The Histone Methyltransferase DOT1L: Discovery of Small-Molecule Inhibitors and its Role in Wnt Signaling.

Abstract

Covalent post translational modifications of histone proteins are an important mechanism of epigenetic gene regulation that modulate chromatin structure. Methylation of histone lysine residues is one of several chemical marks that establish the “histone code” essential for proper temporal and spatial expression of gene programs required for cell fate determination in development. Dysregulation of histone methylation contributes to the development of numerous human diseases, particularly cancer. The sole histone H3 lysine 79 (H3K79) methyltransferase, DOT1L, is required for leukemogenic transformation in a subset of leukemias bearing translocations of the MLL gene. Human leukemias carrying MLL gene rearrangements aberrantly recruit DOT1L to leukemogenic genes leading to increased H3K79 methylation and their transcriptional activation. There are also reports that DOT1L plays a role in Wnt signaling, a pathway frequently dysregulated in colon cancer. Small molecule inhibitors of DOT1L are highly sought for the development of therapeutics in leukemia and as chemical tools to probe the role of DOT1L in other human diseases. We applied several approaches for the identification of DOT1L inhibitors, virtual screening, de novo design, and biochemical screening. Here we present the biochemical, biophysical, and cellular characterization of different classes of DOT1L inhibitors. Several S-adenosylmethionine (SAM) analogues have been identified as DOT1L inhibitors by virtual screening and we developed a novel pathway for synthesis of additional 5’ modified adenosine analogues. Additionally, we identified UMD-7, which inhibits H3K79 methylation by a unique mechanism of histone binding and phenocopies genetic loss of DOT1L. Employing a chemical biology approach the requirement for H3K79 methylation in Wnt signaling was investigated by inhibiting DOT1L with EPZ004777, a selective and potent SAM competitive inhibitor. Our findings indicate that H3K79 methylation is not essential for maintenance or activation of Wnt pathway target gene expression in colon cancer cell lines. Furthermore, H3K79 methylation is not elevated in human colon carcinoma samples in comparison with normal colon tissue. Therefore, our findings indicate that inhibition of DOT1L histone methyltransferase activity is likely not a viable therapeutic strategy in colon cancer.