Targeting Hippo-Dependent and Hippo-Independent YAP1 Signaling for the Treatment of Childhood Rhabdomyosarcoma.

Rhabdomyosarcoma is the most common childhood soft-tissue sarcoma, yet patients with metastatic or recurrent disease continue to do poorly, indicating a need for new treatments. The SRC family tyrosine kinase YES1 is upregulated in rhabdomyosarcoma and is necessary for growth, but clinical trials using single agent dasatinib, a SRC family kinase inhibitor, have failed in sarcomas. YAP1 (YES-associated protein) is highly expressed in rhabdomyosarcoma, driving growth and survival when the upstream Hippo tumor suppressor pathway is silenced, but efforts to pharmacologically inhibit YAP1 have been unsuccessful. Here we demonstrate that treatment of rhabdomyosarcoma with DNA methyltransferase inhibitor (DNMTi) upregulates Hippo activators RASSF1 and RASSF5 by promoter demethylation, activating canonical Hippo signaling and increasing inactivation of YAP1 by phosphorylation. Treatment with DNMTi decreased rhabdomyosarcoma cell growth and increased apoptosis and differentiation, an effect partially rescued by expression of constitutively active YAP (S127A), suggesting the effects of DNMTi treatment are, in part, due to Hippo-dependent inhibition of YAP1. In addition, YES1 and YAP1 interacted in the nucleus of rhabdomyosarcoma cells, and genetic or pharmacologic suppression of YES1 resulted in cytoplasmic retention of YAP1 and decreased YAP1 target gene expression, suggesting YES1 regulates YAP1 in a Hippo-independent manner. Combined treatment with DNMTi and dasatinib targeted both Hippo-dependent and Hippo-independent regulation of YAP1, ablating rhabdomyosarcoma cell growth in vitro and trending toward decreased tumor growth in vivo. These results show that the mechanisms regulating YAP1 in rhabdomyosarcoma can be inhibited by combinatorial therapy of DNMTi and dasatinib, laying the groundwork for future clinical investigations. SIGNIFICANCE: This study elucidates the signaling pathways that regulate the oncogenic protein YAP1 and identifies a combination therapy to target these pathways in the childhood tumor rhabdomyosarcoma.

Integrative Epigenetic Analysis Reveals Therapeutic Targets to the DNA Methyltransferase Inhibitor Guadecitabine (SGI-110) in Hepatocellular Carcinoma.

There is an urgent need to develop more effective therapies for hepatocellular carcinoma (HCC) because of its aggressiveness. Guadecitabine (SGI-110) is a second-generation DNA methyltransferase inhibitor (DNMTi), which is currently in clinical trials for HCC and shows greater stability and performance over first-generation DNMTis. In order to identify potential therapeutic targets of SGI-110 for clinical trials, HCC cell lines (SNU398, HepG2, and SNU475) were used to evaluate the effects of transient SGI-110 treatment by an integrative analysis of DNA methylation, nucleosome accessibility, gene expression profiles, and its clinical relevance by comparison to The Cancer Genome Atlas (TCGA) HCC clinical data. Each HCC cell line represents a different DNA methylation subtype of primary HCC tumors based on TCGA data. After SGI-110 treatment, all cell lines were sensitive to SGI-110 with prolonged antiproliferation effects. Expression of up-regulated genes, including tumor suppressors, was positively correlated with nucleosome accessibility and negatively correlated with gene promoter DNA methylation. Alternatively, expression of down-regulated genes, such as oncogenes, was negatively correlated with nucleosome accessibility and positively correlated with gene body DNA methylation. SGI-110 can also act as a dual inhibitor to down-regulate polycomb repressive complex 2 (PRC2) genes by demethylating their gene bodies, resulting in reactivation of PRC2 repressed genes without involvement of DNA methylation. Furthermore, it can up-regulate endogenous retroviruses to reactivate immune pathways. Finally, about 48% of frequently altered genes in primary HCC tumors can be reversed by SGI-110 treatment. CONCLUSION: Our integrative analysis has successfully linked the antitumor effects of SGI-110 to detailed epigenetic alterations in HCC cells, identified potential therapeutic targets, and provided a rationale for combination treatments of SGI-110 with immune checkpoint therapies.

