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.