Nadine Chapman-Rothe

Heterochromatinization induced by GAA-repeat hyperexpansion in Friedreich's ataxia can be reduced upon HDAC inhibition by vitamin B3

Large intronic expansions of the triplet-repeat sequence (GAA.TTC) cause transcriptional repression of the Frataxin gene (FXN) leading to Friedreichs ataxia (FRDA). We previously found that GAA-triplet expansions stimulate heterochromatinization in vivo in transgenic mice. We report here using chromosome conformation capture (3C) coupled with high-throughput sequencing that the GAA-repeat expansion in FRDA cells stimulates a higher-order structure as a fragment containing the GAA-repeat expansion showed an increased interaction frequency with genomic regions along the FXN locus. This is consistent with a more compacted chromatin and coincided with an increase in both constitutive H3K9me3 and facultative H3K27me3 heterochromatic marks in FRDA. Consistent with this, DNase I accessibility in regions flanking the GAA repeats in patients was decreased compared with healthy controls. Strikingly, this effect could be antagonized with the class III histone deactylase (HDAC) inhibitor vitamin B3 (nicotinamide) which activated the silenced FXN gene in several FRDA models. Examination of the FXN locus revealed a reduction of H3K9me3 and H3K27me3, an increased accessibility to DNase I and an induction of euchromatic H3 and H4 histone acetylations upon nicotinamide treatment. In addition, transcriptomic analysis of nicotinamide treated and untreated FRDA primary lymphocytes revealed that the expression of 67% of genes known to be dysregulated in FRDA was ameliorated by the treatment. These findings show that nictotinamide can up-regulate the FXN gene and reveal a potential mechanism of action for nicotinamide in reactivating the epigenetically silenced FXN gene and therefore support the further assessment of HDAC inhibitors (HDACis) in FRDA and diseases caused by a similar mechanism.

Chromatin H3K27me3/H3K4me3 histone marks define gene sets in high-grade serous ovarian cancer that distinguish malignant, tumour-sustaining and chemo-resistant ovarian tumour cells

In embryonic stem (ES) cells, bivalent chromatin domains containing H3K4me3 and H3K27me3 marks silence developmental genes, while keeping them poised for activation following differentiation. We have identified gene sets associated with H3K27me3 and H3K4me3 marks at transcription start sites in a high-grade ovarian serous tumour and examined their association with epigenetic silencing and malignant progression. This revealed novel silenced bivalent marked genes, not described previously for ES cells, which are significantly enriched for the PI3K (P<10−7) and TGF-β signalling pathways (P<10−5). We matched histone marked gene sets to gene expression sets of eight normal fallopian tubes and 499 high-grade serous malignant ovarian samples. This revealed a significant decrease in gene expression for the H3K27me3 and bivalent gene sets in malignant tissue. We then correlated H3K27me3 and bivalent gene sets to gene expression data of ovarian tumour ‘stem cell-like’ sustaining cells versus non-sustaining cells. This showed a significantly lower expression for the H3K27me3 and bivalent gene sets in the tumour-sustaining cells. Similarly, comparison of matched chemo-sensitive and chemo-resistant ovarian cell lines showed a significantly lower expression of H3K27me3/bivalent marked genes in the chemo-resistant compared with the chemo-sensitive cell line. Our analysis supports the hypothesis that bivalent marks are associated with epigenetic silencing in ovarian cancer. However it also suggests that additional tumour specific bivalent marks, to those known in ES cells, are present in tumours and may potentially influence the subsequent development of drug resistance and tumour progression.

On the determination of the stereochemistry of semisynthetic natural product analogues using chiroptical spectroscopy: desulfurization of epidithiodioxopiperazine fungal metabolites.