Combined epigenetic and differentiation-based treatment inhibits neuroblastoma tumor growth and links HIF2α to tumor suppression.

Neuroblastoma is a pediatric cancer characterized by variable outcomes ranging from spontaneous regression to life-threatening progression. High-risk neuroblastoma patients receive myeloablative chemotherapy with hematopoietic stem-cell transplant followed by adjuvant retinoid differentiation treatment. However, the overall survival remains low; hence, there is an urgent need for alternative therapeutic approaches. One feature of high-risk neuroblastoma is the high level of DNA methylation of putative tumor suppressors. Combining the reversibility of DNA methylation with the differentiation-promoting activity of retinoic acid (RA) could provide an alternative strategy to treat high-risk neuroblastoma. Here we show that treatment with the DNA-demethylating drug 5-Aza-deoxycytidine (AZA) restores high-risk neuroblastoma sensitivity to RA. Combined systemic distribution of AZA and RA impedes tumor growth and prolongs survival. Genome-wide analysis of treated tumors reveals that this combined treatment rapidly induces a HIF2α-associated hypoxia-like transcriptional response followed by an increase in neuronal gene expression and a decrease in cell-cycle gene expression. A small-molecule inhibitor of HIF2α activity diminishes the tumor response to AZA+RA treatment, indicating that the increase in HIF2α levels is a key component in tumor response to AZA+RA. The link between increased HIF2α levels and inhibited tumor growth is reflected in large neuroblastoma patient datasets. Therein, high levels of HIF2α, but not HIF1α, significantly correlate with expression of neuronal differentiation genes and better prognosis but negatively correlate with key features of high-risk tumors, such as MYCN amplification. Thus, contrary to previous studies, our findings indicate an unanticipated tumor-suppressive role for HIF2α in neuroblastoma.

Synergistic inhibitory effects of deferasirox in combination with decitabine on leukemia cell lines SKM-1, THP-1, and K-562.

A multi-center study from the French Myelodysplastic Syndrome (MDS) Group confirmed that iron chelation therapy is an independent prognostic factor that can increase the survival rate of patients who are suffering from transfusion-dependent low-risk MDS. In this study, we aimed to explore this clinical phenomena in vitro, by exploring the synergistic effect of the iron chelator Deferasirox (DFX) and the DNA methyl transferase inhibitor Decitabine (DAC) in the leukemia cell lines SKM-1, THP-1, and K-562. Treatment with both DFX or DAC promoted apoptosis, induced cell cycle arrest, and inhibited proliferation in all three of these cell lines. The combination of DFX and DAC was much greater than the effect of using either drug alone. DFX showed a synergistic effect with DAC on cell apoptosis in all three cell lines and on cell cycle arrest at the G0/G1 phase in K-562 cells. DFX decreased the ROS levels to varying degrees. In contrast, DAC increased ROS levels and an increase in ROS was also noted when the two drugs were used in combination. Treatment of cells with DAC induced re-expression of ABAT, APAF-1, FADD, HJV, and SMPD3, presumably through demethylation. However the combination of DAC and DFX just had strong synergistic effect on the re-expression of HJV.

Chronic myelomonocytic leukemia with double-mutations in DNMT3A and FLT3-ITD treated with decitabine and sorafenib.

Chronic myelomonocytic leukemia (CMML) is a heterogeneous neoplastic hematologic disorder with worse overall survival. Half of CMML have mutations, but case with concomitant mutations of DNA methyltransferase 3A (DNMT3A) and Internal tandem duplications of the juxtamembrane domain of FLT3 (FLT3-ITD) in CMML was not reported before. We reported a 51-year-old man who had CMML with concomitant mutations in DNMT3A and FLT3-ITD.The patient received decitabine and sorafenib combined treatment. In this report, we reviewed DNMT3A mutation and FLT3 mutation, and we reviewed treatment of decitabine and sorafenib. This report is significant. First: This is the first report on CMML with double-mutations of DNMT3A and FLT3-ITD. Second: It shows the importance of targeted drug in combined treatment of CMML.

