Characterization of the Protein-Protein Interactions between Histone Methyltransferase DOT1L and MLL Fusion Proteins towards Developing Small Molecule Inhibitors.

Abstract

The MLL fusion proteins activate target genes in part via recruitment of DOT1L (disruptor of telomeric silencing 1-like), a histone H3 lysine 79 (H3K79) methyltransferase. The resulting hypermethylation of H3K79 at the Hox and MEIS1 loci is a pivotal event for leukemogenesis in acute leukemia, suggesting that the protein-protein interactions (PPIs) between DOT1L and MLL oncogenic fusion proteins represent a potential therapeutic target. The PPIs between DOT1L and the MLL fusion proteins, AF9 and ENL, were characterized. It was determined that full length DOT1L protein binds to AF9 and ENL with Kd of 33 nM and 206 nM respectively. The AF9/ENL binding site in human DOT1L was mapped and 10 amino acids (DOT1L865-874), highly conserved in DOT1L from a variety of species, were identified as essential for binding to AF9/ENL. Alanine scanning mutagenesis studies demonstrated that four conserved hydrophobic residues are crucial for the binding. Functional colony forming unit (CFU) assay showed that the mapped AF9/ENL interacting site is essential for immortalization by MLL-AF9, indicating that the physical interaction between DOT1L and MLL-AF9 is required for transformation by MLL fusions. These results strongly suggest that disruption of interaction between DOT1L and AF9/ENL is a promising therapeutic strategy for MLL leukemias with potentially fewer adverse effects than enzymatic inhibition of DOT1L. High-throughput screening (HTS) approach was employed for the discovery of small-molecule inhibitors targeting the PPI between DOT1L and MLL-fusion proteins. Approximately 101,000 compounds were screened and 39 compounds demonstrated dose-dependent inhibition of AF9-DOT1L interaction. For the first time, four compounds with diverse chemical structure were identified and validated as inhibitors of the DOT1L - AF9 PPI with IC50 values ranging from 10 – 50 µM. This is an important initial proof-of-concept for the development of small molecules targeting C-terminal hydrophobic domain in AF9, indicating it is a “druggable” target. Such inhibitors can serve as chemical probes to understand the biological consequences of inhibiting DOT1L recruitment by MLL-fusion proteins, AF9 and ENL, and the impact on leukemia development, as well as on normal hematopoiesis. Ultimately, this work may result in the development of novel drugs for treatment of MLL acute leukemias.