Isolation and semisynthetic modification of the fungal metabolite chaetocin gave access to a desulfurized analogue of this natural product. Detailed chiroptical studies, comparing experimentally obtained optical rotation values, electronic circular dichroism spectra, and vibrational circular dichroism spectra to computationally simulated ones, reveal the desulfurization of chaetocin to unambiguously proceed with retention of configuration. Consideration of the plausible mechanisms for this process highlighted inconsistencies in the stereochemical assignment of related molecules in the literature. This in turn allowed the stereochemical reassignment of the natural product analogue dethiodehydrogliotoxin.

Current limitations and future opportunities for epigenetic therapies

This article reviews progress in epigenetic therapies that hope to improve the treatment of cancer. Tumors show widespread, aberrant epigenetic changes, leading to changes in the expression of genes involved in all the hallmarks of cancer. These epigenetic changes can potentially be reversed using small-molecule inhibitors of enzymes involved in maintenance of the epigenetic state. DNA-demethylating agents and histone deacetylase inhibitors have shown anti-tumor activity against certain hematological malignancies; however, their activity in solid tumors remains more uncertain. Major challenges remain in delivery of epigenetic therapy, maintenance of a pharmacodynamic response and achievement of a therapeutic index. We believe histone lysine methyl transferases are a highly promising epigenetic target, which has yet to be clinically exploited. Crystallographic studies on histone lysine methyl transferases provide insights into their mechanism and specificity crucial for the design and development of small-molecule inhibitors.

Dual EZH2 and EHMT2 histone methyltransferase inhibition increases biological efficacy in breast cancer cells

BackgroundMany cancers show aberrant silencing of gene expression and overexpression of histone methyltransferases. The histone methyltransferases (HKMT) EZH2 and EHMT2 maintain the repressive chromatin histone methylation marks H3K27me and H3K9me, respectively, which are associated with transcriptional silencing. Although selective HKMT inhibitors reduce levels of individual repressive marks, removal of H3K27me3 by specific EZH2 inhibitors, for instance, may not be sufficient for inducing the expression of genes with multiple repressive marks.ResultsWe report that gene expression and inhibition of triple negative breast cancer cell growth (MDA-MB-231) are markedly increased when targeting both EZH2 and EHMT2, either by siRNA knockdown or pharmacological inhibition, rather than either enzyme independently. Indeed, expression of certain genes is only induced upon dual inhibition. We sought to identify compounds which showed evidence of dual EZH2 and EHMT2 inhibition. Using a cell-based assay, based on the substrate competitive EHMT2 inhibitor BIX01294, we have identified proof-of-concept compounds that induce re-expression of a subset of genes consistent with dual HKMT inhibition. Chromatin immunoprecipitation verified a decrease in silencing marks and an increase in permissive marks at the promoter and transcription start site of re-expressed genes, while Western analysis showed reduction in global levels of H3K27me3 and H3K9me3. The compounds inhibit growth in a panel of breast cancer and lymphoma cell lines with low to sub-micromolar IC50s. Biochemically, the compounds are substrate competitive inhibitors against both EZH2 and EHMT1/2.ConclusionsWe have demonstrated that dual inhibition of EZH2 and EHMT2 is more effective at eliciting biological responses of gene transcription and cancer cell growth inhibition compared to inhibition of single HKMTs, and we report the first dual EZH2-EHMT1/2 substrate competitive inhibitors that are functional in cells.

Approaches to target the genome and its epigenome in cancer

The term epigenetic landscape was coined by CH Waddington to describe how cell fates were established in development, visualized as valleys and ridges directing the irreversibility of cell type differentiation. It is now clear that normal differentiation control breaks down during tumor development and that all tumor types show aberrant regulation of the epigenetic code, including changes in DNA methylation, histone modification and microRNAs. This has led to much interest in the development of epigenetic cancer therapies to target this aberrant epigenetic regulation. Histone deacetylase and DNA methyltransferase inhibitors are now used in the treatment of certain hematological malignancies. However, their more general applicability to solid tumors may be limited by lack of specificity and delivery challenges. Approaches to overcome these limitations and how to develop more specific drugs are discussed. The use of RNAi in the context of genome regulation as well as the possibility to use polyamides and engineered zinc fingers to target master regulators in the future is examined. Ultimately, improved specificity of epigenetic therapies will require increased mapping of the aberrant epigenetic landscape in cancer and cancer-specific target validation using chemical epigenetic approaches.