The Yin-Yang Dynamics of DNA Methylation Is the Key Regulator for Smooth Muscle Cell Phenotype Switch and Vascular Remodeling.

OBJECTIVE: DNA methylation plays an important role in chronic diseases such as atherosclerosis, yet the mechanisms are poorly understood. The objective of our study is to indicate the regulatory mechanisms of DNA methylation in vascular smooth muscle cells (VSMCs) and its roles in atherosclerosis. APPROACH AND RESULTS: In ApoE-/- mice fed a Western diet, DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, significantly attenuated atherosclerotic lesions (20.1±2.2% versus 30.8±7.5%; P=0.016) and suppressed DNA methyltransferase activity and concomitantly decreased global 5-methylcytosine content in atherosclerotic lesions of ApoE-/- mice. Using a carotid ligation model, we found that 5-aza-2'-deoxycytidine also dramatically inhibited neointimal formation (intimal area: 2.25±0.14×104 versus 4.07±0.22×104 µm2; P<0.01). Abnormal methylation status at the promoter of ten-eleven translocation 2, one of the key demethylation enzymes in mammals, was ameliorated after 5-aza-2'-deoxycytidine treatment, which in turn caused an increase in global DNA hydroxymethylation and 5-hydroxymethylcytosine enrichment at the promoter of Myocardin. In vitro, 5-aza-2'-deoxycytidine treatment or DNA methyltransferase 1 knockdown decreased global 5-methylcytosine content and restored Myocardin expression in VSMCs induced by platelet-derived growth factor, thus preventing excessive VSMCs dedifferentiation, proliferation, and migration. Furthermore, DNA methyltransferase 1 binds to ten-eleven translocation 2 promoter and is required for ten-eleven translocation 2 methylation in VSMCs. CONCLUSIONS: The inhibitory effects of DNA demethylation on global 5-methylcytosine content and ten-eleven translocation 2 hypermethylation in atherosclerotic aorta can recover 5-hydroxymethylcytosine enrichment at the Myocardin promoter and prevent VSMC dedifferentiation and vascular remodeling.

Upregulation of Key Molecules for Targeted Imaging and Therapy.

Targeted diagnosis and therapy enable precise tumor detection and treatment. Successful examples for precise tumor targeting are diagnostic and therapeutic radioligands. However, patients with tumors expressing low levels of the relevant molecular targets are deemed ineligible for such targeted approaches. METHODS: We performed a screen for drugs that upregulate the somatostatin receptor subtype 2 (sstr2). Then, we characterized the effects of these drugs on transcriptional, translational, and functional levels in vitro and in vivo. RESULTS: We identified 9 drugs that act as epigenetic modifiers, including the inhibitor of DNA methyltransferase decitabine as well as the inhibitors of histone deacetylase tacedinaline and romidepsin. In vitro, these drugs upregulated sstr2 on transcriptional, translational, and functional levels in a time- and dose-dependent manner. Thereby, their combinations revealed synergistic effects. In vivo, drug-based sstr2 upregulation improved the tumor-to-background and tumor-to-kidney ratios, which are the key determinants of successful sstr2-targeted imaging and radiopeptide therapy. CONCLUSION: We present an approach that uses epigenetic modifiers to improve sstr2 targeting in vitro and in vivo. Translation of this method into the clinic may potentially convert patients ineligible for targeted imaging and therapy to eligible candidates.

Enhanced inhibition of clonogenic survival of human medulloblastoma cells by multimodal treatment with ionizing irradiation, epigenetic modifiers, and differentiation-inducing drugs.

