title: Dual EZH2 and EHMT2 histone methyltransferase inhibition increases biological efficacy in breast cancer cells.
abstract: BACKGROUND
  Many 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  We 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.
authors: Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, Hammersmith Hospital Campus, London, W12 ONN UK.
authors: Edward
authors: E
authors: Curry
authors: Division of Cancer, Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, W12 ONN UK.
authors: Ian
authors: I
authors: Green
authors: Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, Hammersmith Hospital Campus, London, W12 ONN UK.
authors: Nadine
authors: N
authors: Chapman-Rothe
authors: Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, Hammersmith Hospital Campus, London, W12 ONN UK.
authors: Elham
authors: E
authors: Shamsaei
authors: Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, Hammersmith Hospital Campus, London, W12 ONN UK.
authors: Sarah
authors: S
authors: Kandil
authors: Department of Chemistry, Imperial College London, South Kensington Campus, London, SW7 2AZ UK.
authors: Fanny L
authors: FL
authors: Cherblanc
authors: Division of Cancer, Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, W12 ONN UK.
authors: Luke
authors: L
authors: Payne
authors: Division of Cancer, Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London, W12 ONN UK.
authors: Emma
authors: E
authors: Bell
authors: Department of Pharmacology, Emory University, Atlanta, GA 30322 USA.
authors: Thota
authors: T
authors: Ganesh
authors: Department of Chemistry, Imperial College London, South Kensington Campus, London, SW7 2AZ UK.
authors: Nitipol
authors: N
authors: Srimongkolpithak
authors: Department of Chemistry, Imperial College London, South Kensington Campus, London, SW7 2AZ UK.
authors: Joachim
authors: J
authors: Caron
authors: Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7 Canada.
authors: Fengling
authors: F
authors: Li
authors: MRC Clinical Sciences Centre, Hammersmith Hospital Campus, London, W12 0NN UK.
authors: Anthony G
authors: AG
authors: Uren
authors: Department of Chemistry, Emory University, Atlanta, GA 30322 USA.
authors: James P
authors: JP
authors: Snyder
authors: Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7 Canada.
authors: Masoud
authors: M
authors: Vedadi
authors: Department of Chemistry, Imperial College London, South Kensington Campus, London, SW7 2AZ UK.
authors: Matthew J
authors: MJ
authors: Fuchter
authors: Department of Surgery and Cancer, Ovarian Cancer Action Research Centre, Imperial College London, Hammersmith Hospital Campus, London, W12 ONN UK ; Section of Molecular Pathology, Institute of Cancer Research, Sutton, SM2 5NG UK.
authors: Robert
authors: R
authors: Brown