Potent Inhibitor Of DNA Methylation Research Articles

Identification of the Effects of 5-Azacytidine on Porcine Circovirus Type 2 Replication in Porcine Kidney Cells.

Porcine circovirus type 2 (PCV2) is the main pathogen causing post-weaning multisystemic wasting syndrome (PMWS), which mainly targets the body's immune system and poses a serious threat to the global pig industry. 5-Azacytidine is a potent inhibitor of DNA methylation, which can participate in many important physiological and pathological processes, including virus-related processes, by inhibiting gene expression. However, the impact of 5-Aza on PCV2 replication in cells is not yet clear. We explored the impact of 5-Aza on PCV2 infection utilizing PK15 cells as a cellular model. Our objective was to gain insights that could potentially offer novel therapeutic strategies for PCV2. Our results showed that 5-Aza significantly enhanced the infectivity of PCV2 in PK15 cells. Transcriptome analysis revealed that PCV2 infection activated various immune-related signaling pathways. 5-Aza may activate the MAPK signaling pathway to exacerbate PCV2 infection and upregulate the expression of inflammatory and apoptotic factors.

UVR8 interacts with de novo DNA methyltransferase and suppresses DNA methylation in Arabidopsis.

DNA methylation is an important epigenetic gene regulatory mechanism conserved in eukaryotes. Emerging evidence shows DNA methylation alterations in response to environmental cues. However, the mechanism of how cells sense these signals and reprogram the methylation landscape is poorly understood. Here, we uncovered a novel connection between ultraviolet B (UVB) signaling and DNA methylation involving UVB photoreceptor (UVR8) and a de novo DNA methyltransferase (DRM2) in Arabidopsis. We demonstrated that UVB acts through UVR8 to inhibit DRM2-mediated DNA methylation and transcriptional de-repression. Interestingly, DNA transposons with high DNA methylation are more sensitive to UVB irradiation. Mechanistically, UVR8 interacts with and negatively regulates DRM2 by preventing its chromatin association and inhibiting the methyltransferase activity. Collectively, this study identifies UVB as a potent inhibitor of DNA methylation and provides mechanistic insights into how signaling transduction cascades intertwine with chromatin to guide genome functions.

The Identification of Novel Epigenetic Therapies for ALK-Driven Haematological Malignancies

Introduction Through conserved signalling pathways, Anaplastic Lymphoma Kinase (ALK) is well-described in driving haematological malignancies including Anaplastic Large Cell Lymphoma (ALCL) and Diffuse Large B-Cell Lymphoma (DLBCL) and as such presents itself as an amenable therapeutic target. Hence, directed therapeutics (ALK tyrosine kinase inhibitors; TKI) are being used in the treatment of ALK-driven cancers. Unfortunately, findings in the clinic and clinical research studies have taught us that resistance to ALK inhibitors can develop through the activation of ALK signalling bypass tracks. As such there is a need for the development of novel front-line, dual-combination, as well as second-line therapies. Methods A large-scale epigenetic targeted drug library consisting of approximately 300 FDA-approved drugs and novel agents was applied to a number of cell lines representing ALK-driven haematological malignancies: ALCL cell lines (DEL, JB-6, KARPAS-299, SU-DHL-1, SUP-M2) and the DLBCL cell line LM-1. Drugs which caused a >75% decrease in cell viability were classified as 'candidate drugs' and studied further. Results Several of the validated drugs have previously been used/trialled in the clinic for the treatment of various cancers, e.g. aurora kinase (XL-228), topoisomerase (Mitoxantrone HCl) and HDAC (Romidepsin) inhibitors - these functioned as internal controls for the drug screens. However, an assortment of novel drugs was also identified that have not previously been described in the context of the treatment of ALK-driven haematological malignancies; including the FLT3 inhibitor KW2449 which caused a >75% decrease in viability in all the tested cell lines and as such may serve as a novel front line therapy. In addition, a novel DNA methyltransferase (DNMT) inhibitor was identified which is efficacious and resulted in a >90% decrease in viability in all cell lines treated across both disease entities. Furthermore, we investigated the combinatorial potential of the identified DNMT inhibitor with ALK TKIs such as Brigatinib and observed the inhibitor acting synergistically (as per Bliss-Independence calculations) resulting in a further decrease in cell viability. Several cell lines that are resistant to ALK TKIs were also assessed for their sensitivity to the DNMT inhibitor and were shown to be susceptible to this drug, as demonstrated by a significant decrease in cell viability. Figure 1: (A) Viability following drug screen in a representative cell line. Those drugs which led to a >75% change in viability were taken forward for validation, as shown in (B). (C) Candidate drugs identified for each of the cell lines tested, grouped according to their molecular target. Conclusion In conclusion, an epigenetic drug library has been employed to identify novel therapeutic agents for the treatment of ALK-driven haematological malignancies including ALCL and DLBCL. Data reveal a potent inhibitor of DNA methylation as a candidate drug that suppresses the growth of ALK-driven malignancies both alone and in combination with ALK TKIs. Significantly, this identified drug also inhibits the growth of cell lines resistant to directed therapeutics such as ALK TKIs suggesting it has potential clinical use. Disclosures No relevant conflicts of interest to declare.

