxic than hypoxic tumor microenvironment. These data may support further studies and expand the understanding of the DNA methyltransferase inhibitor effects in different tumor contexts.

Abstract PO-015: Hypermethylated genes in leiomyosarcoma can be targeted through guadecitabine treatment

Abstract Leiomyosarcomas (LMS) are aggressive mesenchymal tumors of smooth muscle origin. Understanding the molecular alterations associated with LMS is needed to identify potential therapeutic targets. Epigenetic alterations such as hypermethylation of gene promoter regions with associated transcriptional silencing is a hallmark of most cancers. Epigenetic reprograming is fundamentally linked to tumor initiation, progression and metastasis. Frequent epigenetic alterations have been observed in LMS. Epigenetic drugs such as DNA methyltransferase inhibitors (DNMTi) have been used to reverse aberrant DNA methylation and restore expression of silenced genes. Our previous studies using DNMTi, including guadecitabine, have shown that DNMTi treatment has anti-proliferative and pro-apoptotic effects in in vitro and in vivo models of LMS. Our goal was to characterize the methylation changes following guadecitabine treatment of the leiomyosarcoma cell line SK-UT1. SK-UT1 cells were treated with guadecitabine (0.05 µM-0.1 µM) and methylation was determined using Illumina 450K platform. We filtered the promoter CpG island regions that respond to guadecitabine treatment and were associated with decreased methylation compared to untreated SK-UT1 control (p≤0.05, Δβ≥0.15). In order to assess the relevance of these differentially methylated regions, we compared them to the methylation and expression profiles of LMS patient samples (GSE68312). We focused on the genes that have decreased expression and are associated with increased methylation of the promoter CpG island regions in the patient LMS samples. In conclusion, treatment of SK-UT1 cell line with guadecitabine resulted in decrease of methylation of a subset of promoter CpG island regions that were differentially hypermethylated in LMS patient samples. Further studies are needed to explore the effect of differential methylation on gene expression in guadecitabine treated SK-UT1 cell line. Understanding the effect of DNMTi on LMS is needed to identify potential therapeutic applications targeting the epigenetic pathways. Funding supported by Astex Pharmaceuticals. Citation Format: Nesrin Hasan, Cynthia De Carvalho Fischer, Anup Sharma, Nita Ahuja. Hypermethylated genes in leiomyosarcoma can be targeted through guadecitabine treatment [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-015.

Abstract 3892: Targeting Hippo-dependent and Hippo-independent regulation of the YAP1 oncoprotein in childhood rhabdomyosarcoma

Abstract Background: Patients with metastatic or recurrent rhabdomyosarcoma (RMS), the most common childhood soft tissue sarcoma, continue to do poorly and new treatments are urgently needed. Previously, we showed that the Src family tyrosine kinase YES1 is upregulated in RMS and necessary for growth, but clinical trials using single agent Dasatinib (Src family kinase inhibitor) have failed in sarcomas. YAP1 (YES-associated protein) is also highly expressed in RMS, driving growth and survival when the upstream Hippo tumor suppressor pathway is silenced. Thus far, efforts to pharmacologically inhibit YAP1 have been unsuccessful. We hypothesize that a better understanding of the Hippo-dependent and Hippo-independent (through YES1) regulation of YAP1 in RMS may lead to novel therapeutic approaches. Methods: We studied the effect of DNA methyltransferase inhibitors (DNMTi) on cell growth in human RMS cell lines using live cell imaging and analyzed the YES1-YAP1 interaction by proximity ligation assays. We analyzed YES1 and YAP1 signaling using cell fractionation, immunoblots, qPCR, RNAseq, and genetic loss-of-function. We evaluated changes in DNA methylation by methylation-specific PCR and the Illumina EPIC methylation profiling microarray. To evaluate drug treatments in vivo we utilized orthotopic xenografts in immunocompromised mice. Results: We demonstrate that DNMTi treatment decreases RMS cell growth, and increases apoptosis and myogenic differentiation. DNMTi treatment upregulates Hippo-activators RASSF1 and RASSF5 by promoter demethylation, activates canonical Hippo signaling through LATS1/2, and increases inactivation of YAP1 by phosphorylation. The DNMTi-induced growth inhibition is partially rescued by expression of constitutively active YAPS127A, suggesting the effects of DNMTi treatment are in part due to Hippo-dependent inhibition of YAP1. Additionally, YES1 and YAP1 interact in the nucleus of RMS cells and YES1 shRNA knockdown or pharmacologic inhibition results in YAP1 cytoplasmic retention and decreased YAP1 target gene expression, suggesting YES1 can regulate YAP1 in a Hippo-independent manner. Lastly, to target both Hippo-dependent and Hippo-independent regulations of YAP1 we combined DNMTi treatment with Dasatinib. Combination treatment in vitro ablates RMS cell growth and induces more apoptosis than either drug alone. Combination treatment in orthotopic xenografts in mice results in decreased tumor weight, a trend towards decreased tumor growth, and an increase in myogenic differentiation gene expression. Conclusions: In RMS, YAP1 is regulated by Hippo-dependent and Hippo-independent mechanisms and both can be inhibited by combined treatment of a DNMTi with Dasatinib, laying the groundwork for future clinical investigations of this combination. Citation Format: Katherine Slemmons, Choh Yeung, Josh Baumgart, Jhazeel Martinez Juarez, Amy McCalla, Lee Helman. Targeting Hippo-dependent and Hippo-independent regulation of the YAP1 oncoprotein in childhood rhabdomyosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3892.