BACKGROUND: Medulloblastoma (MB) is the most common pediatric brain tumor. Current treatment regimes consisting of primary surgery followed by radio- and chemotherapy, achieve 5-year overall survival rates of only about 60 %. Therapy-induced endocrine and neurocognitive deficits are common late adverse effects. Thus, improved antitumor strategies are urgently needed. In this study, we combined irradiation (IR) together with epigenetic modifiers and differentiation inducers in a multimodal approach to enhance the efficiency of tumor therapy in MB and also assessed possible late adverse effects on neurogenesis. METHODS: In three human MB cell lines (DAOY, MEB-Med8a, D283-Med) short-time survival (trypan blue exclusion assay), apoptosis, autophagy, cell cycle distribution, formation of gH2AX foci, and long-term reproductive survival (clonogenic assay) were analyzed after treatment with 5-aza-2'-deoxycytidine (5-azadC), valproic acid (VPA), suberanilohydroxamic acid (SAHA), abacavir (ABC), all-trans retinoic acid (ATRA) and resveratrol (RES) alone or combined with 5-aza-dC and/or IR. Effects of combinatorial treatments on neurogenesis were evaluated in cultured murine hippocampal slices from transgenic nestin-CFPnuc C57BL/J6 mice. Life imaging of nestin-positive neural stem cells was conducted at distinct time points for up to 28 days after treatment start. RESULTS: All tested drugs showed a radiosynergistic action on overall clonogenic survival at least in two-outof-three MB cell lines. This effect was pronounced in multimodal treatments combining IR, 5-aza-dC and a second drug. Hereby, ABC and RES induced the strongest reduction of clongenic survival in all three MB cell lines and led to the induction of apoptosis (RES, ABC) and/or autophagy (ABC). Additionally, 5-aza-dC, RES, and ABC increased the S phase cell fraction and induced the formation of gH2AX foci at least in oneout-of-three cell lines. Thereby, the multimodal treatment with 5-aza-dC, IR, and RES or ABC did not change the number of normal neural progenitor cells in murine slice cultures. CONCLUSION: In conclusion, the radiosensitizing capacities of epigenetic and differentiation-inducing drugs presented here suggest that their adjuvant administration might improve MB therapy. Thereby, the combination of 5-aza-dC/IR with ABC and RES seemed to be the most promising to enhance tumor control without affecting the normal neural precursor cells.

CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil.

Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.

Epigenetic Control of the Vasopressin Promoter Explains Physiological Ability to Regulate Vasopressin Transcription in Dehydration and Salt Loading States in the Rat.

The synthesis of arginine vasopressin (AVP) in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus is sensitive to increased plasma osmolality and a decreased blood volume, and thus is robustly increased by both dehydration (increased plasma osmolality and decreased blood volume) and salt loading (increased plasma osmolality). Both stimuli result in functional remodelling of the SON and PVN, a process referred to as functional-related plasticity. Such plastic changes in the brain have recently been associated with altered patterns of DNA methylation at CpG (cytosine-phosphate-guanine) residues, a process considered to be important for the regulation of gene transcription. In this regard, the proximal Avp promoter contains a number of CpG sites and is recognised as one of four CpG islands for the Avp gene, suggesting that methylation may be regulating Avp transcription. In the present study, we show that, in an immortalised hypothalamic cell line 4B, the proximal Avp promoter is highly methylated, and treatment of these cells with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine to demethylate DNA dramatically increases basal and stimulated Avp biosynthesis. We report no changes in the expression of DNA methyltransferases, Dnmt1 and Dnmt3a, whereas there is decreased expression of the demethylating enzyme ten-eleven-translocation 2, Tet2, in the SON by dehydration and salt loading. We found higher methylation of the SON Avp promoter in dehydrated but not salt-loaded rats. By analysis of individual CpG sites, we observed hypomethylation, hypermethylation and no change in methylation of specific CpGs in the SON Avp promoter of the dehydrated rat. Using reporter gene assays, we show that mutation of individual CpGs can result in altered Avp promoter activity. We propose that methylation of the SON Avp promoter is necessary to co-ordinate the duel inputs of increased plasma osmolality and decreased blood volume on Avp transcription in the chronically dehydrated rat.

Emodin enhances the demethylation by 5-Aza-CdR of pancreatic cancer cell tumor-suppressor genes P16, RASSF1A and ppENK.

