Hanghang Zhang

Transcriptional Selectivity of Epigenetic Therapy in Cancer

A central challenge in the development of epigenetic cancer therapy is the ability to direct selectivity in modulating gene expression for disease-selective efficacy. To address this issue, we characterized by RNA-seq, DNA methylation, and ChIP-seq analyses the epigenetic response of a set of colon, breast, and leukemia cancer cell lines to small-molecule inhibitors against DNA methyltransferases (DAC), histone deacetylases (Depsi), histone demethylases (KDM1A inhibitor S2101), and histone methylases (EHMT2 inhibitor UNC0638 and EZH2 inhibitor GSK343). We also characterized the effects of DAC as combined with the other compounds. Averaged over the cancer cell models used, we found that DAC affected 8.6% of the transcriptome and that 95.4% of the genes affected were upregulated. DAC preferentially regulated genes that were silenced in cancer and that were methylated at their promoters. In contrast, Depsi affected the expression of 30.4% of the transcriptome but showed little selectivity for gene upregulation or silenced genes. S2101, UNC0638, and GSK343 affected only 2% of the transcriptome, with UNC0638 and GSK343 preferentially targeting genes marked with H3K9me2 or H3K27me3, respectively. When combined with histone methylase inhibitors, the extent of gene upregulation by DAC was extended while still maintaining selectivity for DNA-methylated genes and silenced genes. However, the genes upregulated by combination treatment exhibited limited overlap, indicating the possibility of targeting distinct sets of genes based on different epigenetic therapy combinations. Overall, our results demonstrated that DNA methyltransferase inhibitors preferentially target cancer-relevant genes and can be combined with inhibitors targeting histone methylation for synergistic effects while still maintaining selectivity. Cancer Res; 77(2); 470-81. ©2016 AACR.

Repositioning FDA-Approved Drugs in Combination with Epigenetic Drugs to Reprogram Colon Cancer Epigenome

Epigenetic drugs, such as DNA methylation inhibitors (DNMTi) or histone deacetylase inhibitors (HDACi), are approved in monotherapy for cancer treatment. These drugs reprogram gene expression profiles, reactivate tumor suppressor genes (TSG) producing cancer cell differentiation and apoptosis. Epigenetic drugs have been shown to synergize with other epigenetic drugs or various anticancer drugs. To discover new molecular entities that enhance epigenetic therapy, we performed a high-throughput screening using FDA-approved libraries in combination with DNMTi or HDACi. As a screening model, we used YB5 system, a human colon cancer cell line, which contains an epigenetically silenced CMV-GFP locus, mimicking TSG silencing in cancer. CMV-GFP reactivation is triggered by DNMTi or HDACi and responds synergistically to DNMTi/HDACi combination, which phenocopies TSG reactivation upon epigenetic therapy. GFP fluorescence was used as a quantitative readout for epigenetic activity. We discovered that 45 FDA-approved drugs (4% of all drugs tested) in our FDA-approved libraries enhanced DNMTi and HDACi activity, mainly belonging to anticancer and antiarrhythmic drug classes. Transcriptome analysis revealed that combination of decitabine (DNMTi) with the antiarrhythmic proscillaridin A produced profound gene expression reprogramming, which was associated with downregulation of 153 epigenetic regulators, including two known oncogenes in colon cancer (SYMD3 and KDM8). Also, we identified about 85 FDA-approved drugs that antagonized DNMTi and HDACi activity through cytotoxic mechanisms, suggesting detrimental drug interactions for patients undergoing epigenetic therapy. Overall, our drug screening identified new combinations of epigenetic and FDA-approved drugs, which can be rapidly implemented into clinical trials. Mol Cancer Ther; 16(2); 397–407. ©2016 AACR.