Abstract A09: Targeting Hippo-dependent and Hippo-independent regulation of the YAP1 oncoprotein in childhood rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. While outcomes for patients with localized disease have improved, patients with metastatic or recurrent disease continue to do poorly; therefore, new treatments are needed. Previously, we showed that the Src family tyrosine kinase YES1 is upregulated in RMS and necessary for growth, but clinical trials using single-agent dasatinib (Src family kinase inhibitor) have not shown clinical activity in sarcomas. YAP1 (YES-associated protein) is also highly expressed in human RMS tumors, driving cell growth and survival. Thus far, efforts to pharmacologically inhibit YAP1 have been unsuccessful. We hypothesize that a better understanding of the Hippo-dependent and Hippo-independent (through YES1) regulation of YAP1 in RMS may lead to novel therapeutic approaches. Methods: We studied the effect of DNA methyltransferase inhibitors (DNMTi) on cell growth in human RMS cell lines using live cell imaging and analyzed the YES-YAP interaction by proximity ligation assays. We analyzed YES1 and YAP1 signaling with cell fractionation, immunoblots, methylation-specific PCR, qPCR, RNAseq, and genetic loss of function. Results: We demonstrate that DNMTi treatment decreases RMS cell growth and increases apoptosis and differentiation. DNMTi treatment activates canonical Hippo signaling through LATS, shown by a LATS activity biosensor, and increases YAP1 phosphorylation. DNMTi treatment also induces RASSF1 re-expression by promoter demethylation, and the DNMTi-induced growth inhibition can be partially rescued by expression of constitutively active YAPS127A. RNAseq analysis after DNMTi treatment also revealed upregulation of RASSF family members RASSF5 and RASSF6 and downregulation of RASSF4. RASSF4 was previously shown to inhibit MST1 and promote tumorigenesis in the alveolar subtype of RMS. These data suggest the effects of DNMTi treatment are in part due to inhibition of YAP1 in a Hippo-dependent manner. DNMTi treatment also decreases YES1 expression and activation, suggesting DNMTis impact both YES1 and YAP1 activity. Furthermore, YES1 and YAP1 interact in the nucleus of RMS cells and shRNA knockdown of YES1 results in YAP1 cytoplasmic retention and decreased expression of YAP1 target genes, suggesting a role for YES1 in regulating YAP1 cellular localization in a Hippo-independent manner. Lastly, to target both Hippo-dependent and Hippo-independent regulation of YAP1 we combine DNMTi treatment with dasatinib, a YES1 kinase inhibitor. Combination treatment in vitro ablates RMS cell growth and leads to a greater induction of apoptosis than either drug alone. We are currently evaluating this combination strategy in RMS xenografts in mice. Conclusions: Overall, we show that YAP1 is regulated in both Hippo-dependent and Hippo-independent mechanisms in RMS and both mechanisms can be inhibited by a combined treatment of DNMTi and dasatinib, laying the groundwork for future clinical investigations of this novel therapy. Citation Format: Katherine K. Slemmons, Choh Yeung, Josh Baumgart, Amy McCalla, Lee Helman. Targeting Hippo-dependent and Hippo-independent regulation of the YAP1 oncoprotein in childhood rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A09.

