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Supplementary Figures 1-4 from Long-term Epigenetic Therapy with Oral Zebularine Has Minimal Side Effects and Prevents Intestinal Tumors in Mice

PDF file - 235K, mRNA expression levels in normal mammary glands, mammary tumors and tumors from zebularine treated mice.

PDF file, 117KB, Protein expression of E-cadherin, as detected by immunocytochemistry.

PDF file - 335K, Differentially down-regulated genes (>1.5-fold differences; P < 0.05) from mammary tumors of the PyMT transgenic mice exposed to zebularine for 48 days.

Zebularine is a novel potent inhibitor of both cytidine deaminase and DNA methylation. We examined the effect of zebularine on mammary tumor growth in genetically engineered MMTV-PyMT transgenic mice that develop mammary tumors at 60 days of age with 100% penetrance. The MMTV-PyMT transgenic mice were randomized at 46 days of age into control (n = 25) and zebularine (n = 25) treatment groups and monitored for parameters of tumor growth. Zebularine was administered at 5 mg/mL in drinking water. We observed a significant delay in the growth of mammary tumors in zebularine-treated mice with a statistically significant reduction (P = 0.0135) in total tumor burden at 94 days of age when the mice were sacrificed. After 48 days of zebularine treatment, the tumors were predominantly necrotic compared with untreated animals. In addition, a high apoptotic index by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay was observed as early as 13 days following treatment. Immunoblot analysis showed depletion of DNMT1 and partial depletion of DNMT3b after zebularine treatment. Microarray analyses of global gene expression identified upregulation of twelve methylation-regulated genes as well as a set of candidate cancer genes that participate in cell growth and apoptosis. In summary, zebularine inhibits the growth of spontaneous mammary tumors and causes early onset of tumor cell necrosis and apoptosis in a genetically engineered mouse model of breast cancer. Defining the parameters of zebularine-mediated tumor inhibition may advance the future development of DNA methyltransferase inhibitors as an effective cancer treatment. Mol Cancer Ther; 11(2); 370–82. ©2011 AACR.

Recent successes in the application of epigenetic drugs for the treatment of myelodysplastic syndrome have raised questions on the safety of long-term administration of DNA methylation inhibitors. We treated preweaned cancer prone ApcMin/+ (Min) mice continuously with the DNA methylation inhibitor zebularine in their drinking water to determine the effects of the drug on normal mouse development as well as cancer prevention. Zebularine caused a tissue-specific reduction in DNA methylation at B1 short interspersed nucleotide elements in the small and large intestines of female Min mice but not in other organs examined after chronic oral treatment. No significant difference in the average weights of mice was observed during the treatment. In addition, analysis of global gene expression of colonic epithelial cells from the females indicated that only 3% to 6% of the genes were affected in their expression. We did not detect toxicity and abnormalities from the histopathologic analysis of liver and intestinal tissues. Lastly, we tested whether prevention of tumorigenesis can be achieved with chronic oral administration of zebularine in Min mice. The average number of polyps in Min females decreased from 58 to 1, whereas the average polyp number remained unaffected in Min males possibly due to differential activity of aldehyde oxidase. Taken together, our results show for the first time that long-term oral administration of zebularine causes a gender-specific abrogation of intestinal tumors while causing a tissue-specific DNA demethylation. Importantly, prolonged treatment of mice with epigenetic drugs resulted in only minor developmental and histologic changes.

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PDF file - 262K, Differentially up-regulated genes (>1.5-fold differences; P < 0.05) from mammary tumors of the PyMT transgenic mice exposed to zebularine for 23 days.

PDF file - 396K, Differentially up-regulated genes (>1.5-fold differences; P < 0.05) from mammary tumors of the PyMT transgenic mice exposed to zebularine for 48 days.

PDF file - 401K, Differentially up-regulated genes (>1.5-fold differences; P < 0.05) from mammary tumors of the PyMT transgenic mice exposed to zebularine for 13 days.

PDF file - 428K, Differentially down-regulated genes (>1.5-fold differences; P < 0.05) from mammary tumors of the PyMT transgenic mice exposed to zebularine for 13 days.

Perspective from Long-term Epigenetic Therapy with Oral Zebularine Has Minimal Side Effects and Prevents Intestinal Tumors in Mice

PDF file, 694KB, Effects of DZNeP/gemcitabine combination on PDAC cells migration.

PDF file - 310K, Differentially down-regulated genes (>1.5-fold differences; P < 0.05) from mammary tumors of the PyMT transgenic mice exposed to zebularine for 23 days.

PDF file - 57K, SuperArray Analysis of Oncogenes, Tumor Suppressors, and Stem Cell-Related Genes in MPM Lines Following Selective PRC2 Knockdown or 5 �M DZNep.

PDF file - 661K, TaqMan Primers, shRNA, and Antibodies.

Supplementary Figure 4 from Combinatorial pharmacologic approaches target EZH2-mediated gene repression in breast cancer cells

PDF file - 102K, Effects of DZNep treatment on cultured mesothelioma cell lines relative to normal mesothelia cells. Pyrosequencing analysis of NBL2 DNA repeat in normal mesothelia (LP9) and MPM cell lines cultured in the presence or absence of DZNep.

Supplementary Fig. S1 from DZNep is a global histone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation

PDf file - 63K, qRT-PCR and immunoblot analysis of EZH2 and EED levels in MPM lines following transduction with shRNA targeting EZH2, EED, or sham sequences. Effects of knock-down of EZH2 and EED on proliferation of MPM lines.