Inhibition of DNA and Histone Methylation by 5-Aza-2'-Deoxycytidine (Decitabine) and 3-Deazaneplanocin-A on Antineoplastic Action and Gene Expression in Myeloid Leukemic Cells.

Epigenetic alterations play an important role in the development of acute myeloid leukemia (AML) by silencing of genes that suppress leukemogenesis and differentiation. One of the key epigenetic changes in AML is gene silencing by DNA methylation. The importance of this alteration is illustrated by the induction of remissions in AML by 5-aza-2′-deoxycytidine (5-AZA-CdR, decitabine), a potent inhibitor of DNA methylation. However, most patients induced into remission by 5-AZA-CdR will relapse, suggesting that a second agent should be sought to increase the efficacy of this epigenetic therapy. An interesting candidate for this purpose is 3-deazaneplanocin A (DZNep). This analog inhibits EZH2, a histone methyltransferase that trimethylates lysine 27 histone H3 (H3K27me3), a marker for gene silencing. This second epigenetic silencing mechanism also plays an important role in leukemogenesis as shown in preclinical studies where DZNep exhibits potent inhibition of colony formation by AML cells. We reported previously that 5-AZA-CdR in combination with DZNep exhibits a synergistic antineoplastic action against human HL-60 AML cells and the synergistic activation of several tumor suppressor genes. In this report, we showed that this combination also induced a synergistic activation of apoptosis in HL-60 cells. The synergistic antineoplastic action of 5-AZA-CdR plus DZNep was also observed on a second human myeloid leukemia cell line, AML-3. In addition, 5-AZA-CdR in combination with the specific inhibitors of EZH2, GSK-126, or GSK-343, also exhibited a synergistic antineoplastic action on both HL-60 and AML-3. The combined action of 5-AZA-CdR and DZNep on global gene expression in HL-60 cells was investigated in greater depth using RNA sequencing analysis. We observed that this combination of epigenetic agents exhibited a synergistic activation of hundreds of genes. The synergistic activation of so many genes that suppress malignancy by 5-AZA-CdR plus DZNep suggests that epigenetic gene silencing by DNA and histone methylation plays a major role in leukemogenesis. Targeting DNA and histone methylation is a promising approach that merits clinical investigation for the treatment of AML.

Zebularine upregulates expression of CYP genes through inhibition of DNMT1 and PKR in HepG2 cells

Drug-induced hepatotoxicity is one of the major reasons cited for drug withdrawal. Therefore, it is of extreme importance to detect human hepatotoxic candidates as early as possible during the drug development process. In this study, we aimed to enhance hepatocyte functions such as CYP gene expression in HepG2 cells, one of the most extensively used cell lines in evaluating hepatotoxicity of chemicals and drugs. We found that zebularine, a potent inhibitor of DNA methylation, remarkably upregulates the expression of CYP genes in HepG2 cells. In addition, we revealed that the upregulation of CYP gene expression by zebularine was mediated through the inhibition of both DNA methyltransferase 1 (DNMT1) and double-stranded RNA-dependent protein kinase (PKR). Furthermore, HepG2 cells treated with zebularine were more sensitive than control cells to drug toxicity. Taken together, our results show that zebularine may make HepG2 cells high-functioning and thus could be useful for evaluating the hepatotoxicity of chemicals and drugs speedily and accurately in in-vitro systems. The finding that zebularine upregulates CYP gene expression through DNMT1 and PKR modulation sheds light on the mechanisms controlling hepatocyte function and thus may aid in the development of new in-vitro systems using high-functioning hepatocytes.

Determinants Of Demethylation and Clinical Response In AML Patients Treated With SGI-110, a Novel Subcutaneous (SQ) Hypomethylating Agent (HMA) In a Phase 1 Study