Prostaglandin E2 Reverses the Effects of DNA Methyltransferase Inhibitor and TGFB1 on the Conversion of Naive T Cells to iTregs

Naturally occurring regulatory T cells (nTregs) are produced under thymic (tTregs) or peripherally induced (pTregs) conditions in vivo. On the other hand, Tregs generated from naive T cells in vitro under some circumstances, such as treatment with transforming growth factor-β (TGFB), are called induced Tregs (iTregs). Tregs are especially characterized by FOXP3 expression, which is mainly controlled by DNA methylation. nTregs play important roles in the suppression of immune response and self-tolerance. The prostaglandin E2 (PGE2) pathway was reported to contribute to regulatory functions of tumor-infiltrating nTregs. In this study, we examined whether PGE2 contributes to the formation of iTregs treated with TGFB1 and 5-aza-2′-deoxycytidine (5-aza-dC), which is a DNA methyltransferase inhibitor. We found that the protein and gene expression levels of FOXP3 and IL-10 were increased in 5-aza-dC and TGFB1-treated T cells in vitro. However, the addition of PGE2 to these cells reversed these increments significantly. In CFSE-based cell suppression assays, we demonstrated that PGE2 decreased the suppressive functions of 5-aza-dC and TGFB1-treated T cells.

Oral Vitamin C Supplementation to Azacitidine in Patients with Myeloid Cancer: Normalization of Plasma Vitamin C Induces Epigenetic Changes

▪IntroductionHematological cancer patients are often vitamin C deficient, and vitamin C is essential for the TET induced conversion of 5-methylcytosine (5mC) to 5-hydroxymethylsytosine (5hmC); the first step in active DNA demethylation. In tissue culture, we have shown that restoration of vitamin C to normal concentrations can potentiate the effect of DNA methyltransferase inhibitors (DNMTis) by activation of DNA demethylation and induction of genes in the viral defense pathway, so called ‘viral mimicry’ (Liu et al. PNAS 2016). Here, we investigated whether oral vitamin C supplementation can correct vitamin C deficiency, enhance the efficacy of active DNA demethylation and induce upregulation of genes in the viral defense pathway in patients with myeloid cancers treated with the DNMTi 5-azacytidine.Study Design and MethodsA randomized, placebo-controlled clinical trial of myelodysplastic syndrome (MDS; n=9), chronic myelomonocytic leukemia (CMML; n=4) and acute myeloid leukemia (AML; n=7) patients was performed during 3 cycles/12 weeks of DNMTi treatment (5-azacytidine, 100mg/m2 day 1 to 5 in a 4-week cycle) supplemented by oral dose of 500mg vitamin C (n=10) or placebo (n=10) daily during the last 8 weeks (Figure 1). Blood samples were drawn on day 1 and 5 of each treatment cycle before 5-azacytidine was administered and on day 28 of the third treatment cycle. Total plasma vitamin C was measured by HPLC in samples that had been acidified by 10% meta-phosphoric acid immediately after blood drawn. Mutational status of the 20 most commonly mutated genes in MDS were conducted by targeted next-generation sequencing. Global levels of 5mC and 5hmC were measured with LC-MS/MS in DNA extracted from MACS-sorted malignant cells and quoted relative to total levels of deoxyguanine. Total RNA-seq was performed on 20 RNA samples from 6 patients; cDNA libraries were prepared using KAPA RNA HyperPrep Kits and sequencing on a NextSeq 500 instrument (Illumina).ResultsFourteen patients were deficient in plasma vitamin C (<23 µM) and 4 of the remaining 6 patients took vitamin supplement at inclusion. Global DNA methylation was significantly higher in patients with severe vitamin C deficiency (<11.4 µM; P=0.004) and borderline significantly higher in DNMTi naïve (n=11) compared to non-naïve patients (P=0.095). At baseline, global 5hmC/5mC levels were lower in the 7 patients with TET2 mutations (n=7; P= 0.013). Oral supplementation restored plasma vitamin C levels to the normal range in all patients in the vitamin C arm (P<0.0005). We show for the first time that active DNA demethylation, estimated as the change in global 5hmC/5mC levels, was significantly increased in patients receiving vitamin C compared to placebo (P=0.041). Preliminary RNA sequencing data show increased upregulation of genes involved in the viral defense pathway, including IRF7 and IFIT1, in vitamin C supplemented patients that were DNMTi naïve at study inclusion.ConclusionsThe increase in active DNA demethylation, indicated by the elevation of 5hmC/5mC levels in myeloid cells from vitamin C supplemented patients compared to placebos, plus the increased expression of viral defense genes in vitamin C treated DNMTi naïve patients, suggest that the efficacy of 5-azacytidine might be increased by oral supplementation of vitamin C. We suggest that normalization of plasma vitamin C by oral supplementation may enhance the biological effects of DNMTis in patients and prompts the investigation of the clinical relevance of vitamin C supplementation to DNMTis in a large randomized placebo controlled trial.Figure 1. Study design. Days 1, 5, and 28: before vitamin C/placebo exposure. Day 32: after short-term vitamin C/placebo exposure. Days 56, 60, and 84: after longer-term vitamin C/placebo exposure. C = cycle, D = day in cycle. [Display omitted] DisclosuresJones:ZYMO Corporation: Consultancy. Grønbæk:Celgene: Membership on an entity's Board of Directors or advisory committees; Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees; Janssen Pharma: Membership on an entity's Board of Directors or advisory committees.