5-Aza-2'-deoxycytidine (5-Aza-CdR) is currently acknowledged as a demethylation drug, and causes a certain degree of demethylation in a variety of cancer cells, including pancreatic cancer cells. Emodin, a traditional Chinese medicine (TCM), is an effective monomer extracted from rhubarb and has been reported to exhibit antitumor activity in different manners in pancreatic cancer. In the present study, we examined whether emodin caused demethylation and increased the demethylation of three tumor-suppressor genes P16, RASSF1A and ppENK with a high degree of methylation in pancreatic cancer when combined with 5-Aza-CdR. Our research showed that emodin inhibited the growth of pancreatic cancer Panc-1 cells in a dose- and time-dependent manner. Dot-blot results showed that emodin combined with 5-Aza-CdR significantly suppressed the expression of genome 5mC in PANC-1 cells. In order to verify the effect of methylation, methylation-specific PCR (MSP) and bisulfite genomic sequencing PCR (BSP) combined with TA were selected for the cloning and sequencing. Results of MSP and BSP confirmed that emodin caused faint demethylation, and 5-Aza-CdR had a certain degree of demethylation. When emodin was combined with 5-Aza-CdR, the demethylation was more significant. At the same time, fluorescent quantitative PCR and western blot analysis results confirmed that when emodin was combined with 5-Aza-CdR, the expression levels of P16, RASSF1A and ppENK were increased more significantly compared to either treatment alone. In contrast, the expression levels of DNA methyltransferase 1 (DNMT1) and DNMT3a were more significantly reduced with the combination treatment than the control or either agent alone, further proving that emodin in combination with 5-Aza-CdR enhanced the demethylation effect of 5-Aza-CdR by reducing the expression of methyltransferases. In conclusion, the present study confirmed that emodin in combination with 5-Aza-CdR enhanced the demethylation by 5-Aza-CdR of tumor-suppressor genes p16, RASSF1A and ppENK by reducing the expression of methyltransferases DNMT1 and DNMT3a.

A high-throughput small molecule screen identifies synergism between DNA methylation and Aurora kinase pathways for X reactivation.

X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened ∼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by "priming" cells with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.

Is there a role for therapy after transplant?

Despite the steady increase in the number of stem cell transplants performed since 1980 and improvements in survival rates, disease relapse remains the major cause of death after HLA matched sibling and unrelated donor transplants for acute myeloid leukemia (AML). Given this situation, maintenance therapy after transplant may be an appropriate strategy to reduce the relapse rate and prolong survival. A number of agents are being investigated as maintenance therapy after stem cell transplant in AML patients, including azacitidine, decitabine, and other agents. This paper focuses on the role of maintenance treatment to reduce the risk of relapse after transplant.

Decitabine and Sorafenib Therapy in FLT-3 ITD-Mutant Acute Myeloid Leukemia.

BACKGROUND: Acute myeloid leukemia (AML) characterized by Feline McDonough Sarcoma-like tyrosine kinase-3 (FLT-3) internal tandem duplication (ITD) mutations have poor outcomes. Treatment options are limited, because these mutations confer resistance to conventional chemotherapy. FLT-3 inhibitors such as sorafenib have been studied as a single agent and in combination with conventional chemotherapy or azacytidine with fair responses. PATIENTS AND METHODS: Here we describe our preclinical and clinical experience with the combination of the DNA hypomethylating agent, decitabine and sorafenib for the treatment of FLT-3 ITD-mutant AML. RESULTS: In vitro treatment of the human FLT-3 ITD-mutant AML cell line, MV4-11, with both drugs significantly improved growth inhibition over single-agent therapy and resulted in synergistic antitumor effects (combination index < 1). A case series of 6 patients treated with off protocol combination of decitabine and sorafenib demonstrated overall responses in 5 patients (83%) with a median survival of 155 days. Four of the 5 patients (80%) with relapsed/refractory AML achieved complete responses with incomplete count recovery. The combination was also well tolerated. CONCLUSION: Further investigation is warranted to confirm these responses.

Myelodysplastic syndromes: 2015 Update on diagnosis, risk-stratification and management.