Abstract Introduction SGI-110 is a dinucleotide of decitabine and deoxyguanosine and is a potent inhibitor of DNA methylation in-vitro and in-vivo. A Phase 1 study established the biologically effective dose of SGI-110 in MDS/AML patients as 60 mg/m2 SQ daily x 5 and demonstrated clinical responses that correlated with hypomethylation induction. Here, we analyze AML patients treated at pharmacologically effective doses of SGI-110 to explore determinants of hypomethylation and response. Methods We studied patients with relapsed/refractory AML treated at therapeutic dose ranges of SGI-110 (36 mg/m2-125 mg/m2). DNA methylation pre/post treatment (for pharmacodynamics [PD]) was measured by bisulfite-pyrosequencing for the LINE-1 repetitive element and the INS6 CpG island gene promoter which is highly methylated in all somatic tissues. Gene expression at baseline and after treatment was measured by qPCR. Results We analyzed samples from 27 patients with AML. Median age was 64, (range, 29–86), 18 were Males (67%), 13 (48%) had poor risk cytogenetics at study entry and 59% had prior exposure to a hypomethylating agent. Overall, peak LINE-1 demethylation generally occurred on Day 8 and correlated strongly with INSL6 demethylation (R=0.95, p<0.001). In individual patients, peak LINE-1 demethylation ranged from +1% to -39%. We next examined baseline expression of a panel of genes (CDA, P15, P21, DNMT3B, DNMT3A, DNMT1, CTCF) as possible predictors of SGI-110 response. High expression of DNMT3b (but not DNMT1) was associated with a trend for reduced demethylation (R=-0.20, p>0.05). Unsupervised classification grouped the patients into four clusters: A (N=2), B (N=6), C (N=10), and D (N=9). Cluster D is characterized by high DNMT3b expression, low P15 expression, low CDA expression and reduced demethylation (demethylation average -10.9% in cluster D compared to -22.7% in clusters B and C, p=0.06). Next, we examined SGI-110 mediated induction of gene expression for the P15, P21, DNMT3B and CTCF genes. P15 was significantly induced in patients who were treated on the daily x 5 regimen (p=0.03) but not in patients receiving the weekly x 3 regimen. In this group of 14 patients with induced P15, P15 induction peaked on Day 8 and averaged a 2.4-fold increase. P15 induction was associated with a trend for increased demethylation on Day 8 (R=0.28) and on Day 29 (R=0.37), p>0.05 for both. Of the 27 patients, 5 showed major clinical responses (2 CR, 3 CRi/CRp). LINE-1 and INSL6 demethylation averaged -21.1% and -16.4% in responders compared to -13.1% and -11.3% in non-responders. A three gene classifier score (low CDA, low P15 and high DNMT3B) was associated with low LINE-1 demethylation (R=0.43, p=0.025) as well as resistance to SGI-110 (mean score 6.2 in non-responders compared to 0.5 in responders, p=0.047). Finally, peak induction of P15 was similar in responders and non-responders, but sustained induction (at Day 29) was higher in responders (3.1 fold) than in non-responders (1.0 fold). While a genetic signature of response could not be identified among those 8 genes that were examined by exome sequencing, mutations in IDH1 or IDH2 were identified in 5 patients. One of these patients was positive for substitution R132H of IDH1 (described to induce epigenetic alterations and may predict poor clinical outcome in AML) and achieved a CR in response to SGI-110 treatment. The TP53 polymorphism NM_000546:c.C215G:p.P72R, (associated with differential response to chemotherapy in AML), was identified in 9 subjects, 3 of whom responded to SGI-110. Conclusions At therapeutic doses of SGI-110, we identified a gene expression signature (high DNMT3B, low P15, and low CDA) that differentiates responders from non-responders to SGI-110 and we find trends for associations between demethylation and response, as well as sustained P15 induction and response. These associations will be further investigated in the ongoing Phase 2 study of SGI-110 in AML. Disclosures: Chung: Astex Pharmaceuticals Inc.: Research Funding. Off Label Use: SGI-110. Taverna:Astex Pharmaceuticals Inc.: Employment. Lyons:Astex Pharmaceuticals Inc.: Employment. Hao:Astex Pharmaceuticals Inc.: Employment. Azab:Astex Pharmaceuticals Inc.: Employment. Kantarjian:Astex Pharmaceuticals Inc.: Research Funding. Kropf:Astex Pharmaceuticals Inc.: Research Funding. Issa:Astex Pharmaceuticals Inc.: Honoraria, Research Funding.

Abstract A128: Zebularine, a DNA methyltransferase inhibitor, increases apoptosis and decreases cell proliferation, cell cycle arrest and clonogenic capacity on childhood medulloblastoma cell lines.