The correlation between DNA methylation and transcriptional expression of human dopamine transporter in cell lines

This study aims to investigate the relationship between DNA methylation and expression of human dopamine transporter (hDAT). We examined methylation status of hDAT in cells with various hDAT expression levels, including two dopaminergic neural cell lines (SK-N-AS and SH-SY-5Y) and one non-dopaminergic cell line (HEK293) by bisulfite sequencing PCR(BSP). The effects of DNA methyltransferase inhibitor 5-aza-dC or/and histone deacetylase inhibitor (HDACi, sodium butyrate, NaB) on the DNA methylation status and mRNA expression levels of hDAT were examined. The results revealed marked hypomethylation of the two promoter regions (-1214 to -856bp and -48 to 439bp, the first base of exon 1 was taken as +1 bp)of hDAT in SK-N-AS (4.7%±2.0mC and 3.5%±1.0mC, respectively) compared with SH-SY-5Y (88.0%±4.4%mC and 81.1%±8.8%mC) and HEK293 (90.7%±2.4mC and 84.4%±8.6% mC) cell lines, indicating a cell-specific methylation regulation of hDAT. 5-aza-dC and NaB decreased hypermethylation,while increase hDAT expression in SH-SY-5Y cells and recovered hDAT mRNA expression in HEK293 cells. DNA methylation enabled the cell-specific differential expression of the hDAT gene. hDAT silencing was reversed by the introduction of DNA hypomethylation via 5-aza-dC or/and NaB.

Abstract 4045: Decreased Fanconi anemia gene expression contributes to efficacy of PARP and DNMT inhibitor combination therapy in triple negative breast cancer

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) have efficacy in a sub-set of triple negative breast cancers (TNBCs) with inherited mutations in DNA double strand break repair (DSBR) genes, such as the BRCA1/2 genes in homologous recombination (HR), through synthetic lethality. However, PARP inhibitors have failed for the majority of sporadic TNBCs with intact BRCA1 genes. Therefore, novel targeted therapies must be explored. We have recently reported that PARPi Talazoparib in combination with DNA methyl transferase inhibitors (DNMTi)s azacytidine (AZA) or decitabine (DAC) have efficacy in sporadic TNBCs in vitro and in vivo. PARPi act not only to catalytically inhibit PARP but also to trap PARP at DNA single strand breaks (SSB), leading to suicidal DNA-protein crosslinks (DPC). We also showed that Talazoparib in combination with AZA/DAC increases PARP trapping in DNA, leading to increased and persistent levels of lethal DSBs, suggesting that DSBR may also be impaired with this treatment. HR plays an important role in resolving DPC in mammalian cells and Fanconi anemia (FA)-dependent HR has been previously shown to resolve AZA-induced replication lesions. Herein, we determined whether HR is impaired with combination drug treatment using a chromosomally integrated GFP-based reporter in the TNBC cell line, MDA-MB-231. HR activity was significantly (p&amp;lt;0.05) decreased with AZA or AZA/Talazoparib combination treatment, suggesting that AZA mediates decreased HR activity. We next questioned whether subsets of HR genes are indirectly down-regulated by the epigenetic reprogramming effects of DNMTis, thereby contributing to the efficacy of DNMTi and PARPi combination therapy. Microarray analysis was performed in multiple TNBC cell lines (including MDA-MB-231, MDA-MB-468, and SUM159PT) post DNMTi treatment and showed significant (p&amp;lt;0.05) decreases in expression of FANCD2, FANCC, and FANCE that could potentially generate a synthetic lethality when combined with PARPi, as has recently been reported. Decreased FA gene expression was validated by qPCR of mRNA and western analysis of proteins. Moreover, treatment of TNBC with DNA crosslinking agents, which require FA-dependent repair, have increased sensitivity post DNMTi treatment. These data suggest that decreased FA gene expression contributes to the efficacy of PARPi/DNMTi treatment in TNBC. Work is now underway to determine whether depletion of FA proteins can increase PARP trapping in PARPis treated TNBCs, leading to increased levels of cytotoxic DSBs. Citation Format: Lena J. McLaughlin, Huili Li, Pratik Nagaria, Stephen B. Baylin, Cynthia A. Zahnow, Feyruz V. Rassool. Decreased Fanconi anemia gene expression contributes to efficacy of PARP and DNMT inhibitor combination therapy in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4045. doi:10.1158/1538-7445.AM2017-4045