Interferon-γ signaling in melanocytes and melanoma cells regulates expression of CTLA-4

CTLA4 is a cell surface receptor on T cells that functions as an immune checkpoint molecule to enforce tolerance to cognate antigens. Anti-CTLA4 immunotherapy is highly effective at reactivating T-cell responses against melanoma, which is postulated to be due to targeting CTLA4 on T cells. Here, we report that CTLA4 is also highly expressed by most human melanoma cell lines, as well as in normal human melanocytes. Interferon-γ (IFNG) signaling activated the expression of the human CTLA4 gene in a melanocyte and melanoma cell-specific manner. Mechanistically, IFNG activated CTLA4 expression through JAK1/2-dependent phosphorylation of STAT1, which bound a specific gamma-activated sequence site on the CTLA4 promoter, thereby licensing CBP/p300-mediated histone acetylation and local chromatin opening. In melanoma cell lines, elevated baseline expression relied upon constitutive activation of the MAPK pathway. Notably, RNA-seq analyses of melanoma specimens obtained from patients who had received anti-CTLA4 immunotherapy (ipilimumab) showed upregulation of an IFNG-response gene expression signature, including CTLA4 itself, which correlated significantly with durable response. Taken together, our results raise the possibility that CTLA4 targeting on melanoma cells may contribute to the clinical immunobiology of anti-CTLA4 responses.Significance: These findings show that human melanoma cells express high levels of the immune checkpoint molecule CTLA4, with important possible implications for understanding how anti-CTLA4 immunotherapy mediates its therapeutic effects. Cancer Res; 78(2); 436-50. ©2017 AACR.

Abstract 5064: Identifying novel potential epigenetic anti-cancer drugs from natural compounds using a phenotypic-based screening

Epigenetic aberrations such as DNA hypermethylation and repressive chromatin are validated targets for cancer chemotherapy. Since epigenetic modifications are reversible, the goal of epigenetic therapy is to reverse the abnormal alternations in cancer cells and induce tumor suppressor gene reactivation, leading to cancer cell differentiation and cell death. Many known anti-cancer drugs are derived from natural compounds and there have been reports of natural compounds modulating epigenetic activity. To explore this idea, our lab developed a phenotypic-based system (YB5) by stably transfecting SW48 cells with a vector containing GFP driven by a methylated and silenced CMV promoter. GFP re-expression can be achieved by known epigenetic drugs that lead to demethylation or induce active chromatin marks in the CMV promoter. By screening an NDL-3040 natural compounds library and grouping the compounds based on chemical structures, we identified two main drug classes. We then synthesized 77 new analogs based on class #1’s lead’s structure and 23 were positive in the YB5 system. The most potent analog (HH2) can induce ~60% GFP+ cells upon 500nM treatment after 96hr. All the positive hits can also be validated in two other cancer cell lines (MCF7 and HCT116). Consistent with GFP reactivation, endogenous hypermethylated genes (MGMT, RARβ, etc) can also be re-expressed upon drug treatment. We then performed RNA-seq analysis to identify global gene expression changes following drug treatment. We observed that most genes (2964 genes) were upregulated upon HH1 treatment (10uM) and that many of the upregulated genes were expressed in normal tissues but repressed in cancer, indicating that they might be potential tumor suppressor genes (TSGs). Consistent with this, 94 TSGs could be reactivated upon 10uM drug treatment. These drug target upregulated genes were also enriched for hypermethylation. By performing connectivity mapping using RNA-seq, we identified X as the class #1 drug target. The on-target effect could be further validated by using other selective X inhibitors as well as a dominant negative X construct. Consistent with drug inhibition, dominant-negative X can also reactivate drug targeted hypermethylated genes. Additionally, when we overexpressed wild-type X, we saw that GFP induction as well as endogenous gene reactivations can be inhibited. Strikingly, by using GFP induction as readout to optimize drugs, we found that the in vitro IC50 against X for our top lead compound (HH2) is only 5nM and it is at least 22-fold selective for X over other X family members. Thus, a novel epigenetic drug class derived from natural compounds was identified and can be developed by targeting silenced gene expression. Citation Format: Hanghang Zhang, Noel J.-M Raynal, Takahiro Sato, Yasuyuki Okamoto, Judith Garriga, Benjamin Garcia, George Morton, Wayne Childers, Marlene A. Jacobson, Stephen B. Baylin, Xavier Grana, Magid Abou-Gharbia, Jean-Pierre J. Issa. Identifying novel potential epigenetic anti-cancer drugs from natural compounds using a phenotypic-based screening [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 5064. doi:10.1158/1538-7445.AM2017-5064

Abstract B50: A phenotypic screen to identify novel potential epigenetic anticancer drugs from natural compounds

