Search

In cancer, epigenetic modifications are strongly altered and are responsible for gene expression aberrations. In a drug screening initiative, we recently reported that proscillaridin, a cardiac glycoside (CG), exhibits unsuspected epigenetic and anticancer activities. To understand CG's epigenetic mechanisms of action, we performed RNA sequencing analysis, which showed proscillaridin effects on global gene expressions in acute lymphoblastic leukemia cells (MOLT-4). Genes associated with apoptosis and cell differentiation were upregulated whereas master transcription factors and oncogenic pathway genes were downregulated. Mechanistic studies revealed that proscillaridin decreased histone 3 acetylation, which correlated with histone acetyltransferase (KATs) downregulation (CBP, P300, TIP60, GCN5 and MOZ). Acetylome studies by mass spectrometry showed an acetylation loss in chromatin regulators, the oncogene C-MYC and its associated proteins. Proscillaridin induced C-MYC transcript and protein degradation. Moreover, in a panel of cancer cell lines, we measured that cancer cells sensitivity to proscillaridin treatment was positively correlated with C-MYC protein levels. Conversely, proscillaridin did not affect C-MYC protein level in low C-MYC expressing cancer cell lines. For the first time, we showed that CGs target histone acetyltransferases and C-MYC oncogene in high C-MYC expressing cancers. We propose that CGs can be repurposed as new epigenetic drugs in high C-MYC expressing cancers. Citation Format: Elodie M. Da Costa, Gregory Armaos, Annie Beaudry, Chantal Richer, Maxime Caron, Pascal St-Onge, Jeffrey Johnson, Nevan Krogan, Yuka Sai, Michael Downey, Daniel Sinnett, Serge McGraw, Noël J. Raynal. Targeting histone acetyltransferases to reprogram high C-MYC expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1381.

Epigenetic modifications play a key role in establishing and maintaining gene expression. In cancer, they are highly altered and responsable of gene expression deregulation. Epigenetic drugs have the ability to reset cancer cell epigenome producing cancer cell differentiation and apoptosis. In a drug screening initiative, we recently reported a series of FDA-approved drugs with unsuspected epigenetic and anticancer activities. Here, we tested in a secondary screen the activity of these drugs against acute lymphoblastic leukemia (ALL) cell lines (MOLT-4 and NALM-6). We found that Proscillaridin A, a cardiac glycoside used for heart failure treatment, was the most active with IC50 values in the low nanomolar range, suggesting drug repositioning potential. Proscillaridin treatments induced a significant decrease in RNA and protein levels of C-MYC, a master oncogenic driver in ALL. Shortly after proscillaridin A treatment, C-MYC exhibited a 75% reduction in lysine acetylation, a post-translational modification known to prevent its degradation. Loss of acetylation was associated with down-regulation of lysine acetyltransferases CBP, P300 and Tip60, which also correlated with a reduction in histone 3 and 4 acetylation levels (H3K14ac, H3K9ac, H3K27ac and H4K5ac). Preliminary analysis with si-RNA experiments reveal that independent HAT activities are not responsible of C-MYC downregulation. RNA sequencing and gene set enrichment analysis in proscillaridin-treated ALL cells (5 nM for 48h) showed that genes associated with cell differentiation and apoptosis pathways were up-regulated whereas down-regulated genes were associated with C-MYC target genes. Altogether, our findings show that acetylation through lysine acetyltransferase down-regulation simultaneously induces loss of C-MYC and H3 acetylation leading to epigenetic reprogramming in ALL cells. This drug repositioning strategy, using proscillaridin A, has the potential to reprogram cancer cells that are driven by MYC overexpression or hyperactivation. Citation Format: Elodie M. Da Costa, Gregory Armaos, Simon Jacques-Ricard, Annie Beaudry, Pascal St-Onge, Maxime Caron, Daniel Sinnett, Serge McGraw, Noël J. Raynal. Loss of C-MYC and chromatin acetylation induce epigenetic reprogramming in acute lymphoblastic leukemia [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 1375. doi:10.1158/1538-7445.AM2017-1375

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, Noël J.-M Raynal, Takahiro Sato, Yasuyuki Okamoto, Judith Garriga, Benjamin Garcia, George Morton, Wayne Childers, Marlene A. Jacobson, Stephen B. Baylin, Xavier Graña, 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