Effect of DNMT inhibitor on bovine parthenogenetic embryo development

DNA methylation catalyzed by DNA methyltransferase (DNMT) family plays an important role during mammal preimplanted embryo development. However, the effects of RG108, a DNMT inhibitor (DNMTi), on DNMT in the development of bovine preimplanted embryos are not fully elucidated. In this study, we investigated the role of RG108 on the development, dynamics of gene-specific DNA methylation and transcription of bovine parthenogenetic preimplantation embryos. We found that Dnmt1 and Dnmt3b showed highly transcription in parthenogenetic 2-cell embryos, and then the transcription levels decreased during the following development stages, whereas Dnmt3a was always maintained at a lower transcription level during bovine parthenogenetic preimplantation embryo development. Treatment with RG108 blocked the development of bovine parthenogenetic preimplantation embryos and induced hypomethylation in the embryos. RG108 decreased the methylation level of the Nanog gene promoter region, which caused activation of the Nanog gene in 8-cell embryos and increased the transcription level. RG108 also induced the hypomethylation of the repeat elements (satellite I and α-satellite), which may cause genome instability, increasing the number of apoptotic cells in the blastocysts and also the transcription level of the apoptotic gene Bax. These results indicate that RG108, a DNMT inhibitor (DNMTi), inhibits the development of bovine parthenogenetic preimplantation embryos, suggesting that the DNMT is necessary for bovine parthenogenetic preimplanatation embryo development.

DNA methyltransferase inhibition accelerates the immunomodulation and migration of human mesenchymal stem cells.

DNA methyltransferase (DNMT) inhibitors regulate target gene expression through epigenetic modifications, and these compounds have primarily been studied for cancer therapy or reprogramming. However, the effect of DNMT inhibitors on the immunomodulatory capacity of human mesenchymal stem cells (hMSCs) has not been investigated. In the present study, we treated hMSCs with 5-azacytidine (5-aza), a DNMT inhibitor, and confirmed that the inhibitory effects on mononuclear cell proliferation and cell migration toward activated T cells were increased. To identify the immunomodulatory factors stimulated through 5-aza treatment, we investigated the changes in promoter methylation patterns using methylation arrays and observed that the promoters of immunomodulatory factors, COX2 and PTGES, and migration-related factors, CXCR2 and CXCR4, were hypomethylated after 5-aza treatment. In addition, we observed that the COX2-PGE2 pathway is one of the main pathways for the enhanced immunosuppressive activity of hMSCs through 5-aza treatment. We also determined that the migration of hMSCs toward ligands for CXCR2/CXCR4 was increased after 5-aza treatment. Moreover, using an experimental colitis model, we showed that 5-aza pre-treatment could enhance the therapeutic effect of MSCs against immune-related diseases.

Tet1-mediated DNA demethylation regulates neuronal cell death induced by oxidative stress.

