Abstract IA016: Characterization of the protein-protein interactions involved in hDOT1L epigenetic regulation on biochemical, structural and functional level towards developing new therapeutic intervention
Authors: Nikolovska-Coleska Zaneta 
Published: December 2022

MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. More than 90 different MLL translocation partners have been identified with the most common fusion partners, AF9, ENL, AF4, and AF10. AF9 and ENL are closely related members of the YEATS domain protein family and have been identified as part of several reported complexes including Super Elongation Complex (SEC) and DotCom Complex. AF9 and ENL, through their C-terminal hydrophobic domain, known as ANC1 Homology Domain (AHD), recruit the histone H3K79 methyltransferase hDOT1L to the MLL target genes, constitutively activating transcription of pro-leukemic targets. We have characterized the protein−protein interactions between hDOT1L and MLL fusion oncogenic proteins, AF9 and ENL, on the biochemical, biophysical, and functional level. This study mapped a 10 amino-acid region (aa 865− 874), highly conserved in hDOT1L from a variety of species, as an interacting site with the AHD domain of AF9/ENL proteins and demonstrated that these PPIs are essential for cellular immortalization mediated by MLL-AF9. Applying genetic approach and studying the effects of blocking the AF9-hDOT1L PPIs or hDOT1L enzymatic activity, it was demonstrated complete disruption of hDOT1L recruitment to critical target genes, Hoxa9 and Meis1, in cells expressing hDOT1L mutants with an impaired AF9 binding site. This was followed by significant decrease in H3K79me2 in the promoter regions of these genes, cell differentiation and induction of apoptosis. To identify the potential therapeutic benefits of this novel targeting approach, the effect of these different hDOT1L mutants was investigated on adult murine hematopoiesis. The in vivo studies demonstrated that genetic interventions that result in loss of hDOT1L enzymatic activity rapidly depletes hematopoietic stem and progenitor cells. In contrast, blocking the AF9-hDOT1L PPIs allowed for sustained hematopoiesis. These studies provided an important proof of concept for the potential therapeutic advantage of inhibiting the AF9-hDOT1L interaction and disrupting the integrity of the MLL-fusion complexes. In addition, these results demonstrated that hDOT1L recruitment can be abolished by a single point mutant (I867A), highlighting the crucial role of the hydrophobic interactions and giving insights for further drug discovery. We have reported the first peptidomimetics targeting PPIs between MLL-AF9 and hDOT1L, providing evidence for the druggability assessment of this novel potential therapeutic approach. The AF9/ENL C-terminal domain, is an intrinsically disordered protein, representing a challenge for structural studies. Applying X-ray crystallography, we have successfully determined the 3D structure of the AF9 AHD domain in complex with peptidomimetics establishing a platform for structure-based design of novel, optimized, potent inhibitors, the current focus of our research efforts.

Citation Format: Zaneta Nikolovska-Coleska. Characterization of the protein-protein interactions involved in hDOT1L epigenetic regulation on biochemical, structural and functional level towards developing new therapeutic intervention. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA016.