DISEASE OVERVIEW: The myelodysplastic syndromes (MDS) are a very heterogeneous group of myeloid disorders characterized by peripheral blood cytopenias and increased risk of transformation to acute myelogenous leukemia (AML). MDS occurs more frequently in older males and in individuals with prior exposure to cytotoxic therapy. DIAGNOSIS: Diagnosis of MDS is based on morphological evidence of dysplasia upon visual examination of a bone marrow aspirate and biopsy. Information obtained from additional studies such as karyotype, flow cytometry, or molecular genetics is complementary but not diagnostic. Risk-stratification: Prognosis of patients with MDS can be calculated using a number of scoring systems. In general, all these scoring systems include analysis of peripheral cytopenias, percentage of blasts in the bone marrow, and cytogenetic characteristics. The most commonly used system still is probably the International Prognostic Scoring System (IPSS). IPSS is being replaced by the new revised score IPSS-R. RISK-ADAPTED THERAPY: Therapy is selected based on risk, transfusion needs, percent of bone marrow blasts, and more recently cytogenetic and mutational profiles. Goals of therapy are different in lower risk patients than in higher risk. In lower risk, the goal is to decrease transfusion needs and transformation to higher risk disease or AML, as well as to improve survival. In higher risk, the goal is to prolong survival. Current available therapies include growth factor support, lenalidomide, hypomethylating agents, intensive chemotherapy, and allogeneic stem cell transplantation. The use of lenalidomide has significant clinical activity in patients with lower risk disease, anemia, and a chromosome 5 alteration. 5-Azacitidine and decitabine have activity in higher risk MDS. 5-Azacitidine has been shown to improve survival in higher risk MDS. A number of new molecular lesions have been described in MDS that may serve as new therapeutic targets or aid in the selection of currently available agents. Additional supportive care measures may include the use of prophylactic antibiotics and iron chelation. Management of progressive or refractory disease: At the present time there are no approved interventions for patients with progressive or refractory disease particularly after hypomethylating based therapy. Options include participation in a clinical trial or cytarabine based therapy and stem cell transplantation.

Decitabine Enhances Lymphocyte Migration and Function and Synergizes with CTLA-4 Blockade in a Murine Ovarian Cancer Model.

The lack of second-line treatment for relapsed ovarian cancer necessitates the development of improved combination therapies. Targeted therapy and immunotherapy each confer clinical benefit, albeit limited as monotherapies. Ovarian cancer is not particularly responsive to immune checkpoint blockade, so combination with a complementary therapy may be beneficial. Recent studies have revealed that a DNA methyl transferase inhibitor, azacytidine, alters expression of immunoregulatory genes in ovarian cancer. In this study, the antitumor effects of a related DNA methyl transferase inhibitor, decitabine (DAC), were demonstrated in a syngeneic murine ovarian cancer model. Low-dose DAC treatment increases the expression of chemokines that recruit NK cells and CD8(+) T cells, promotes their production of IFNγ and TNFα, and extends the survival of mice bearing subcutaneous or orthotopic tumors. While neither DAC nor immune checkpoint blockade confers durable responses as a monotherapy in this model, the efficacy of anti-CTLA-4 was potentiated by combination with DAC. This combination promotes differentiation of naïve T cells into effector T cells and prolongs cytotoxic lymphocyte responses as well as mouse survival. These results suggest that this combination therapy may be worthy of further consideration for improved treatment of drug-resistant ovarian cancer.

Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation.

The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5 hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5 mC) and 5 hmC at a CCGG site within the 5' γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5 mC and 5 hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5 hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5 hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5 hmC and negatively correlated with 5 mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5 hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter.

Potentiation of growth inhibition and epigenetic modulation by combination of green tea polyphenol and 5-aza-2'-deoxycytidine in human breast cancer cells.