Prospects for Epigenetic Compounds in the Treatment of Autoimmune Disease

There is growing evidence for a role for epigenetic mechanisms in the development of autoimmune diseases. In most cases ofautoimmune disease the precise epigenetic mechanism involved remains to be resolved, however DNA hypomethylation accompanied by hypoacetylation ofhistone H3/H4 is commonly observed. Due to the reversible nature of epigenetic marks their maintenance enzymes such as DNA methyltransferases (DNMTs), histone deacetylases (HDACs) and histone lysine methyltransferases (HKMT) are attractive drug targets. Small molecule inhibitors of histone modification and DNA methylation maintenance are increasingly becoming available and will be useful chemical biological tools to dissect epigenetic mechanisms in these diseases. However, although epigenetic therapies used in cancer treatment are a promising starting point for the exploration of autoimmune disease treatment, there is a requirement for more specific and less toxic agents for these chronic diseases or for use as chemopreventative agents.

Abstract A162: Targeting chromatin: Small molecules reactivating H3K27me3 silenced genes.

There is a need for the development of compounds allowing more targeted and efficient reversal of aberrant epigenetic silencing in cancer. Recently, the discovery of histone lysine methyltransferases (HKMT) being involved in tumorgenesis as well as tumor stem cell like maintenance has sparked interest in this particular class of enzymes. We have set-up a cell based assay (MDA-MB-231) in order to identify small molecule inhibitors which are able to re-express endogenous genes where EZH2 has been shown to be involved in the silencing process. In order to identify compounds which inhibit a specific chromatin remodeler class (silencing of chromatin), we chose two DNA unmethylated EZH2 target genes (KRT17, FBXO32), which we would be expect to be re-expressed and one DNA methylated EZH2 target gene (RUNX3) which we would expect to be unaffected in case of an HKMT inhibitor. SiRNA to EZH2 increases expression of KRT17 and FBXO32, but not RUNX3. We have assayed compounds chemically related to a known HKMT inhibitor and identified compounds that up-regulate KRT17 as well as FBXO32 but fail to act on RUNX3. These compounds inhibit tumor cell growth in the low microMolar range. Chromatin immunoprecipitation (ChIP) experiments verified a decrease in silencing marks (H3K27me3, H3K9me3) and importantly an increase in active chromatin marks (e.g. H3K4me2, H3K4me3) at the promoter region of KRT17 and FBXO32. Of note, the H3K27me3 demethylase JMJD3 also showed increased binding at the promoter region which correlated with the presence of H3K4me2/3. In conclusion, we have identified compounds that induce re-expression of genes, reverse H3K27me3 mediated gene silencing and induce inhibition of tumor cell growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A162.

Abstract B165: Structural and biological studies on analogues of the natural product histone methyltransferase inhibitor chaetocin.

Epigenetic mechanisms play a critical role in the regulation of gene expression, and how such regulation becomes aberrant in cancer. Along with DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), histone methyltransferases (HKMTs) have recently appeared as new promising drug targets for epigenetic cancer therapies. While several HDAC and DNMT inhibitors have been clinically validated, to date only few HKMT inhibitors have been reported. Chaetocin, a fungal metabolite belonging to the 3,6-epidithio-diketopiperazine (ETP) class was first described as a specific inhibitor of the histone methyltransferase SU(VAR)3–9.(1) Like other fungal metabolites of the ETP class however, it exhibits a broad range of antibacterial and cytostatic activity, including a remarkable cytotoxicity against HeLa cells. Since the broad cytotoxicity of the ETPs is due to the presence of the disulfide bridge of the 3,6-epidithio-diketopiperazine (causing protein crosslinking, reactive oxygen species generation, or zinc chelation), we reasoned that access to semi-synthetic derivatives devoid of such functionality, would provide valuable insight into the chemical biology of this natural product, particularly its reported HKMT activity. We developed conditions to extract and purify the natural product culture (Chaetomium virescens var. thielavioideum), followed several semi-synthetic approaches to obtain chaetocin analogues. Comparison of experimental and computationally simulated chiroptical spectroscopy allowed unambiguous structural characterization of our products. Evaluation of our compounds in a number of in vitro HKMT assays revealed a total loss in inhibitory potency upon modification of the disulfide bridge. These studies thus demonstrate the ETP functionality of chaetocin to be responsible for its reported HKMT activity. In conclusion, we have synthetically prepared distinct and novel chaetocin analogues from the natural product and explored their HKMT activity, implicating the ETP functionality as the active pharmacophore. Reference : 1. Greiner, D.; Bonaldi, T.; Eskeland, R.; Roemer, E.; Imhof, A. Nat. Chem. Biol. 2005, 1, 143. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B165.