Epigenetic aberrations such as DNA hypermethylation and repressive chromatin are validated targets for cancer chemotherapy. Since epigenetic modifications are reversible, the goal of epigenetic therapy is to reverse the abnormal alternations in cancer cells and induce tumor suppressor genes reactivation, leading to cancer cell differentiation and cell death. Thus, epigenetic enzymes are attractive drug targets in the field of drug discovery. Many known anti-cancer drugs are derived from natural compounds and there have been reports of natural compounds modulating epigenetic activity. Therefore, it would be of interest to screen natural compounds as potential epigenetic drugs. We screened 3040 natural compounds and derivatives by measuring GFP expression in the YB5 cell line, a colon cancer cell line generated by stably transfecting SW48 cells with a vector containing GFP driven by a methylated and silenced CMV promoter. GFP re-expression can be achieved by known epigenetic drugs that lead to demethylation or induce active chromatin marks in the CMV promoter. After 24hr treatment with the natural compounds, FACS analysis was used to check the GFP expression levels. After the primary screening (average Z9 factor = 0.6), we set a stringent criterion that GFP induction value should be more than the average of all drugs +3 standard deviations in order to be considered as positive. 33 hits were positive (positive rate = 1.1%) among which 18 hits were validated through 24hr dose curves, fluorescence microscopy and qPCR. We then grouped the positive hits based on chemical structures. In class #1, 3 positive hits were discovered from the primary screening using NDL-3040 library, then HH1 was discovered as a potent top lead from a secondary screening using 93 analogs. The Moulder Center then synthesized 56 new analogs based on the lead9s structure and 14 are positive in the YB5 system. The most potent analog (HH2) can induce 10% GFP+ cells upon 5uM treatment after 24hr. This potency is in the same range as that obtained with decitabine (a DNMT inhibitor). All the positive hits can also be validated in two other cancer cells (MCF7 and HCT116). Consistent with GFP reactivation, these drugs can also reactivate many hypermethylated genes (CDH13, MGMT, SYNE1, RRAD, PYGM etc) in the YB5 cell line. These compounds also synergize with Decitabine in terms of GFP induction and many endogenous hypermethylated reactivation (RARβ, SYNE1, RRAD, FAM184A etc). For this class, we found no effects on DNA methylation, HDAC activity or effects on known histone methyltransferases/demethylases (HMT/HDM) using biochemistry-based assays. Global histone methylation and acetylation level changes were determined using mass spectrometry and we found upregulation of H3K79me2 levels. Proliferation assays showed differential sensitivity of a panel of colon cancer cell lines compared to normal cells (IMR90). These drugs can also lead to G2/M arrest and GFP positive cells are more likely to be arrested than GFP negative cells. Thus, a novel epigenetic drug class derived from natural compounds was identified and can be developed by targeting silenced gene expression. Citation Format: Hanghang Zhang, Noel J.-M Raynal, Takahiro Sato, Yasuyuki Okamoto, Benjamin Garcia, George Morton, Wayne Childers, Marlene A. Jacobson, Stephen B. Baylin, Magid Abou-Gharbia, Jean-Pierre J. Issa. A phenotypic screen to identify novel potential epigenetic anticancer drugs from natural compounds. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B50.

Abstract 4701: A phenotypic cell-based screen to identify novel potential epigenetic anti-cancer drugs from natural compounds

