Pathologies such as human cancer result largely from the inappropriate silencing or activation of genes. It is well established that gene expression can be partly controlled by modulating the access of the transcriptional machinery to target genes through chemical modifications of DNA sequences or histones, the proteins that package DNA. These modifications are mediated by cellular enzymes, including DNA methyltransferases, histone acetyl transferases (HATs), histone deacetylases (HDACs), histone methyltransferases (HMTs), histone demethylases, and histone kinases. Epigenetic enzymes function during development, helping orchestrate complex transcriptional programs that control differentiation pathways. During adult life, these enzymes continue to influence transcription by maintaining tissue-specific epigenetic and transcriptional patterns as well as by acting as coregulators for transcription factors. In many cancers, the regulation of transcriptional processes is altered partly because of the abnormal expression and/or function of epigenetic enzymes resulting in the silencing of tumor suppressor genes or in equivalent events that lead to unchecked cellular growth [1
]. Indeed, it has been estimated that epigenetic changes are at least ten to forty times more frequent in cancers than genetic mutations [1
]. Furthermore, specific mutations in HATs and HMTs have been found in relation to the cancer phenotype, suggesting them as potential targets for therapy [4
]. Thus, effective treatment of cancer will, at least in part, necessitate the chemical targeting of the cancer epigenome.
Over the last two decades, drugs have been identified that modulate the pathways mediated by a subset of epigenetic enzymes. Because cancer cells often have abnormally silenced tumor suppressor genes or overexpressed oncogenes, mediated by epigenetic pathways, these drugs have been studied in preclinical contexts. Of the known compounds, however, only a few have shown success in clinical settings, with toxicities observed for most other compounds due to their global, unspecific effects on cell function [5
]. This has led to new drug discovery and drug development efforts at industrial and academic laboratories over the last few years. These programs have used cell-based, in vitro
, in silico
, or yeast systems to identify novel drugs or have developed second-generation compounds structurally related to already known inhibitors [10
Altogether, more recent studies have yielded several new compounds that target epigenetic enzymes, primarily histone deacetylase family members and enzymes that modulate methylation [15
]. Some of these compounds offer limited benefit over existing drugs, since they are structurally closely related to known inhibitors of epigenetic enzymes, are unspecific, or lack substantial in vivo
activity, due at least in part to limitations in drug screen design. To date, there are only a few epigenetic drugs approved by the FDA, including: 5-azacytidine and its deoxy derivative decitabine, both DNA methyltransferase inhibitors used for the treatment of myelodysplastic syndromes, vorinostat, and recently romidepsin, HDAC inhibitors used for the treatment of cutaneous T cell lymphoma [23
]. There is, therefore, a persistent need to increase the number and diversity of available anticancer epigenetic modulators and to develop innovative, improved approaches for drug discovery.
Because screens that use in vitro
or in silico
approaches may lead to hits that prove to be toxic, insoluble, or inefficient when taken to the in vivo
setting, using systems in which drugs are directly tested in cells, as was done for some of the original HDAC inhibitors [10
], can save time and effort on followup studies of drugs that are only effective in vitro
or would require substantial chemical optimization. Here, we report the characterization and use of a cell-based assay in which a locus containing an easily quantifiable marker, green fluorescent protein (GFP), is epigenetically silenced, and derepressed chemically by known epigenetic modulators targeting both histone acetylation and DNA methylation. We have now successfully used this system, the Locus Derepression assay (LDR) [27
], to screen the NCI's structural diversity library to identify novel compounds with epigenetic activity. Four confirmed hits from the screen were further investigated for their anticancer properties and their ability to inhibit histone deactylases. We found that two of our hits potently blocked the viability of both lung cancer and melanoma cells and that one of them caused cancer cells to accumulate in the G2/M phase, preventing cell cycle progression. A third hit inhibited deacetylase activity in vitro
and in cells, but on its own had little toxicity, while a fourth compound selectively inhibited the viability of melanoma cells compared to lung cancer cells.