Abstract PL05-04: Epigenetic marks as biomarkers and their mechanisms of maintenance as targets in ovarian cancer.

All types of cancer show wide-spread aberrant epigenetic changes, leading to changes in expression of genes involved in all the classic hallmarks of cancer, as well as genes involved in how tumors will respond to chemotherapy. These epigenetic changes can be reversed, at least temporarily, using small molecule inhibitors of maintenance of the epigenetic state. DNA demethylating agents and histone deacetylase inhibitors have shown activity against certain haematological malignancies, however their activity in solid tumors remains more uncertain (1). For successful treatment of solid tumors with epigenetic therapies, major challenges remain in the delivery of epigenetic therapy, the maintenance of a pharmacodynamic response and the achievement of a therapeutic index. In addition, the development of robust predictive biomarkers linked to an understanding of the underlying biology will be key to improved epigenetic therapy approaches. Defining the epigenetic profile of individual cancer subtypes may allow epigenetic therapies to be targeted to those groups of patients who are most likely to benefit from these types of treatment. Furthermore, targeting drug resistant or tumor sustaining subpopulations with epigenetic therapies may be vital, especially given the key role of epigenetic mechanisms in maintenance of a stem cell state. Ovarian cancer is the most lethal gynaecological cancer. Although at least 70% of patients respond to platinum-based chemotherapy, the majority of patients eventually relapse. We have shown that methylation of multiple promoter CpG islands (CGIs) at genes in the Wnt pathways are associated with progression-free survival (PFS) of epithelial ovarian cancer (EOC) patients (2). We have taken this approach further by systematically profiling DNA methylation at promoter CGIs of 9 further key pathways/families (AKT/mTOR, BRCA1/2, Redox, p53, FA families, igLON families, HR, NHEJ and MMR) in EOCs to identify methylation biomarkers predicting early disease relapse and response to platinum-based chemotherapy. Late-stage EOCs prospectively collected through a SGCTG cohort study were analysed by differential methylation hybridisation (DMH) (n=150) and association with PFS evaluated by Cox model using DMH ratios as a continuous variable. Methylation of 30 loci were correlated with PFS (p NKD1, PRD×2 and VEGFB. Validation of the prognostic values of loci identified were then sought in an independent patient cohort of high-grade serous ovarian tumors (n=311) collected by The Cancer Genome Atlas (TCGA) study and methylation analysed using Illumina Infinium assay. Among the 30 loci identified by DMH as significantly prognostic, only 19 loci were evaluable in the TCGA cohort. Of these 19 loci, promoter methylation of 10 loci remain significantly prognostic in this independent cohort and using this alternative method of methylation analysis. Further analysis using logistic regression model found patients with higher methylation of VEGFB, GP×4 and RAD54L were more likely to have poor response to platinum-based chemotherapy (p We have shown that subpopulations of tumor sustaining cells can be isolated from human ovarian tumors and cell lines which have stem cell like properties, including expression of stem cell markers, growth as spheroids in anchorage independent manner and as xenografts in NOD/SCID mice (3). Such subpopulations are more resistant to chemotherapeutic drugs such as carboplatin and appear to be enriched for following carboplatin-based chemotherapy in patients. This highlights the importance of drugs that can target this drug resistant