Epigenetic therapy by DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza 2'dC) is clinically effective in acute myeloid leukemia; however, it has shown limited results in treatment of breast cancer and has significant toxicity to normal cells. Green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) has anti-cancer and DNA demethylating properties with no significant toxicity toward normal cells. Therefore, the objective of this study was to evaluate the therapeutic efficacy of a combination of non-toxic, low dose of 5-aza 2' dC with EGCG, on growth inhibition of breast cancer cells. Human breast cancer cell lines (MCF-7, MDA-MB 231) and non-tumorigenic MCF-10A breast epithelial cells were treated with either 5-aza 2' dC, EGCG, or their combination for 7 days. Cell growth inhibition was determined by cell count, cell viability, cell cycle, and soft agar assay, whereas genes expression changes were determined by quantitative real-time PCR and/or Western blot analysis. Histone modifications and global DNA methylation changes were determined by Western blot and RAPD-PCR, respectively. The results revealed significantly greater inhibition of growth of breast cancer cells by co-treatment with 5-aza 2' dC and EGCG compared to individual treatments, whereas it has no significant toxicity to MCF-10A cells. This was further confirmed by gene expression analysis. Changes in DNA methylation and histone modifications were also greater in cells with combination treatment. Findings of this study suggest that potentiation of growth inhibition of breast cancer cells by 5-aza 2' dC and EGCG combination treatment, at least in part, is mediated by epigenetic mechanism.

Interactive actions of Bdnf methylation and cell metabolism for building neural resilience under the influence of diet.

Quality nutrition during the period of brain formation is a predictor of brain functional capacity and plasticity during adulthood; however it is not clear how this conferred plasticity imparts long-term neural resilience. Here we report that early exposure to dietary omega-3 fatty acids orchestrates key interactions between metabolic signals and Bdnf methylation creating a reservoir of neuroplasticity that can protect the brain against the deleterious effects of switching to a Western diet (WD). We observed that the switch to a WD increased Bdnf methylation specific to exon IV, in proportion to anxiety-like behavior, in Sprague Dawley rats reared in low omega-3 fatty acid diet, and these effects were abolished by the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine. Blocking methylation also counteracted the reducing action of WD on the transcription regulator CTCF binding to Bdnf promoter IV. In vitro studies confirmed that CTCF binding to Bdnf promoter IV is essential for the action of DHA on BDNF regulation. Diet is also intrinsically associated to cell metabolism, and here we show that the switch to WD downregulated cell metabolism (NAD/NADH ratio and SIRT1). The fact that DNA methyltransferase inhibitor did not alter these parameters suggests they occur upstream to methylation. In turn, the methylation inhibitor counteracted the action of WD on PGC-1α, a mitochondrial transcription co-activator and BDNF regulator, suggesting that PGC-1α is an effector of Bdnf methylation. Results support a model in which diet can build an "epigenetic memory" during brain formation that confers resilience to metabolic perturbations occurring in adulthood.

Human DNA methyltransferase gene-transformed yeasts display an inducible flocculation inhibited by 5-aza-2'-deoxycytidine.

Mammalian DNA methyltransferases (DNMTs) play an important role in establishing and maintaining the proper regulation of epigenetic information. However, it remains unclear whether mammalian DNMTs can be functionally expressed in yeasts, which probably lack endogenous DNMTs. We cotransformed the budding yeast Saccharomyces cerevisiae with the human DNMT1 gene, which encodes a methylation maintenance enzyme, and the DNMT3A/3B genes, which encode de novo methylation enzymes, in an expression vector also containing the GAL1 promoter, which is induced by galactose, and examined the effects of the DNMT inhibitor 5-aza-2'-deoxycytidine (5AZ) on cell growth. Transformed yeast strains grown in galactose- and glucose-containing media showed growth inhibition, and their growth rate was unaffected by 5AZ. Conversely, 5AZ, but not 2'-deoxycytidine, dose-dependently interfered with the flocculation exhibited by DNMT-gene transformants grown in glucose-containing medium. Further investigation of the properties of this flocculation indicated that it may be dependent on the expression of a Flocculin-encoding gene, FLO1. Taken together, these findings suggest that DNMT-gene transformed yeast strains functionally express these enzymes and represent a useful tool for in vivo screening for DNMT inhibitors.