Abstract Medulloblastoma (MB) is the most common malignant brain tumors in childhood, accounting for 20 % of all primary pediatric intracranial tumors. Although conventional therapies can cure a large proportion of patients with medulloblastoma, the majority of survivors suffer from long-term side effects, including developmental and neurological deficits. Thus, new approaches and therapeutic drugs are needed. Epigenetic drugs such as inhibitors of DNA methyltransferases (iDNMTs) have shown antineoplastic effects in different tumors. Zebularine (ZB) is a potent inhibitor of DNA methylation and has been associated with induction of apoptosis and enhancing tumor chemo- and radiosensitivity. However, its effects on childhood MB have not been previously reported. Herein, this study shows the preliminary data from the effects of ZB on cell proliferation, cell cycle, clonogenic capacity and apoptosis of childhood MB cells. DAOY, ONS-76, UW-402 and UW-473 MB cell lines were used in all functional studies. Cell proliferation by XTT® assay, clonogenic capacity, cell cycle and apoptosis by flow cytometry using propidium iodide and anexin-V staining were performed using different doses of ZB (50-800µM). Statistical analysis was performed by one or two-way ANOVA and Bonferoni post-hoc. ZB decreased cell proliferation and clonogenic capacity in all cell lines in a time- and dose-dependent manner, respectively (P< 0.05). ZB also caused apoptotic cell death in MB cells, where the percentage of apoptotic cells significantly increased after treatment compared control (P<0.05), reaching 60% on DAOY cells. Since the dose of 200uM, the drug showed a cell cycle arrest in S-phase in UW-473 and UW-402 cell lines and in G2/M phase in ONS-76 and DAOY cells after 72h of exposure. These results indicate that ZB may be a promising drug for MB treatment and it confirms the potential of demethylating drugs in cancer treatment. Others studies and novel assays are being conducted to better understand these findings. Financial Support: FAPESP (process no. 2011/22440-7) Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A128. Citation Format: Augusto F. Andrade, Kleiton S. Borges, Veridiana K. Suazo, Angel Maurício Castro-Gamero, Rosane G. P. Queiroz, Carlos A. Scrideli, Luiz Gonzaga Tone. Zebularine, a DNA methyltransferase inhibitor, increases apoptosis and decreases cell proliferation, cell cycle arrest and clonogenic capacity on childhood medulloblastoma cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A128.

Identification of hypermethylation in hepatocyte cell adhesion molecule gene promoter region in bladder carcinoma.

Background: Epigenetic regulation such as aberrant hypermethylation of CpG islands in promoter plays a key role in tumorigenesis. 5-Aza-2'-deoxycytidine (5-aza-CdR) which is a potent inhibitor of DNA methylation can reverse the abnormal hypermethylation of the silenced tumor suppressor genes (TSGs). It has been reported that hepatocyte cell adhesion molecule (hepaCAM) acts as a tumor suppressor gene and expression of its mRNA and protein were down-regulated in bladder cancer. Over-expression of hepaCAM can inhibit cancer growth and arrest renal cancer cells at G0/G1 phase. In this study, we investigated the methylation status of hepaCAM gene, as well as the influence of 5-aza-CdR on expression of hepaCAM gene in bladder cancer cells. Methods: CpG islands in hepaCAM promoter and methprimers were predicted and designed using bioinformatics program. Methylation status of hepaCAM promoter was evaluated in bladder cancer tissues and two cell lines (T24 and BIU-87) by Methylation-specific PCR; Western blot and Immunofluorescence were used to detect expression of hepaCAM protein after 5-aza-CdR treatment; Flow cytometry assay was performed to determine effectiveness of 5-aza-CdR on cell cycle profile. Results: CpG island in promoter of hepaCAM gene was hyper-methylated both in bladder carcinoma tissues and cell lines (T24 and BIU-87). Otherwise, aberrant methylation of its promoter was associated with its decreased expression. Hypermethylation of hepaCAM gene was reversed and expression of its mRNA and protein were re-activated in two cell lines by DNA methyltransferases inhibitor 5-aza-CdR. Flow cytometry assay demonstrated that 5-aza-CdR can inhibit growth of cancer cells by arresting cancer cells at G0/G1 phase. Conclusion: Abnormal hypermethylation in CpG island of hepaCAM promoter is involved in absence of hepaCAM gene expression when bladder cancer occurs. Re-activation of hepaCAM gene by 5-aza-CdR can inhibit growth of cancer cells and arrest cells at G0/G1 phase.

Decitabine, independent of apoptosis, exerts its cytotoxic effects on cell growth in melanoma cells

Decitabine is a synthesized cytosine analog that is a potent inhibitor of DNA methylation. There have been a few reports on the in vitro anti-melanoma effect of decitabine or its functional mechanisms. We investigated the anti-proliferation effect of decitabine on the cultured murine melanoma cell line K1735M2. MTT assay showed that decitabine had strong inhibition on melanoma K1735M2 in a time- and dose-dependent manner in vitro. Morphological observation showed that decitabine could induce melanoma K1735M2 cells to produce dendrite-like structures with the increase of decitabine concentration and incubation time. Decitabine could effectively induce K1735M2 cells to differentiate in vitro. Additionally, decitabine could induce a dose-dependent G2/M cell cycle arrest in K1735M2 cells. We provided experimental evidences that the anti-proliferation effect of decitabine on murine K1735M2 melanoma cells was associated predominately with G2/M cell cycle arrest and the induction of differentiation rather than apopotosis.