Acute lymphoblastic leukemia (ALL) represents approximately 25% of all pediatric cancers diagnosed every year. In about 80% of cases, pediatric patients will attain an event-free 5-year survival. Unfortunately, patients who are resistant to treatment or who relapse have a poor prognosis. Hence, novel therapeutic approaches are necessary to increase survival rates. Epigenetic alterations, such as DNA methylation and histone modifications, are involved in disease development, progression, and in particular, resistance to treatment. These reversible alterations represent additional targets in ALL. Recently, we discovered candidate epigenetic drugs in FDA-approved drug libraries. We hypothesize that one drug in particular, the cardiac glycoside proscillaridin A, has both epigenetic and anti-cancerous properties in preclinical models of ALL and can therefore be repositioned for the treatment of the disease. To test our hypothesis, we treated two ALL cell lines Nalm-6 (pre-B ALL) and Molt-4 (T-ALL) in vitro with clinically relevant concentrations of proscillaridin A and analyzed cell growth, cell cycle, gene expression and chromatin modifications. We observed dose-dependent growth inhibition in all cell lines, with IC50 values of 3.0 and 2.3 nM in Nalm-6 and Molt-4, respectively. Our results using BrdU staining indicate a block in the G2/M phase of the cell cycle. By western blot, we detected a reduction in histone acetylation levels and in histone modifying enzymes CBP and Tip60, as well as the c-myc oncogene. These promising results illustrate the perspective of using the cardiac glycoside proscillaridin A as a novel epigenetic drug for the treatment of relapsed or refractory ALL. Citation Format: Gregory Armaos, Simon Jacques-Ricard, Elodie Da Costa, Annie Beaudry, Noël J-M Raynal. Proscillaridin A effects on chromatin modifications and oncogene degradation in acute lymphoblastic leukemia. [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 4526.

Pediatric neuroblastoma is one of the most common extra cranial cancers in children. Despite an improvement in survival with the currently available therapies, neuroblastoma with an amplification of the transcription factor MYCN have a very poor prognosis. New therapeutic approaches must be developed to increase the survival of patients. One such approach is epigenetic drug therapy. Neuroblastoma, like many other pediatric cancers, contains several epigenetic alterations at the level of DNA methylation and histone modifications. In a screening of FDA approved drugs, we discovered some molecules having characteristics of epigenetic drugs that were unknown until now. Our study seeks to demonstrate the efficacy of these molecules in the treatment of neuroblastoma cell lines. Following preliminary tests, one of the molecules approved by the FDA stood out: disulfiram, a medication approved for the treatment of chronic alcoholism. We treated neuroblastoma cell lines (MYCN amplified: IMR-32, N91 and SK-N-DZ; MYCN non-amplified: SK-N-AS and SK-N-SH) for 48 hours with disulfiram at clinically relevant concentrations (from 10 nM to 50 μM). Our results demonstrate a 50% growth inhibition (IC50) of 50nM for the cell lines tested. In addition, after analysis by flow cytometry, we found a cell cycle block in G2/M. We also observed a decrease in the transcription factor MYCN and a reduction in acetylation of several histone marks by Western blots analysis. Further studies are underway to determine the mechanism of action of disulfiram and we also plan on doing RNA sequencing and ChIP sequencing. This study will evaluate the efficacy of disulfiram alone as well as in combination with other epigenetic drugs, such as DNA methyltransferase inhibitor decitabine, for the treatment of neuroblastoma. Citation Format: Simon Jacques-Ricard, Noel Raynal, Gregory Armaos, Elodie Da Costa, Annie Beaudry. New epigenetic mechanism of disulfiram targeting chromatin remodeling in neuroblastoma. [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 3200.

Sarcoma accounts for 15% of all pediatric cancers diagnosed every year. Refractory patients have a poor prognosis with a 5-year survival rate of 10%, demonstrating the need for development of new therapeutic approaches. Epigenetic events, including DNA methylation and post-translational histone modifications (such as histone acetylation), are responsible for silencing tumor suppressor genes and overexpressing oncogenes. In sarcomas, a better understanding of epigenetic alterations is needed to develop new epigenetic targeted therapies. Several epigenetics drugs have demonstrated anticancer activity against sarcoma cell lines but the identification of a specific target is still missing. We demonstrated that targeting histone acetylation may represent a specific epigenetic mark in pediatric sarcomas. We identified that proscillaridin A, a newly defined epigenetic drug, has epigenetic and anticancer effects on osteosarcoma (U2OS) and rhabdomyosarcoma (RD) cell lines. Our findings show a decrease of histone 3 acetylation on specific lysine residues after a 48h-treatment with proscillaridin A at 5 nM, a clinically relevant dose. These effects correlate with downregulation of histone acetylation associated proteins such as bromodomain BRD4 and oncogene C-MYC. A 48h-treatment with proscillaridin A produces a G2/M block and a reduction in the clonogenic potential of both U2OS and RD cells with IC50 value of approximately 6 nM. Interestingly, remaining colonies of RD cells fully lost their abilities to growth after drug removal, suggesting a long-term epigenetic reprograming. We demonstrated that proscillaridin A epigenetic effects occurred through calcium signaling since its mechanism of action may be dependent on calcium-calmodulin kinase activity.These promising results illustrate the epigenetic and anticancer potential of proscillaridin A against pediatric sarcoma. Additional experiments are currently ongoing in order to further characterize the mechanism of action of proscillaridin A in vitro and in vivo. Citation Format: Elodie Da Costa, Gregory Armaos, Simon Jacques-Ricard, Annie Beaudry, Noël Raynal. Chromatin alterations induce C-MYC and BRD4 downregulation in pediatric sarcoma. [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 LB-157.