Epigenetic aberrations such as DNA hypermethylation and repressive chromatin are validated targets for cancer chemotherapy. Since epigenetic modifications are reversible, the goal of epigenetic therapy is to reverse the abnormal alternations in cancer cells and induce tumor suppressor genes reactivation, leading to cancer cell differentiation and cell death. Thus, epigenetic enzymes are attractive drug targets in the field of drug discovery. Many known anti-cancer drugs are derived from natural compounds and there have been reports of natural compounds modulating epigenetic activity. Therefore, it would be of interest to screen natural compounds as potential epigenetic drugs. In an effort to identify novel targets that can reactivate hypermethylated silenced genes, our lab developed a phenotypic-based system, YB5. YB5 is a colon cancer cell line generated by stably transfecting SW48 cells with a vector containing GFP driven by a methylated and silenced CMV promoter. GFP re-expression can be achieved by known epigenetic drugs that lead to demethylation or induce active chromatin marks in the CMV promoter. By screening an NDL-3040 library and grouping the molecules based on chemical structures, we were able to identify three main drug classes. The Moulder Center then synthesized 60 new analogs based on class #19s lead9s structure and 15 are positive in the YB5 system. The most potent analog can induce 10% GFP+ cells upon 500nM treatment. All the positive hits can also be validated in two other cancer cells (MCF7 and HCT116). Consistent with GFP reactivation, endogenous hypermethylated genes can be reactivated upon drug treatment. Also, GFP positive cells show higher endogenous gene reactivation than unsorted and GFP negative cells. By performing RNA-seq analysis upon class #1 top lead treatment followed by connectivity mapping, we identified X as the class #1 drug target. The on-target effect can be validated by using other selective X inhibitors as well as a dominant negative X construct. Consistent with drug inhibition, dominant-negative X can also reactivate drug targeted hypermethylated genes. Proliferation assays showed differential sensitivities of a panel of colon cancer cell lines compared to normal cells. These drugs can also lead to G2/M arrest and GFP positive cells are more likely to be arrested than GFP negative cells. Besides class #1 drugs, a novel class of LSD1 inhibitors was identified and the most active drug can induce 15% GFP at 5uM. Consistent with LSD1 inhibition, many known LSD1 target genes can also be upregulated. Like known LSD1 inhibitors, these compounds significantly inhibited proliferation of AML cells. We also identified some known natural compounds that have epigenetic activities, including arsenic trioxide, cardiac glycosides, and toyocamycin. Thus, many novel epigenetic drug classes derived from natural compounds were identified and can be developed by targeting silenced gene expression. Citation Format: Hanghang Zhang, Noel J.-M Raynal, Takahiro Sato, Yasuyuki Okamoto, Judit Garriga, George Morton, Wayne Childers, Marlene A. Jacobson, Stephen B. Baylin, Xavier Grana, Magid Abou-Gharbia, Jean-Pierre J. Issa. A phenotypic cell-based screen to identify novel potential epigenetic anti-cancer drugs from natural compounds. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4701.

Abstract B42: Decitabine specifically targets genes that gain DNA methylation and lose expression in cancer and can be combined with histone methylation inhibitors for increased efficacy

A major question facing the use of epigenetic therapies in cancer is the specificity in reactivating gene expression. In addition, combined targeting of DNA and histone methylation remains largely unexplored despite the promising synergistic effects observed from combining DNA methyltransferase inhibitors with HDAC inhibitors. To address these questions, we performed RNA-seq in colon cancer (SW48) and breast cancer (MCF7) models treated with a DNA methyltransferase inhibitor (Decitabine/DAC) either alone or in combination with a LSD1 inhibitor (S2101), G9a inhibitor (UNC0638), EZH2 inhibitor (GSK343) or a HDAC inhibitor (Depsipeptide). Additionally, we performed ChIP-seq (H3K4me2, H3K9me2, and H3K27me3) and DNA methylation analysis (DREAM) on SW48 cells. Both low dose (100nM) and intermediate dose (1uM) DAC were very specific in upregulating genes with high levels of DNA methylation (86% and 78% of upregulated genes in SW48 cells, respectively). DAC alone and in combination with S2101, UNC0638, or GSK343 preferentially upregulated genes that were expressed in normal colon and silenced in SW48 cells (443, 718, 728, 1095 genes, respectively) compared to genes that didn9t change expression (194, 343, 424, 479 genes, respectively). Additionally, the combination therapies specifically upregulated genes that were not DNA methylated in normal colon but gained methylation in cancer (91%, 85%, 87%, and 79% of upregulated genes, respectively). The combination therapies also preferentially upregulated genes that lost expression in cancer and were silenced by H3K9me2 and/or H3K27me3 (121, 204, 277, 380 genes, respectively) compared to genes that didn9t change expression in cancer but were silenced by histone methylation (42, 82, 163, 167 genes, respectively). In contrast, Depsipeptide and the combination of Depsipeptide with DAC induced major expression changes in all genes (2167 and 2938 genes upregulated that were expressed in normal colon and silenced in SW48, compared to 1807 and 2389 genes upregulated with no change in expression, respectively). Furthermore, DAC in combination with S2101, UNC0638, or GSK343 induced a significant number of synergistic genes that were only upregulated with the combination therapy and not with either monotherapy (>490 genes for each inhibitor combination in both cancer models). These synergistic genes showed limited overlap between the three combination therapies (only 7.64% of genes commonly upregulated in YB5, 7.31% in MCF7), and formed distinct clusters in a hierarchical cluster analysis. IPA analysis demonstrated that these genes are enriched in critical cancer regulation pathways such as cell proliferation, cell death, and cell movement. Consistent with this analysis, the combination therapies were able to decrease cancer cell proliferation more effectively than monotherapy. Overall, these results demonstrate that DNA methyltransferase inhibitors preferentially target cancer relevant genes, and can be combined with epigenetic inhibitors targeting histone methylation to increase efficacy while still maintaining specificity. Citation Format: Takahiro Sato, Matteo Cesaroni, Anthony Tran, Jozef Madzo, Yasuyuki Okamoto, Hanghang Zhang, Shoghag Panjarian, Jaroslav Jelinek, Jean-Pierre Issa. Decitabine specifically targets genes that gain DNA methylation and lose expression in cancer and can be combined with histone methylation inhibitors for increased efficacy. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B42.

