Abstract SY08-01: Protein methyltransferases as a target class for cancer drug discovery
Authors: Copeland Robert A. 
Published: July 2012

Abstract
The protein methyltransferases (PMTs) constitute a class of enzymes that catalyze the methylation of lysine or arginine residues on histones and other proteins. Methylation of specific residues on histones leads to localized chromatin remodeling that in turn controls gene transcription. A number of PMTs have been shown to be genetically altered in cancers through, for example, gene amplification, chromosomal translocations and point mutations; in several cases the enzymatic activity of the genetically altered PMT has been shown to be critical for malignancy. We are approaching drug discovery efforts against these enzymes in two distinct, but interrelated manners. First, we are approaching the PMTs as a target class, taking advantage of common aspects of the enzymology of these enzymes to develop a broad platform of biochemical assays with which to search for small molecule inhibitors. To facilitate these efforts, we have performed a comprehensive survey of the human genome to define the constituency and structural relatedness among the active sites of these enzymes. We find that there are 96 putative PMTs within humans and that the target class divides into two families, largely based on methyl-accepting amino acid preference: the protein lysine methyltransferase family (PKMTs) and the protein arginine methyltransferase family (PRMTs). Currently, our cross-screening panel of biochemical assays consists of 12 representative members of the PMT class, including both PKMTs and PRMTs. Potent, selective inhibitors of most of the enzymes in this cross-screening panel have been identified through these efforts. Our second approach targets specific enzymes that are genetically altered in cancer, and for which strong evidence of a pathogenic role of enzymatic activity has been established. The enzymes DOT1L and EZH2 provide two representative examples of this second approach. Patients suffering from mixed lineage leukemia (MLL) contain chromosomal translocations affecting the MLL gene, resulting in fusion of part of the MLL gene product with partners from the AF and ENL families of proteins. In the context of this translocation, DOT1L is ectopically recruited to aberrant gene locations where is methylates lysine 79 of histone H3, leading to increased expression of leukemogenic genes. We have identified a potent, selective inhibitor of DOT1L, EPZ004777. This compound is a ca. 80 pM, SAM-competitive inhibitor of DOT1L activity that demonstrates &gt; 1000-fold selectivity against other PMTs. In cells the compound leads to selective, concentration-dependent inhibition of H3K79 methylation and accompanying changes in gene transcription. The compound selectively kills leukemia cells bearing the MLL translocation without effect on the proliferation of nontranslocated cells. The biochemical and biological characteristics of this compound suggest that selective inhibition of DOT1L activity is a viable mechanism of therapeutic intervention in MLL. EZH2 is the catalytic subunit of the multiprotein polycomb repressive complex 2 (PRC2), which catalyzes methylation of H3K27 and resulting suppression of gene transcription. An increased level of trimethylated H3K27, catalyzed by the enzymatic activity of PRC2, has been suggested to drive a number of tumor types. Recently, point mutations of tyrosine 641 in EZH2 were reported in subsets of patients with non-Hodgkin lymphomas (NHL). Patients bearing these mutations were always found to be heterozygous, expressing both a wild-type (WT) and mutant EZH2 message. Initial biochemical analysis suggested that these mutants resulted in a loss-of-function phenotype. We performed a detailed set of biochemical studies on these mutants. While the mutant EZH2 forms were unable to methylate an unmodified H3K27-representing peptide substrate, they all were active methyltransferases when provided with natural oligonucleosomes as substrate. The nucleosomes isolated from natural sources are heterogeneous with respect to the methylation state of H3K27. We therefore investigated the effect of H3K27 methylation status on catalysis by the mutant EZH2 forms. The wild-type enzyme is most effective in catalyzing the first methylation reaction, leading from lysine to monomethyllysine; it is less effective at catalyzing the second reaction leading to dimethyllysine and least effective at catalyzing the third reaction leading to trimethyllysine at H3K27. In stark contrast, all of the mutant forms of EZH2 displayed the opposite pattern of substrate utilization; they were ineffective as H3K27 monomethyltransferases but were quite effective as trimethyltransferases. The steady state kinetic parameters for the WT and mutant enzymes suggested that when both forms are present in a cell (i.e., heterozygous) enzymatic coupling occurs, leading to enhanced levels of trimethylated H3K27. The pattern of H3K27 methylation predicted by the kinetic simulations described well the pattern observed in mutant-bearing NHL cells. These data suggest that enzymatic coupling between WT and mutant EZH2 is required for the elevated trimethylation of H3K27 that drives lymphomagenesis in these cells. Consistent with this hypothesis, application of potent, selective EZH2 inhibitors to cells leads to a selective killing of lymphoma cells that are heterozygous for the EZH2 mutants at concentrations that are inconsequential to lymphoma cells that are homozygous for WT EZH2. The data for both DOT1L and EZH2 inhibitors suggest that potent, selective inhibitors of genetically altered PMTs may be a general approach to targeted therapies in patients for which the genetic alteration drives disease.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY08-01. doi:10.1158/1538-7445.AM2011-SY08-01