Abstract 5489: Using genetic and chemical approaches to probe the mechanism of DOT1L recruitment in MLL fusion leukemia

Abstract

Abstract Mixed lineage leukemia (MLL) is an aggressive form of leukemia where the MLL gene is translocated and fused to more than 80 different nuclear, cytoplasmic and membrane proteins. MLL-AF9 is one of the most common MLL-fusions. This fusion partner is known to recruit several multiprotein complexes including the AEP, SEC and DOT1L complexes leading to transcriptional activation. In particular, the recruitment of Disruptor of telomeric silencing 1-like (DOT1L), a H3K79 histone methyltransferase, is essential for leukemogenesis by multiple MLL fusion proteins. We mapped the binding site to a 10 amino acid segment (865 - 874) on DOT1L where AF9 binds. Peptides derived from this region that show dose-dependent disruption of this interaction. We also demonstrated that DOT1L mutants lacking these 10 residues did not support transformation by MLL-AF9. Encouraged by these results, we generated both genetic and chemical tools to elucidate the role of DOT1L recruitment to the MLL fusion partners and the mechanism of leukemogenic inhibition by disrupting the protein-protein interaction (PPI) between MLL-AF9 and DOT1L. Fl/Fl DOT1L MLL-AF9 Cre+ cell lines were generated with different constructs of DOT1L. These constructs consisted of DOT1L mutants lacking the 10 amino acid binding site, a I867A point mutant known to block DOT1L binding, and an enzymatic mutant known to yield a catalytically inactive protein. As control cell lines, MLL-AF6, a MLL-fusion containing a cytoplasmic protein, and E2A-HLF, a non-DOT1L dependent fusion, were generated to demonstrate the specific effects of generated DOT1L mutant constructs. Both DOT1L PPI mutants impaired the transformation by MLL-AF9 and induced cell death by inducing apoptosis and cell cycle arrest similarly to enzymatic inhibition. These results established a foundation for discovering small-molecule inhibitors that disrupting the AF9-DOT1L as potential disease-specific therapies that target chromatin modifications in this highly aggressive leukemia. A high throughput screening was conducted identifying several different chemical classes of small molecules that bind to the AF9 C-terminal hydrophobic binding site and disrupt the PPI between DOT1L and MLL-AF9 fusion protein. Identified small molecule inhibitors were validated with series of biochemical, functional and cell-based assays. Validated compounds selectively inhibit the growth of the DOT1L dependent murine cells and induce cell death in a similar manner to the genetic approach. The small molecules also showed specificity in killing human MLL-fusion cell lines in comparison to non-MLL fusion leukemia. These results show that blocking the recruitment of DOT1L by AF9 using both genetic and chemical tools eliminate MLL-AF9 mediated immortalization emphasizing an essential function for this interaction in leukemogenesis and warrant further development of the identified small-molecule inhibitors. Citation Format: Sierrah M. Grigsby, James Ropa, Justin Serio, Chenxi Shen, Jennifer Chase, Ivan Maillard, Andrew Muntean, Zaneta Nikolovska-Coleska. Using genetic and chemical approaches to probe the mechanism of DOT1L recruitment in MLL fusion leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5489. doi:10.1158/1538-7445.AM2017-5489