Upregulation of MAGE-A1, MAGE-A3, NY-ESO-1 and MHC Molecules on Neuroblastoma Cells Following Exposure to Decitabine and Interferon-γ

AbstractAbstract 5179MAGE-A1, MAGE-A3, and NY-ESO-1 are cancer-testis (CT) antigens expressed on a number of malignant solid tumors, including neuroblastoma (NB), but many tumor cell lines down-regulate the expression of CT antigens as well as major histocompatibility (MHC) antigens, precluding recognition by antigen specific T cells. If expression of cancer antigens as well as MHC molecules could be enhanced pharmacologically, the efficacy of CT antigen specific immunotherapy could be improved. DNA methylation is an epigenetic mechanism used by cells to decrease the expression of several genes, including tumor suppressor genes. Decitabine (5-aza-2′-deoxycytidine, DAC) is a potent inhibitor of DNA methylation, and the doses associated with the demethylating action of this drug are much lower than those required for cytotoxicit. We have demonstrated that the expression of MAGE-A1, MAGE-A3, and NY-ESO-1 can be upregulated on neuroblastoma cells following exposure to the DAC. When these cell lines are cultured in the presence of DAC at concentrations similar to levels achieved with clinical administration, we were able to achieve expression of NY-ESO-1, MAGE-A1, or MAGE-A3 in 11/11 neuroblastoma cell lines. Concentrations of 1uM DAC were associated with strongest expression of these antigens, which was optimal after 72 hours of drug exposure. Culture of NB cell lines with IFN-γ was also associated with an increased expression of either MHC Class I or II by cytofluorometry, and Class I upregulated in 10/11 of these cell lines, although to varying degrees. MAGE-A1, MAGE-A3, and NY-ESO-1 specific CTL were cultured from volunteer donors by stimulating peripheral blood mononuclear cells with dendritic cells pulsed with overlapping peptide mixes derived from full length proteins, and these CTL lysed HLA partially matched, DAC treated neuroblastoma as well as glioblastoma cell lines. These studies confirm that DAC and IFN-γ can be used to increase the expression of CT antigens and MHC molecules on neuroblastoma cells, and pre-treatment with these agents makes tumor cell lines more susceptible to CTL mediated killing. Disclosures:No relevant conflicts of interest to declare.

3-Deazauridine enhances the antileukemic action of 5-aza-2′-deoxycytidine and targets drug-resistance due to deficiency in deoxycytidine kinase

New approaches should be sought to treat high-risk acute lymphoblastic leukemia (ALL). Since aberrant DNA methylation plays an important role in leukemogenesis of ALL, it can be targeted by 5-aza-2′-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylation. 5-AZA-CdR is a prodrug that is activated by deoxycytidine kinase (DCK). Leukemic cells lacking DCK are drug-resistant. In a previous phase I study, we reported that 5-AZA-CdR could induce remissions in ALL. However, some patients developed drug-resistance due to deficiency in DCK. These observations aroused our interest in 3-deazauridine (3-DU), a CTP synthetase inhibitor that is effective against leukemic cells deficient in DCK. In this report, we observed that 3-DU enhanced the in vitro antineoplastic action of 5-AZA-CdR on human leukemic cells by increasing its incorporation into DNA. Using an optimized dose-schedule we showed that this combination could cure some mice bearing L1210 leukemia, even in the presence of a subpopulation of drug-resistant (L1210/ARA-C) leukemic cells lacking DCK. 3-DU alone also cured some mice with L1210/ARA-C leukemia. In a pilot study on 3 relapsed patients with advanced ALL, the combination of 5-AZA-CdR and 3-DU produced a marked reduction in leukemic blasts, confirming our preclinical observations. Furthermore, after several treatments with these agents all three patients developed drug-resistance to 5-AZA-CdR as determined by an in vitro drug sensitivity test. In two patients we showed by enzymatic analysis that the drug-resistance was due to deficiency in DCK. Our preclinical and clinical results provide a strong rationale to further investigate the combination of 5-AZA-CdR and 3-DU for the treatment of advanced ALL.

Abstract LB-296: Synergistic anticancer action of inhibitor of DNA methylation 5-aza-2-deocycytidine and EZH2 histone methylase inhibitor 3-deazaneplanocin A