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 #1's lead's 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, Noël J.-M Raynal, Takahiro Sato, Yasuyuki Okamoto, Judit Garriga, George Morton, Wayne Childers, Marlene A. Jacobson, Stephen B. Baylin, Xavier Graña, 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.

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 Z' 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 lead's 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, Noël 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.

Background: Epigenetic aberrations, including DNA hypermethylation of tumor suppressor genes are a common feature in solid tumors. Thus, targeting epigenetic pathways is a promising approach for cancer chemotherapy. However, only occasional proof-of-principle responses can be seen in solid tumors. The activity of hypomethylating drugs, such as decitabine could be improved by a treatment combination. Preclinical studies have suggested that adding genistein, a natural isofavone may synergize with demethylating drugs and improve treatment results. Methods: Here, we describe a phase I/IIa dose-escalation study of decitabine with a fixed dose of genistein. Primary objective of the phase I trial was to determine the Maximum Tolerated Dose (MTD) and to evaluate pharmacokinetic parameters in patients with advanced solid tumor. The objective of the phase IIa was to assess its efficacy in lung cancer. Decitabine was administered over 10-hours at increasing doses (60, 120, 240 mg/m2) with continuous administration of genistein 300 mg/day orally. Treatment was repeated every 4 to 6 weeks. Results: For the phase I, ten patients with advanced solid tumors were recruited. The recommended dose of decitabine (for the phase IIa) was 120 mg/m2 with neutropenia as limiting side effect. Decitabine at 120 mg/m2 and genistein produced plasma levels of 0.62 ± 0.06 μM and 8.5 ± 5.6 μM, respectively. The drug combination was well tolerated and produced stable disease for more than 7 months (7-14 months) in 50% of the patients. One patient had a 50% reduction in tumor burden after 6 months of therapy. In the phase IIa trial against advanced lung cancer, patients progressed rapidly, within 2 months of treatment mainly because of the apparition of new lesions in the liver. Conclusion: The combination of decitabine and genistein is well-tolerated in advanced solid tumors and shows interesting anticancer effects. This study on decitabine and genistein warrants future investigations to address this issue of liver metastasis in advanced lung cancer. Citation Format: Noël J. Raynal, Elie Kassouf, Luc Daigneault, Patrick Colin, Isabelle Plante, Guylaine Lassonde, Richard Momparler, Michel Charbonneau, Normand Blais. Results of a phase I/IIa dose-escalation study of the combination of decitabine and genistein against advanced solid tumors. [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 CT311. doi:10.1158/1538-7445.AM2015-CT311

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, Noël 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

Targeting epigenetic pathways to induce tumor suppressor gene reactivation is a promising approach for cancer therapy. Epigenetic drugs produce impressive responses in some patients; however, treatment options are limited to a small number of drugs. We screened drug libraries for epigenetic activity using a live cell-based assay. We found that around 1% of US-FDA approved drugs have significant epigenetic activity detected by GFP reactivation of a DNA methylated and silenced promoter in colon cancer cells. Newly identified drugs, most prominently cardiac glycosides, induced tumor suppressor gene reactivation and showed selective anticancer apoptosis induction. These drugs did not change DNA methylation or histone acetylation globally but induced gene reactivation and cancer cell killing through calcium signaling leading to nuclear exclusion of chromatin repressors. Our data identify new epigenetic drugs that can be rapidly repurposed for cancer clinical trials and raise questions about the safety of commonly used medications. Citation Format: Noël J. Raynal, Justin T. Lee, Youjun Wang, Judith Garriga, Gabriel Malouf, Sarah Dumont, Elisha J. Dettman, Vazganush Gharibyan, Saira Ahmed, Woonbok Chung, Wayne E. Childers, Magid Abou-Gharbia, Ryan A. Henry, Andrew Andrews, Jaroslav Jelinek, Ying Cui, Stephen B. Baylin, Donald L. Gill, Jean-Pierre J. Issa. Discovery of new epigenetic drugs among FDA-approved drug libraries. [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 380. doi:10.1158/1538-7445.AM2014-380

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, Noël 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