Epigenetic regulations including DNA methylation and demethylation play critical roles in neural development. However, whether DNA methylation and demethylation may play a role in neuronal cell death remains largely unclear. Here we report that the blockade of DNA methyltransferase inhibits apoptosis of cerebellar granule cells and cortical neurons in response to oxidative stress. We found that knockdown of ten-eleven translocation methylcytosine dioxygenase (Tet1), a critical enzyme for DNA demethylation, significantly increase apoptosis of cerebellar granule cells induced by hydrogen peroxide. Moreover, cerebellar granule cells from tet1null mice appeared to be more sensitive to oxidative stress, suggesting the critical role of Tet1 in neuronal cell death. We further showed that the expression of Klotho, an antiaging protein, in cerebellar granule cells is tightly regulated by DNA methylation. Finally, we found that knockdown of Klotho diminished the rescue effects of DNA methyltransferase inhibitors and Tet1 on neuronal cell death induced by oxidative stress. Our work revealed the role of Tet1-mediated DNA demethylation on neuronal protection against oxidative stress and provided the molecular mechanisms underlying the epigenetic regulation of neuronal cell death, suggesting the role of Klotho in regulating neuronal cell death in response to oxidative stress.

Processes of DNA methylation are involved in the mechanisms of amnesia induction and conditioned food aversion memory reconsolidation.

We studied the role of DNA methylation in the mechanisms of amnesia in edible snails, which was induced by impairment of conditioned food aversion memory reconsolidation with NMDA glutamate receptor antagonist. The effects of DNA methyltransferase inhibitors were shown to depend on the stage of amnesia. At the early stage of amnesia (day 3 after induction), injections of methyltransferase inhibitors in combination with conditioned food stimulus (reminder) were followed by memory recovery. Application of inhibitors in the absence of the reminder was ineffective. Methyltransferase inhibitors were ineffective at the late stage of amnesia (day 10). Our results suggest that the presentation of reminding conditioned stimuli is followed by reactivation of amnesia. Methylation or demethylation of DNA in nerve cells serves as one of the key mechanisms for amnesia.

Influence Of DNA Methyltransferese Inhibitors On The Anti-Leukemic Effect Of Umbilical Cord Blood Derived NK Cells Against Acute Myeloid Leukemia

Natural killer (NK) cell-based immunotherapy is a promising adjuvant, relatively non-toxic therapy approach for AML. However, further improvement of NK cell-based therapy is needed to increase the clinical effect. In this regard, NK cells generated ex vivo from hematopoietic progenitor cells (HPC) may have significant clinical benefits over enriched NK cells from adult donors, including the ability to choose an appropriate killer-cell immunoglobuline-like receptor (KIR)-ligand or KIR B haplotype alloreactive donor, as well as the capacity to reach high therapeutic dosages. Previously, we reported a GMP-compliant, cytokine/heparin-based culture protocol for the ex vivo generation of highly active NK cells from CD34+ HPC isolated from cryopreserved umbilical cord blood (UCB) units. Expansion in closed, large-scale bioreactors yields a clinically relevant dose of NK cells with high purity and cytolytic activity against AML cells in vitro. Currently, a clinical phase I trial with these HPC-NK cells is ongoing in our hospital. Trafficking studies in NOD/SCID/IL2Rgnull (NSG) mice demonstrated that these HPC-NK cells migrate to the bone marrow (BM) as well as to lymphoid organs where in vivo expansion and maturation can take place. Analysis of the chemokine receptor expression profile of UCB-NK cells matched in vivo findings. Particularly, a firm proportion of UCB-NK cells functionally expressed CXCR4, what could trigger BM homing in response to its ligand CXCL12. In addition, high expression of CXCR3 and CCR6 supported the capacity of UCB-NK cells to migrate to inflamed tissues via the CXCR3/CXCL10-11 and CCR6/CCL20 axis. Importantly, a single HPC-NK cell infusion combined with supportive IL-15 administration was shown to efficiently inhibit growth of K562 leukemia cells implanted in the femur of NSG mice, resulting in significant prolongation of mice survival. Furthermore, we investigated whether modulation by the DNA methyltransferase (DNMT) inhibitors Azacytidine (Aza) and Decitabine (Deci) could further potentiate the antileukemic effect of HPC-NK cells against AML cells. In concordance with previous reports, we observed a dose-dependent effect of Aza and Deci on the growth of the AML cell lines THP1 and KG1a. In subsequent NK cell killing assays, we used clinical relevant low drug concentrations to pre-treat AML cells that did not affect HPC-NK cell viability and cytolytic function. Interestingly, increased killing of pre-treated THP1 and KG1a cells by HPC-NK cells could be observed, which was correlated with an increase in the NKG2D ligand ULBP2, the DNAM-1 ligands CD112 and CD155 as well as TRAIL-R2. Notably, maintenance of low-dose DNMT inhibitors during the KG1a/NK co-culture resulted in pronounced AML growth inhibition. To examine the effect of DNMT inhibitors in vivo, THP1.LucGFP-bearing NSG mice were treated with increasing dose of both agents, which were administered according to current standard protocols applied in humans. Data indicated that treatment with Aza or Deci at dosage equivalent in human to 12.5 and 5 mg/m2 respectively was well tolerated with minimal and/or transient weight loss, and efficiently reduced the progression of THP-1.LucGFP cells in vivo. Currently, we explore whether HPC-NK cells and DNMT inhibitors can work together to combat AML in our xenograft models. These preclinical studies may provide a rationale to investigate the possible additive and/or synergistic anti-AML effects of adoptive HPC-NK cell transfer in combination with these DNMT inhibitors in AML patients. Disclosures: Tordoir: Glycostem Therapeutics: Employment. Spanholtz:Glycostem Therapeutics: Employment.