Abstract The purpose of this study was to investigate the antineoplastic action of epigenetic therapy using 5-aza-2′-deoxycytidine (5-AZA-CdR) in combination with 3-deazaneplanocin A (DZNep). Aberrant epigenetic modifications play an important role in oncogenesis. DNA hypermethylation, which can silence tumor suppressor genes (TSGs), can be reversed by treatment with 5-AZA-CdR, a potent inhibitor of DNA methylation. Overexpression of EZH2 methyltransferase (HMT) can also contribute to neoplastic transformation by silencing differentiation genes and TSGs. Recent studies have shown that DZNep, an inhibitor of HMT, can reactivate certain TSGs and has activity as an antineoplastic agent. Inhibitors of DNA methylation and histone deacetylation are reported to produce synergistic reactivation of silent TSGs and synergistic anticancer activity. Our objective was to determine if this type of “cross-talk” also exists between inhibitors of DNA methylation and inhibitors of HMT. Our experimental approach was to treat leukemic and tumor cells with 5-AZA-CdR and/or DZNep and determine cell survival by in vitro colony assays. Treatment of human HL-60 myeloid leukemic cells for 24 h with 50 nM 5-AZA-CdR or 500 nM DZNep produced a loss of clonogenity (LC) of 50.0 ± 5.8% and 75.6 ± 4.15, respectively. 5-AZA-CdR in combination with DZNep at the same concentrations of each agent produced a LC of 99 ± 1% (n =3). The interaction between these two different types of epigenetic agents was clearly synergistic. We also performed colony assays with 5-AZA-CdR plus DZNep on human A549 lung carcinoma cells. These epigenetic agents in combination also produced a synergistic LC on these tumor cells. The epigenetic action of 5-AZA-CdR plus DZNep on malignant cells of different phenotype suggests that this type of treatment may show effectiveness in most types of cancer. Our current objective is to verify the synergistic interaction between inhibitors of DNA methylation and HMT in mouse models with cancer. We are also investigating action of these agents alone and in combination on expression of cancer-related genes in order to understand the molecular basis of the synergistic anticancer activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-296.

Abstract 5447: A novel inhibitor of DNA methylation

Abstract We previously reported the discovery of XB05, a synthetic small molecule with antiproliferative activity. XB05 was found to be a strong inhibitor of DNMT1 activity in cell-free and cell-based assays. However, screening in the NCI 60 tumor cell lines and analysis using COMPARE indicated a novel mechanism of action for XB05, different from standard demethylating agents such as 5-azacytidine (azaC) and decitabine. Here, we report on new research to further characterize XB05 activity. We have examined the effects of XB05 on clonogenicity, promoter methylation, gene expression, and DNMT1 protein. XB05 was found to inhibit colony formation of HCT116 colon carcinoma cells in a dose-dependent manner with a sub-micromolar IC50. Western blots of nuclear extracts from HCT116 cells treated for 72 h with 100 nM XB05 or 10 µM azaC (as positive control) showed greatly decreased levels of DNMT1, suggesting that binding to XB05 can induce degradation of DNMT1 (previous research indicates direct drug-enzyme binding). Analysis of CDKN2A promoter methylation by bisulfite modification and sequencing demonstrated heavy methylation in untreated HCT116 cells. This was reversed by treatment of with 100 nM XB05 or 10 µM azaC, resulting in re-expression of the gene (which encodes tumor suppressor, p16), as shown by qRT-PCR. Experiments to assess promoter methylation using multiplex arrays and to examine gene expression changes using DNA microarrays were recently completed and data analysis is ongoing. To evaluate in vivo activity, we treated mice bearing subcutaneous HCT116 xenografts by IP injection of XB05 with multiple doses of up to 10 mg/kg. There was no evidence of toxicity as judged by body weights and gross necropsies. The tumors of mice treated with XB05, although not significantly smaller than in control animals, were characterized by extensive necrosis in the center of the tumor, leading to a “hollow” tumor in the majority (12/20) of mice. No similar necrosis was observed in vehicle-treated or azaC-treated mice, suggesting that this effect is treatment related. Because induced necrosis is seen with vascular disrupting agents, we also examined the effect of XB05 on endothelial cells in an in vitro assay, and observed reproducible inhibition of endothelial tube formation at 400 nM or higher. In summary, our new data confirm that XB05 is a potent inhibitor of DNA methylation in cultured cancer cells, and provide the first evidence of in vivo activity, which may involve effects on the tumor vasculature, as well as the tumor itself. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5447.

Proteomic analysis of liver cancer cells treated with 5‐Aza‐2′‐deoxycytidine (AZA)

Abstract5‐Aza‐2′‐deoxycytidine (AZA) is a potent inhibitor of DNA methylation that exhibits anti‐tumor activity in a variety of tumor cells via reactivation of tumor suppressor genes. However, few studies have been done on the biological and clinical significance of AZA in human hepatocellular carcinoma. To identify potential genes that may be aberrantly methylated and confer growth advantage to neoplastic cells and to better understand the molecular mechanism(s) underlying AZA anti‐tumor activity, a proteomics approach was used to annotate global gene expression changes of HepG2 cell line pre‐ and post‐treatment with AZA. A total of 56 differentially expressed proteins were identified by 2D gel analysis, 48 of which were up‐regulated while the remaining 8 were down regulated. Among the identified proteins, eight of these showed marked changed proteins, including seven up‐regulated proteins: glutathione S‐transferase P, protein DJ‐1, peroxiredoxin‐2, UMP‐CMP kinase, cytochrome c‐type heme lyase, enhancer of rudimentary homolog, profilin‐1, and one down‐regulated protein, heat‐shock protein β−1. The possible implication of these proteins in hepatocarcinogenesis is discussed. We tested two up‐regulated proteins, glutathione S‐transferase P and peroxiredoxin‐2, using RT‐PCR and their expression was consistent with the results obtained in the protein level. Both of these genes were methylated when methylation‐specific PCR was used against their promoter regions. Following treatment with AZA, the gene promoter regions were found to be unmethylated, concomitant with overexpression of the proteins compared to HepG2 cells without treatment. These data provide useful information in evaluating the therapeutic potential of AZA for the treatment of HCC. Drug Dev Res 69, 2009. © 2009 Wiley‐Liss, Inc.

