Abstract A158: Therapeutic strategies for targeting histone methyltransferase DOT1L in MLL rearranged leukemia.
Authors: Gibbons Garrett , Shen Chenxi , Muntean Andrew , Jo Stephanie , Hess Jay , Nikolovska-Coleska Zaneta 
Published: June 2012

MLL fusion proteins activate target genes in part via recruitment of the histone-modifying enzyme, DOT1L (disruptor of telomeric silencing 1-like), a unique histone methyltransferase with selective activity for histone H3 lysine 79 (H3K79). The resulting hypermethylation of H3K79 at the HOX and MEIS1 loci appears to be pivotal for leukemogenesis in both AML and ALL. These findings emphasize the central role of DOT1L in leukemogenesis and strongly suggest that pharmacologic inhibition of DOT1L could be of therapeutic benefit for a number of ALL and AML subtypes. Applying a biochemical screening approach, two classes of small molecule DOT1L substrate competitive inhibitors were discovered. They selectively inhibit the cell growth in leukemic cells with MLL-translocation, in comparison with leukemic cells without MLL translocation, resulting in a dose and time dependent decrease of cellular H3K79 methylation, decrease in expression of MLL-target genes HOXA9 and MEIS1, inducing G0/G1 cell cycle arrest, an increase in expression of differentiation markers and death by apoptosis.

Although the DOT1L inhibitors showed no toxicity on normal bone marrow, knockout studies show that DOT1L deficiency is associated with chromosomal instability and fatal progressive anemia. This suggested that developing therapeutic strategies other than global inhibition of DOT1L activity may be necessary. Therefore we characterized the protein-protein interaction (PPI) between DOT1L and ENL/AF9 and assessed its importance on MLL fusion protein-mediated transformation. We found that ENL and AF9 C-terminal domains bind to DOT1L with Kd of 238 nM and 111 nM respectively. We have mapped the binding site in DOT1L and found that only 10 amino acid of DOT1L are involved in interaction with ENL and AF9. Synthetic DOT1L 10mer peptide binds ENL/AF9 and disrupts the interaction between DOT1L/ENL and DOT1L/AF9. Alanine scanning mutagenesis studies showed that four hydrophobic residues are detrimental for the binding of DOT1L to both proteins ENL/AF9. To examine the critical role of the DOT1L-AF9 interaction in transformation by MLL fusion protein, colony forming unit (CFU) assay was performed using MLL-AF9 transformed cells lacking endogenous DOT1L. The colony forming potential of the MLL-immortalized cells was completely abolished by introducing DOT1L construct lacking 10 amino acid AF9 interacting residues (DOT1L (Δ10aa)), while the wild type DOT1L construct was able to restore CFU formation. Importantly global levels of H3K79 methylation were equivalent, but only the DOT1L (Δ10aa) construct failed to restore CFU formation. These results demonstrate that DOT1L interaction with MLL-AF9 in addition to H3K79 methylation is required for transformation of MLL-AF9. This suggests that selective disruption of this PPI is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL-fusion protein associated leukemias.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A158.