Genome-wide effects of DNA methyltransferase inhibitor on gene expression in double-stranded RNA transfected porcine PK15 cells

Double-stranded RNA (dsRNA) is produced in host cells during viral replication. The effects of DNA demethylation on gene expression in dsRNA transfected swine cells are unclear. The study aims to profile the transcriptome changes which are induced by DNA methyltransferase inhibitor (Aza-CdR) in porcine PK15 cells transfected with viral-like dsRNA (Poly(I:C)). A total of 44, 76 and 952 differentially expressed genes (DEGs) were detected in the cells treated by Poly(I:C) plus Aza-CdR (P+A), Poly(I:C) (P) or Aza-CdR (A) alone compared to the controls (C). Immune response-related pathways are observed in the comparison of A vs. C and P vs. C, and the genes in the pathways were recovered in the comparison of (P+A) vs. C. GO analysis indicated that Aza-CdR has negative regulatory effects on viral reproduction. The results suggest that the stimulant of Poly(I:C) could be regressed by Aza-CdR. These observations provide new insights into the epigenetic regulatory effects on viral replication.

Abstract 4624: Identification of novel DNA methylation markers to track patient's response to DNA demethylation agents.

Abstract The successful use of DNA methyltransferase inhibitors (DNMTis) in Myelodysplastic Syndromes (MDS) therapy has brought epigenetic drugs to the forefront of cancer management. However it has been difficult to find a consistent association between patients’ outcome and DNA methylation changes. This could be attributed to a variety of factors including the choice of DNA methylation markers to track the response to treatment. LINE-1 methylation changes are widely studied as a marker predictive of genome-wide DNA methylation changes; however recent reports have shown that its methylation levels are variable across tissues. In addition, we have found that LINE-1 remethylation after DNMTis withdrawal occurs faster than that of other regions, suggesting that LINE-1 methylation changes might not reflect the overall demethylation effects of DNMTis. Thus, it is imperative to find more sensitive DNA methylation markers to accurately track the methylation change that occurs after DNMTi treatment. Ideally, such markers could be applied to various tumor types and to samples collected by invasive and non-invasive methods. To find improved DNA methylation markers, we took advantage of the well-established Infinium DNA methylation platform and found 1429 probes, which were consistently methylated in both normal/tumor bladder tissue samples as well as white blood cells from healthy donors. The remethylation pattern of those 1439 probes was investigated in the T24 bladder cancer and HL60 leukemia cell lines treated with 5-Aza-CdR for 24 hours. Probe consensus clustering yielded a group of 79 probes that significantly responded to the demethylation treatment and remained demethylated beyond 30 days. In silico analysis of the DNA methylation patterns of the top two probes show consistent hypermethylation in both normal and tumor samples. As a proof of principle, we tested the DNA demethylation levels of these two markers in urine sediments from 7 MDS patients treated with Azacitidine using pyrosequencing. Our results showed that the two markers were significantly demethylated; in contrast, LINE-1 methylation showed no clear decreasing trend. Demethylation of these two markers was also observed in peripheral blood samples from MDS patients treated with Azacitidine. We are in the process of testing more samples to find the association between marker demethylation and patient's outcome. In summary, we have identified and validated two DNA methylation markers, which unlike LINE-1, show consistent demethylation in response to the DNMTi treatment irrespective of the type of sample tested. The wider range of demethylation provided by these markers may offer a more accurate representation of the patient's response to treatment. Citation Format: Xiaojing Yang, Han Han, Marianne B. Treppendahl, Yvonne C. Tsai, Casey O'Connell, Dan Weisenberger, Fides Lay, Kirsten Grønbæk, Gangning Liang, Peter A. Jones. Identification of novel DNA methylation markers to track patient's response to DNA demethylation agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4624. doi:10.1158/1538-7445.AM2013-4624