Inhibition of DNA Methyltransferase Activates Tumor Necrosis Factor α–Induced Monocytic Differentiation in Acute Myeloid Leukemia Cells

Transcriptional silencing via promoter methylation of genes important for cell growth and differentiation plays a key role in myeloid leukemogenesis. We find that clinically achievable levels of 5-aza-2'-deoxycytidine (5-AZA-dC), a potent inhibitor of DNA methylation, can modify chromatin and restore the ability of tumor necrosis factor alpha (TNFalpha) to induce monocytic differentiation of the acute myeloid leukemia cells NB4 and U937. Although 5-AZA-dC cannot fully induce differentiation, we show that 5-AZA-dC acts directly on TNFalpha-responsive promoters to facilitate TNFalpha-induced transcriptional pathways leading to differentiation. 5-AZA-dC regulates the expression of Dif-2, a TNFalpha target gene, by deacetylating chromatin domains in a methylation-dependent manner. Chromatin immunoprecipitation analyses of the Dif-2 promoter show histone hyperacetylation and a recruitment of the nuclear factor-kappaB transcription factor in response to 5-AZA-dC. Furthermore, 5-AZA-dC plus TNFalpha enhances the level of phosphorylated RNA Pol II at the Dif-2 promoter via synergistic recruitment of TFIIH. We conclude that nonspecific changes in chromatin can allow a specific transcriptional inducer to overcome blocks in leukemic cell differentiation. Our results support the concept of low doses of 5-AZA-dC acting in combination with other agents to target epigenetic changes that drive malignant growth in leukemic cells. [Cancer Res 2009;69(1):55-64].

Preclinical evaluation of the antineoplastic action of 5-aza-2'-deoxycytidine and different histone deacetylase inhibitors on human Ewing's sarcoma cells

BackgroundMost patients with advanced Ewing's sarcoma (EWS) respond poorly to conventional chemotherapy, indicating the need for new treatment approaches. Epigenetic events, such as promoter hypermethylation and chromatin histone deacetylation, silence the expression of tumor suppressor genes (TSGs) and play an important role in tumorigenesis. These epigenetic changes can be reversed by using 5-aza-2'-deoxycytidine (5AZA-CdR), a potent inhibitor of DNA methylation, in combination with an inhibitor of histone deacetylase (HDAC).ResultsHere, we used a clonogenic assay to evaluate the in vitro antineoplastic activity of 5AZA-CdR in combination with different HDAC inhibitors on EWS cells. We observed that the HDAC inhibitors, MS-275, trichostatin-A, phenylbutyrate, LAQ824 and depsipeptide, enhanced the antineoplastic action of 5AZA-CdR on EWS cells. The combination of 5AZA-CdR and MS-275 showed marked synergy, and was correlated with significant reactivation of the expression of two TSGs, E-cadherin and tumor suppressor lung cancer-1 (TSLC1), in a EWS cell line.ConclusionThese results suggest the value of future clinical studies investigating the combination of 5AZA-CdR and MS-275 in patients with advanced EWS.

Synergistic Effect of 5-Aza-2′-Deoxycytidine and Genistein in Combination Against Leukemia

5-Aza-2'-deoxycytidine (5-AZA-CdR), a potent inhibitor of DNA methylation, is an effective agent for the treatment of leukemia. The aim of this study was to investigate the antileukemic activity of this epigenetic agent in combination with genistein, a nontoxic isoflavone with chemopreventive activity. The combined treatment produced a synergistic loss of clonogenicity in human myeloid (HL-60) and lymphoid (MOLT-3) leukemic cell lines. Genistein alone showed a significant antileukemic activity against murine 5-AZA-CdR-resistant cells, and this effect was enhanced when used in combination with 5-AZA-CdR. The combined treatment also produced a synergistic increase in life span of mice with L1210 leukemia. These results suggest that genistein may have the potential to increase the clinical efficacy of 5-AZA-CdR for the treatment of leukemia.