PDF file - 63K, Genes Simultaneously Modulated by DZNep in MES1 and H28.

PDF file - 661K, Photomicrographs of NMES1 and NMES2 normal pleura cultures, and MES1 through MES4 mesothelioma cell lines established at the NCI.

Supplementary Figure 1 from Combinatorial pharmacologic approaches target EZH2-mediated gene repression in breast cancer cells

Supplementary Table 3 from Combinatorial pharmacologic approaches target EZH2-mediated gene repression in breast cancer cells

PDF file - 97K, Supplementary Materials and Methods.

PDF file, 659K, Supplemental Figure 1. Treatment with DZNep depletes expression of PRC2 proteins in MCL MO2058 and Z-138 cells. Supplemental Figure 2. Treatment with DZNep upregulates mRNA expression of EZH2 target genes in MCL cells.A-C. Supplemental Figure 3. Treatment with DZNep does not alter hsa-miR-101 expression in MCL cells. Supplemental Figure 4. Effects of treatment with DZNep or PS in MCL Z-138 cells Supplemental Figure 5. Treatment with PS significantly enhances the anti-MCL activity of DZNep in JeKo-1 and Z-138 cells.

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Polycomb protein EZH2-mediated gene silencing is implicated in breast tumorigenesis through methylation of histone H3 on Lysine 27 (H3K27). We have previously shown that S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A can modulate histone methylation and disrupt EZH2 complex. Here, we used 3-deazaneplanocin A, together with other chromatin remodeling agents, as well as RNA interference–mediated EZH2 depletion, to probe the role of EZH2 in coordination with other epigenetic components in gene regulation in breast cancer cells. Through genome-wide gene expression analysis, coupled with extensive chromatin immunoprecipitation analysis of histone modifications, we have identified a variety of gene sets that are regulated either by EZH2 alone or through the coordinated action of EZH2 with HDAC and/or DNA methylation. We further found that tumor antigen GAGEs were regulated by distinct epigenetic mechanisms in a cell context–dependent manner, possibly reflecting mechanistic heterogeneity in breast cancer. Intriguingly, we found that EZH2 regulates a remarkable cohort of genes whose functions are highly enriched in immunoresponse and autocrine inflammation network, and that their transcriptional activation upon EZH2 perturbation is cancer specific, revealing a potential novel role of EZH2 in regulating cancer immunity. These findings show the complexity and diversity of epigenetic regulation in human cancer and underscore the importance for developing combinatorial pharmacologic approaches for effective epigenetic gene reactivation. [Mol Cancer Ther 2009;8(12):3191–202]

Supplementary Table 1 from Combinatorial pharmacologic approaches target EZH2-mediated gene repression in breast cancer cells

Supplementary Table 2 from Combinatorial pharmacologic approaches target EZH2-mediated gene repression in breast cancer cells

Supplementary Table 4 from Combinatorial pharmacologic approaches target EZH2-mediated gene repression in breast cancer cells

PDF file - 62K, Demographic Data Pertaining to the Patients from whom NCI-SB-MES 1-4 lines Were Established.

Purpose: A deregulated epigenome contributes to the transformed phenotype of mantle cell lymphoma (MCL). This involves activity of the polycomb repressive complex (PRC) 2, containing three core proteins, EZH2, SUZ12, and EED, in which the SET domain of EZH2 mediates the histone methyltransferase activity. We determined the effects of 3-deazaneplanocin A (DZNep), an S-adenosylhomocysteine hydrolase inhibitor, and/or pan-histone deacetylase inhibitor panobinostat (PS) on cultured and primary MCL cells.Experimental Design: Following treatment with DZNep and/or PS, apoptosis and the levels and activity of EZH2 and PRC2 proteins in cultured and primary MCL cells were determined.Results: Treatment with DZNep depleted EZH2, SUZ12, and 3MeK27H3 in the cultured human MCL cells. DZNep also increased expression of p21, p27, and FBXO32, whereas it depleted Cyclin D1 and Cyclin E1 levels in MCL cells. In addition, DZNep treatment induced cell-cycle arrest and apoptosis in cultured and primary MCL cells. Furthermore, as compared with treatment with each agent alone, cotreatment with DZNep and PS caused greater depletion of EZH2, SUZ12, 3MeK27H3, and Cyclin D1 levels, whereas it induced greater expression of FBXO32, p16, p21, and p27. Combined treatment with DZNep and PS synergistically induced apoptosis of cultured and primary MCL cells while relatively sparing normal CD34 + cells. Cotreatment with DZNep and PS also caused significantly greater inhibition of tumor growth of JeKo-1 xenografts in NOD/SCID mice.Conclusions: These preclinical in vitro and in vivo findings show that cotreatment with DZNep and PS is an active combined epigenetic therapy worthy of further in vivo testing against MCL. Clin Cancer Res; 18(22); 6227–38. ©2012 AACR.

Supplementary Figure 3 from Combinatorial pharmacologic approaches target EZH2-mediated gene repression in breast cancer cells

Supplementary Figure and Table Legends 1-4 from Combinatorial pharmacologic approaches target EZH2-mediated gene repression in breast cancer cells

PDF file - 245K, EZH2 expression in tumors correlates with cell line expression.

Supplementary Figure 2 from Combinatorial pharmacologic approaches target EZH2-mediated gene repression in breast cancer cells