Abstract 2657: Target specificity of epigenetic therapy in cancer

A major question facing the use of epigenetic therapies in cancer is specificity in modulating gene expression. In addition, combined targeting of DNA and histone methylation remains largely unexplored despite the promising synergistic effects observed from combining DNA methyltransferase inhibitors with HDAC inhibitors. To address these questions, we performed RNA-seq, DNA methylation analysis and ChIP-seq (H3K4me2, H3K9me2, and H3K27me3) to study the effects of inhibitors of DNA methyltransferases (DAC), histone deacetylases (Depsi), histone demethylases (KDM1A inhibitor S2101), and histone methylases (EHMT2 inhibitor UNC0638 and EZH2 inhibitor GSK343) in three different cancer models (colon cancer, breast cancer, and leukemia). In colon cancer cells (YB5), DAC affected 3% of the transcriptome and 93% of the effect was gene upregulation. DAC had a greater effect on genes expressed in normal tissues and silenced in cancer (443 genes) compared to genes that do not change in cancer (194 genes). 90% of DAC targets genes showed no promoter DNA methylation in normal colon but gained methylation in cancer. Depsi changed the expression of 35% of the transcriptome and showed little specificity for gene upregulation or silenced genes. S2101, UNC0638, and GSK343 had limited effects on their own ( Citation Format: Takahiro Sato, Matteo Cesaroni, Shoghag Panjarian, Anthony Tran, Jozef Madzo, Yasuyuki Okamoto, Hanghang Zhang, Xiaowei Chen, Jaroslav Jelinek, Jean-Pierre J. Issa. Target specificity of epigenetic therapy in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2657.

Abstract 3519: A phenotypic screen to discover novel epigenetic anticancer drugs from natural compounds