subpopulation. Ovarian tumor sustaining cells over-express EZH2 (3), a histone lysine methyltransferase (HKMT) and key component of the polycomb repressive complex, PRC2. SiRNA knock-down of EZH2 alone or in combination with EZH1, leads to reduced spheroid and tumor growth of ovarian tumor sustaining cells. We have used a cell based assay to identify small molecules which are able to re-express endogenous genes where EZH2 has been shown to be involved in the epigenetic silencing process. We have identified compounds that up-regulate EZH2 target genes, but not DNA methylated, EZH2 target genes. Chromatin immunoprecipitation (ChIP) experiments verified a decrease in silencing marks (H3K27me3, H3K9me3) and importantly an increase in active chromatin marks (e.g. H3K4me2, H3K4me3) at promoter regions. In ES cells, bivalent chromatin domains, containing H3K4me3 and H3K27me3 marks, silence developmental genes while keeping them poised for later activation following differentiation (4). We have identified gene sets associated with H3K27me3 and H3K4me3 marks at transcription start sites in a high grade ovarian serous tumor to test for correlations with epigenetic silencing and malignancy characteristics. This revealed novel silenced bivalent marked genes not described previously for ES cells, which are significantly enriched for the PI3K (p −7 ) and the TGF-beta signalling pathways (p −5 ). We matched histone marked gene sets to gene expression sets of 8 normal fallopian tube and 499 high grade serous malignant ovarian samples which revealed a significant decrease in gene expression for the H3K27me3 and bivalent gene sets, which occurred largely independent of DNA methylation. Next, we correlated H3K27me3 and bivalent gene sets to gene expression data of ovarian tumor stem cell like sustaining cells versus non-sustaining cells, which showed a significantly lower expression for the H3K27me3 and bivalent gene sets in the tumor sustaining cells. Similarly, comparison of matched chemo-sensitive and chemo-resistant ovarian cell lines showed again a significantly lower expression of H3K27me3/bivalent marked genes in the chemo-resistant compared to the chemo-sensitive cell line. Our analysis supports the hypothesis that bivalent marks are associated with epigenetic silencing in ovarian cancer (5), but suggests that additional tumor specific bivalent marks to those known in ES cells are present in tumor at presentation that can potentially influence the subsequent acquisition of drug resistance and tumor progression. References : 1. Graham, J, Kaye SB and Brown R (2009) The promises and pitfalls of epigenetic therapies in solid tumors Eur J Cancer, 45 : 1129–1136, 2009. 2. Dai W, Teodoridis JM, Zeller C, Graham J, Hersey J, Flanangan JM, Stronach E, Siddiqui N, Paul J, Brown R (2011) Systematic CpG Islands Methylation Profiling of Genes in the Wnt Pathway in Epithelial Ovarian Cancer Identifies Biomarkers of Progression-Free Survival Clin Cancer Res, 17:4052–62. 3. Rizzo S, Hersey JM, Mellor P, Dai W, Santos-Silva A, Liber D, Luk L, Titley I, Carden CP, Box G, Hudson DL, Kaye SB, and Brown R (2011) Ovarian Cancer Stem Cell-Like Side Populations Are Enriched Following Chemotherapy and Overexpress EZH2 Mol Cancer Therapeutics, 10:325–335. 4. Bernstein, B. E., T. S. Mikkelsen, et al. (2006). “A bivalent chromatin structure marks key developmental genes in embryonic stem cells.” Cell, 125: 315–26. 5. Widschwendter, M., Fiegl, H., Egle, D., Mueller-Holzner, E., Spizzo, G., Marth, C., Weisenberger, D. J., Campan, M., Young, J., Jacobs, I., and Laird, P. W. (2007) Epigenetic stem cell signature in cancer. Nat Genet, 39 : 157–158. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PL05-04.