Delivery of 5-Aza-2′-Deoxycytidine to Cells Using Oligodeoxynucleotides

The major goal of epigenetic therapy is to reverse aberrant promoter hypermethylation and restore normal function of tumor suppressor genes by the use of chromatin-modifying drugs. Decitabine, or 5-aza-2'-deoxycytidine (5-aza-CdR), is a well-characterized drug that is now Food and Drug Administration approved for the treatment of myelodysplastic syndrome. Although 5-aza-CdR is an extremely potent inhibitor of DNA methylation, it is subject to degradation by hydrolytic cleavage and deamination by cytidine deaminase. We show that short oligonucleotides containing a 5-aza-CdR can also inhibit DNA methylation in cancer cells at concentrations comparable with 5-aza-CdR. Detailed studies with S110, a dinucleotide, showed that it works via a mechanism similar to that of 5-aza-CdR after incorporation of its aza-moiety into DNA. Stability of the triazine ring in aqueous solution was not improved in the S110 dinucleotide; however, deamination by cytidine deaminase was dramatically decreased. This is the first demonstration of the use of short oligonucleotides to provide effective delivery and cellular uptake of a nucleotide drug and protection from enzymatic degradation. This approach may pave the way for more stable and potent inhibitors of DNA methylation as well as provide means for improving existing therapeutics.

Inhibition of DNA methylation by caffeic acid and chlorogenic acid, two common catechol-containing coffee polyphenols

We studied the modulating effects of caffeic acid and chlorogenic acid (two common coffee polyphenols) on the in vitro methylation of synthetic DNA substrates and also on the methylation status of the promoter region of a representative gene in two human cancer cells lines. Under conditions that were suitable for the in vitro enzymatic methylation of DNA and dietary catechols, we found that the presence of caffeic acid or chlorogenic acid inhibited in a concentration-dependent manner the DNA methylation catalyzed by prokaryotic M.SssI DNA methyltransferase (DNMT) and human DNMT1. The IC50 values of caffeic acid and chlorogenic acid were 3.0 and 0.75 microM, respectively, for the inhibition of M.SssI DNMT-mediated DNA methylation, and were 2.3 and 0.9 microM, respectively, for the inhibition of human DNMT1-mediated DNA methylation. The maximal in vitro inhibition of DNA methylation was approximately 80% when the highest concentration (20 microM) of caffeic acid or chlorogenic acid was tested. Kinetic analyses showed that DNA methylation catalyzed by M.SssI DNMT or human DNMT1 followed the Michaelis-Menten curve patterns. The presence of caffeic acid or chlorogenic acid inhibited DNA methylation predominantly through a non-competitive mechanism, and this inhibition was largely due to the increased formation of S-adenosyl-L-homocysteine (SAH, a potent inhibitor of DNA methylation), resulting from the catechol-O-methyltransferase (COMT)-mediated O-methylation of these dietary catechols. Using cultured MCF-7 and MAD-MB-231 human breast cancer cells, we also demonstrated that treatment of these cells with caffeic acid or chlorogenic acid partially inhibited the methylation of the promoter region of the RARbeta gene. The findings of our present study provide a general mechanistic basis for the notion that a variety of dietary catechols can function as inhibitors of DNA methylation through increased formation of SAH during the COMT-mediated O-methylation of these dietary chemicals.

Evaluation of a 7-Day Continuous Intravenous Infusion of Decitabine: Inhibition of Promoter-Specific and Global Genomic DNA Methylation

The nucleoside analog 5-aza-2'-deoxycytidine (5-aza-CdR, decitabine) is a potent inhibitor of DNA methylation in vitro. Cellular treatment with this agent induces the re-expression of methylation-silenced genes. It remains unclear to what extent this compound inhibits DNA methylation in vivo. A clinical study was designed to examine the molecular effects and toxicity of a continuous 1-week intravenous infusion of decitabine in solid tumor patients. Ten patients with refractory solid tumors were included in this study. Decitabine was administered at 2 mg/m(2)/d [DOSAGE ERROR CORRECTED] via continuous infusion for 168 hours. Quantitative polymerase chain reaction and high performance liquid chromatography were utilized to measure promoter-specific and global DNA methylation in peripheral-blood cells before and after treatment. Transient grade III/IV neutropenia (two patients) and grade II thrombocytopenia (one patient) was observed at the lowest planned dose step (2 mg/m2/d for 7 days). Nonhematologic toxicities were not observed. Quantitative polymerase chain reaction demonstrated significant MAGE-1 promoter hypomethylation by 14 days after the start of treatment in all 13 treatment cycles examined. Significant genomic DNA hypomethylation was also seen by day 14 in 11 of 13 treatment cycles analyzed. Genomic DNA methylation reverted to baseline levels by 28 to 35 days after the start of treatment, demonstrating that inhibition of DNA methylation by decitabine is transient. A 168-hour continuous infusion of decitabine is well tolerated and results in the inhibition of promoter-specific and genomic DNA methylation in vivo. This treatment schedule is suitable for evaluation of decitabine in combination with agents whose activity may be enhanced by the reversal of DNA methylation-mediated gene silencing.