5-Aza-2′-deoxycytidine inhibited PDGF-induced rat airway smooth muscle cell phenotypic switching

Airway smooth muscle (ASM) cell phenotypic switching played an important role in airway remodeling in asthma. In vitro platelet-derived growth factor (PDGF) induced ASM cell phenotypic switching from a mature to pro-remodeling phenotype, but the mechanism remained incompletely understood. This study was to explore the effect of DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (Aza-CdR) on PDGF-induced rat ASM cell phenotypic switching and biological behaviors. Rat airway smooth muscle (RASM) cells were obtained by primary explant techniques. Western blot, 3-dimensional gel contraction, transwell and wound healing assay, and MTT were applied to detect cell phenotypic switching, contractility, migration and proliferation, respectively. Cytoskeleton rearrangement was observed by immunofluorescence. Results showed Aza-CdR inhibited PDGF-induced down-regulation of contractile markers in RASM cells and increased cell contractility. Aza-CdR inhibited PDGF-induced RASM cell migration by abrogating cell morphology change and cytoskeletal reorganization and attenuated the effect of PDGF on proliferating cell nuclear antigen expression and cell cycle progression, ultimately cell proliferation. PDGF-induced DNA methyltransferase 1 (DNMT1) expression was mediated by activation of PI3K/Akt and ERK signaling in RASM cells. Selective depletion of DNMT1 protein by Aza-CdR inhibited PDGF-induced RASM cell phenotypic switching, revealing DNMT1-mediated DNA methylation was implicated in asthmatic ASM remodeling. We proposed for the first time that DNMT1 played a key role in PDGF-induced RASM cell phenotypic switching and Aza-CdR is promising in intervening ASM remodeling in asthma. Although study of abnormal DNA methylation in PDGF-stimulated ASM cells is in its infancy, this work contributes to providing new insights into the mechanism of ASM remodeling and may be helpful for developing effective treatments for airway remodeling in asthma.

The effects of 5-azacytidine on the function and number of regulatory T cells and T-effectors in myelodysplastic syndrome

Expansion of regulatory T cells occurs in high-risk myelodysplastic syndrome and correlates with a poor prognosis. DNA methyltransferase inhibitors, particularly 5-azacytidine, have been shown to increase the survival of patients with high-risk myelodysplastic syndrome. It is not entirely clear whether this improvement in patients' survival is related to the effects of DNA methyltransferase inhibitors on the immune system and/or the direct effect of these drugs on the dysplastic clone. In this study we investigated the effect of 5-azacytidine on the function and proliferation capability of regulatory T cells and T-helper cells. The number and function of CD4(+) T-cell subsets in 68 patients with intermediate-2/high-risk myelodysplastic syndrome were serially assessed at diagnosis and following treatment. The in-vitro effects of 5-azacytidine on CD4(+) T-cell subsets isolated from both healthy donors and patients with myelodysplastic syndrome were also investigated. The number of peripheral blood regulatory T cells was significantly higher in myelodysplastic syndrome patients than in healthy donors and responders to treatment (P=0.01). The absolute numbers of T-helper 1 and T-helper 2, but not T-helper 17, cells were significantly reduced following 12 months of treatment (P=0.03, P=0.03). The in vitro addition of 5-azacytidine to CD4(+) T cells reduced the proliferative capacity of regulatory T cells (P=0.03). In addition, the 5-azacytidine-treated regulatory T cells had reduced suppressive function and produced larger amounts of interleukin-17. The FOXP3 expression in 5-azacyti-dine-treated T-effectors was also increased. Interestingly, these FOXP3(+)/interleukin-17(+) cells originated mainly from effector T cells rather than regulatory T cells. Our data suggest that 5-azacytidine has profound effects on CD4(+) T cells, which correlate with disease status after treatment. Furthermore, despite the demethylation of the FOXP3 promoter and increased FOXP3 expression following 5-azacytidine treatment, these phenotypic regulatory T cell-like cells lack the regulatory function and cytokine profile of regulatory T cells. These findings are important in correlating the clinically relevant immunomodulatory effects of 5-azacytidine.