Abstract 3878: A highly sensitive assay for the discovery of inhibitors of the histone methyltransferase DOT1L to treat MLL-rearranged leukemia
Authors: Watson Venita G. , Drake Katherine M. , Napper Andrew D. 
Published: August 2012

Abstract
MLL-rearranged leukemia is an aggressive form of cancer characterized by specific chromosomal rearrangements of the mixed lineage leukemia (MLL) gene. In normal cells, MLL, a histone methyltransferase, methylates the lysine 4 residue on histone 3 (H3K4) and thereby regulates the expression of genes which control hematopoietic development. In leukemic cells, chromosomal rearrangements fuse the N-terminus of MLL with one of more than 50 partner proteins, giving rise to oncogenic fusion proteins that lack H3K4 methyltransferase activity. These fusion proteins inappropriately recruit DOT1L, which methylates H3K79, producing an abnormal methylation pattern and activation of genes normally suppressed during differentiation. Elevated expression of HOXA9 in particular leads to a block in normal hematopoetic differentiation and the onset of leukemia. A solid body of evidence has shown DOT1L to be crucial for the development and maintenance of MLL-rearranged leukemias; consequently, it is a promising target for therapeutic intervention. We have focused on developing an assay suitable for high-throughput screening (HTS) to detect compounds that inhibit DOT1L enzymatic activity. DOT1L catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to H3K79 and generates S-adenosylhomocysteine (SAH) as a byproduct. The enzyme displays a very low rate of methylation of histone octamers or nucleosomes and no activity towards individual histones or histone-derived peptides. Thus DOT1L HTS requires a highly sensitive assay that is able to accommodate protein-protein and protein-DNA complexes as substrates. An assay using radiolabeled SAM has sufficient sensitivity, but is relatively laborious and not well-suited for HTS. We tested several previously reported non-radioactive assay formats, but none enabled robust detection of DOT1L activity. Therefore, we developed a coupled enzyme assay in which SAH is converted to homocysteine and adenosine using SAH hydrolase. In a second enzyme-coupled step, adenosine kinase generates AMP and ADP from the SAH-derived adenosine and added ATP. Finally, ADP is detected using the luciferase-based Promega ADP-Glo assay. The luminescent signal provides a highly sensitive measure of ADP at a concentration as low as 10 nM. The readout is directly proportional to SAH production; thus DOT1L inhibitors are identified by a reduction in this signal. We report development of the assay, including careful optimization of the activity of adenosine kinase to enhance sensitivity, and assay validation demonstrating DOT1L inhibition by the non-selective SAM-competitive inhibitor sinefungin, and pilot HTS of a 2,000-compound library.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3878. doi:1538-7445.AM2012-3878