Epigenetics is the study of heritable changes in gene expression that are not caused by changes in DNA sequence. Since epigenetic modifications are reversible, the goal of epigenetic therapy is to reverse the abnormal alternations in cancer cells and induce tumor suppressor genes reactivation, leading to cancer cell differentiation and cell death. Thus, epigenetic enzymes are attractive drug targets in the field of drug discovery. Many known anti-cancer drugs are derived from natural compounds and there have been reports of natural compounds modulating epigenetic activity. Therefore, it would be of interest to screen natural compounds as potential epigenetic drugs. We screened 3040 natural compounds and derivatives by measuring GFP expression in the YB5 cell line, a colon cancer cell line generated by stably transfecting SW48 cells with a vector containing GFP driven by a methylated and silenced CMV promoter. GFP re-expression can be achieved by known epigenetic drugs that lead to demethylation or induce active chromatin marks in the CMV promoter. After 24hr treatment, FACS analysis was used to check the GFP expression levels. After the primary screening (average Z’ factor = 0.6), we set a stringent criterion that GFP induction value should be more than the average of all drugs mean +3 standard deviations in order to be considered as positive. 33 hits were positive (positive rate = 1.1%) among which 18 hits were validated through 24hr dose curves, fluorescence microscopy and qPCR. We then grouped the positive hits based on chemical structures. Two classes were selected for further studies. For class#1 compounds, the most active drug induced 20% GFP at 10uM. For this class, we found no effects on DNA methylation, HDAC activity or effects on known histone methyltransferases/demethylases (HMT/HDM) using biochemistry-based assays. Global histone acetylation level was determined using mass spectrometry and we saw upregulation of H4K16ac levels. Proliferation assays showed differential sensitivity of a panel of colon cancer cell lines compared to normal cells (IMR90). These drugs also reactivated two endogenously hypermethylated genes (CDH13 and MGMT). For class#2 compounds, the most active drug activated 15% GFP at 10uM. By using a HMT/HDM biochemistry assay panel, class #2 was determined to inhibit LSD1. qPCR analysis demonstrated upregulation of endogenous LSD1 target genes. Furthermore, like known LSD1 inhibitors, these compounds significantly inhibited cell proliferation of AML cells. Both drug classes can synergize with decitabine (a DNMT inhibitor) to reactivate different tumor suppressor genes. Thus, two novel epigenetic drug classes derived from natural compounds were discovered, with activities on LSD1 and H4K16 acetylation. Citation Format: Hanghang Zhang, Noel Raynal, Takahiro Sato, Yasuyuki Okamoto, Ryan Henry, Andrew J. Andrews, George Morton, Wayne Childers, Marlene A. Jacobson, Magid Abou-Gharbia, Jean-Pierre J. Issa. A phenotypic screen to discover novel epigenetic anticancer drugs from natural compounds. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3519. doi:10.1158/1538-7445.AM2015-3519

Abstract 378: Discovering potential epigenetic anti-cancer drugs derived from natural compounds

Introduction and Aims: Epigenetics is the study of heritable changes in gene expression that are independent of changes in DNA sequence. The main biochemical modifications that govern epigenetics are DNA methylation and post-translational histone modifications. Small molecules modulating the activity of the DNA/chromatin-modifying enzymes have been shown to be able to revert malignant cells to a more normal epigenetic state. Thus, the opportunity for developing potential epigenetic drugs is of great interest in the fields of cancer biology and drug discovery. Natural compounds are bioactive, have variable structures, and many known anti-cancer drugs are derived from natural compounds. In addition, there have been reports of natural compounds modulating epigenetic activity. Therefore, it would be of interest to screen natural compounds as potential epigenetic drugs. Methods: We used the YB5 cell line, a colon cancer cell line generated by our lab by stably transfecting SW48 cells with a vector containing GFP driven by a methylated and silenced CMV promoter. GFP re-expression can be achieved by epigenetic drugs that lead to demethylation or induce active chromatin marks in the CMV promoter. After 24hr treatment with the natural compounds, FACS analysis was used to check the GFP expression levels. After the primary screening, top hits were validated by doing dose curves, fluorescence microscopy and qPCR. In addition, hypermethylated genes were checked by qPCR and methylation levels of the CMV promoter were determined by pyrosequencing. Results: We have screened 3040 natural compounds, with 33 potential positive hits as measured by an increase in GFP expression (more than 2.64% of GFP+) after a 24-hour treatment. Among these, 19 hits have been validated through dose curves, fluorescence microscopy and qPCR. Two hits have synergistic effects with Decitabine (a DNA methyltransferase inhibitor). By using a biochemistry-based assay, we have determined that none of the positive hits are HDAC inhibitors. In addition, none of the hits decreased methylation levels of the CMV promoter after 24hr treatment. These hits can be classified into several different categories based on their structures. Four class representative top hits were selected to do further analysis. These four hits can reactivate two hypermethylated genes (CDH13 and WIF1) and upregulate p21. A combination of Decitabine and the four top hits can upregulate hypermethylated gene expression levels synergistically in different patterns. Conclusions: We have identified 33 natural compounds that have potential epigenetic activity. Based on the structural analysis, gene reactivation patterns, as well as synergistic effects analysis, these top hits may work via different mechanisms. Further experiments are being carried out to identify other potential target genes of the top hits, study the biological effects of the hits and discover their potential mechanisms of action. Citation Format: Hanghang Zhang, Noel J-M RAYNAL, Marlene A. Jacobson, Jean-Pierre Issa. Discovering potential epigenetic anti-cancer drugs derived from natural compounds. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 378. doi:10.1158/1538-7445.AM2014-378